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The annexin A2 heterotetramer as a host cell cofactor in human papillomavirus infection

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The annexin A2 heterotetramer as a host cell cofactor in human papillomavirus infection

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Content i

The annexin A2 heterotetramer as a host cell
cofactor in human papillomavirus infection

Julia Rose Taylor

A dissertation submitted for the degree of

Doctor of Philosophy
in Medical Biology

Faculty of the USC Graduate School
University of Southern California

May 2019
ii
Dedication

This thesis is dedicated to anyone who has donated biologically, financially, or
emotionally to the advancement of biomedical research. And to my rock, Thomas.

iii
Acknowledgements

To Dr. Kast, Diane, my committee, my family, my friends (C&K), my mom, and to
Thomas: I will be forever grateful for your continued guidance and support.

iv
List of Abbreviations

A2t Annexin A2/S100A0 heterotetramer
A2ti A2t inhibitors
A2ti-1 First generation A2ti
A2ti-2 Second generation A2ti
Ab Antibody
AnxA2 Annexin A2
BME Beta-mercaptoethanol
bp Base pairs
BSA Bovine serum albumin
BTV Bluetongue virus
CDK Cyclin dependent kinase
CFDA-SE Carboxyfluorescein diacetate succinimidyl ester
CMV Cytomegalovirus
CSFV Classical swine fever virus
DMSO Dimethyl sulfoxide
dsDNA Double stranded DNA
EDTA Ethylene-diamine-tetra acetic acid
EGFR Epidermal growth factor receptor
ER Endoplasmic reticulum
EV71 Enterovirus 71
FBS Fetal bovine serum
FITC-BSA Fluorescein isothiocyanate-conjugated BSA
GFP Green fluorescent protein
gRNA Guide RNA
HCV Hepatitis C virus
HFMD Hand, foot, and mouth disease
HIV Human immunodeficiency virus type 1
v
HNC Head and neck cancer
HPV Human papillomavirus
HPV16 HPV type 16
HSPGs Heparan sulfate proteoglycans
IAV Influenza A virus
IBV Infectious bronchitis virus
IC 50 Half maximal inhibitory concentration
IMDM Iscove’s Modified Dulbecco’s Medium
ITC Isothermal titration calorimetry
KO Knock-out
L1 HPV16 major capsid protein L1
L2 HPV16 minor capsid protein L2
M Matrix protein M
MFI Mean fluorescence intensity
MOI Multiplicity of infection
Ms Mouse
MV Measles virus
MVEs Multivesicular endosomes
NS Non-structural
OBSL1 Obscurin-like 1 protein
p.i. Post-infection
PBS Phosphate buffered saline
PBST phosphate buffered saline with tween
PFA Paraformaldehyde
PI Propidium iodide
PRRSV Porcine reproductive and respiratory syndrome virus
PsV Pseudovirion
rAnxA2 Recombinant AnxA2
RaV Rabbit vesivirus
vi
Rb Rabbit
RSV Respiratory syncytial virus
s.d. Standard deviation
SLPI Secretory leukocyte protease inhibitor
ssRNA Single stranded RNA
TCID50 50% tissue culture infective dose
TEMs Tetraspanin enriched microdomains
TGN Trans-Golgi network
transferrin-AF488 Alexa Fluor® 488-labeled transferrin
vDNA Viral DNA
VLPs Virus-like particles
WT Wild type

vii
Table of Contents
DEDICATION II
ACKNOWLEDGEMENTS III
LIST OF ABBREVIATIONS IV
TABLE OF CONTENTS VII
CHAPTER 1 - AN INTRODUCTION TO HPV, ANNEXIN A2, AND THE
HOST-PATHOGEN INTERACTION 1
1.1 HPV and disease 1
1.2 HPV entry and intracellular trafficking 2
1.3 HPV and the annexin A2/S100A10 heterotetramer (A2t) 4
CHAPTER 2 - SMALL MOLECULE INHIBITORS OF
HETEROTETRAMERIC ANNEXIN A2/S100A10 PREVENT HPV16
INFECTION, IN VITRO 8
2.1 Introduction 8
2.2 Results 9
A2ti bind to A2t and are non-toxic to HeLa cells 9
A2ti block HPV16 infection in HeLa cells 12
A2ti treatment reduces HeLa cell proliferation 13
2.3 Discussion 14
2.4 Methods 15
Cell culture (HeLa), reagents, and pseudovirion (PsV) production 15
Cytotoxicity assay 16
HPV16 pseudovirus infection assay 16
Cell proliferation assay 16
viii
CHAPTER 3 - GENETICALLY ENGINEERING CELLS TO FURTHER
INVESTIGATE A2T IN HPV INFECTION 17
3.1 Introduction 17
3.2 Results 18
CRISPR/Cas9 gene editing effectively knocks out S100A10
and AnxA2 protein expression 18
AnxA2 and A2t are essential for the infection of HPV16 in HeLa cells 20
S100A10 and AnxA2 are not required for efficient cell proliferation 20
3.3 Discussion 23
3.4 Methods 25
Cell culture 25
Antibodies 25
CRISPR/Cas9 gene editing 25
Pseudovirus infection and infection rescue assay 26
Cell proliferation analyses 26
CHAPTER 4 - HETEROTETRAMERIC ANNEXIN A2/S100A10 (A2T) IS
ESSENTIAL FOR ONCOGENIC HUMAN PAPILLOMAVIRUS
TRAFFICKING AND CAPSID DISASSEMBLY, AND PROTECTS
VIRIONS FROM LYSOSOMAL DEGRADATION 28
4.1 Introduction 28
4.2 Results 31
Annexin A2 (AnxA2) and heterotetrameric AnxA2/S100A10 (A2t) are
essential for the infection of high-risk oncogenic HPV in HeLa cells 31
Restoration of A2t through AnxA2 knock-in restores HPV16 infectivity 34
A2t is not required for HPV cell surface attachment 36
Bulk HPV internalization is not affected by S100A10 KO and A2t KO,
and lysosomal degradation is increased 39
HPV endocytosis remains clathrin-independent in S100A10 KO and A2t KO cells
42
ix
A2t controls the progression of HPV-containing early endosomes to late
multivesicular endosomes (MVEs), inhibits capsid disassembly, and
AnxA2 forms a complex with tetraspanin CD63 during HPV trafficking 44
4.3 Discussion 50
4.4 Methods 54
Cell culture, CRISPR/Cas9 gene editing, and proliferation analysis 54
Pseudovirion and virus-like particle production 55
Antibodies 55
Pseudovirus infection and infection rescue assay 57
Cell surface binding assay 57
Virus internalization assays 58
Lysosome degradation assay via Western blot 60
Endocytic uptake assays 60
Immunofluorescence microscopy 60
Statistics 61
CHAPTER 5 - DISCUSSION 62
5.1 Introduction 62
5.2 Broader implications 62
Annexin A2 as an emerging viral host factor 64
Annexin A2 and human papillomavirus (HPV) 67
Annexin A2 and enterovirus 71 (EV71) 67
Annexin A2 and respiratory syncytial virus (RSV) 67
Annexin A2 and cytomegalovirus (CMV) 68
Annexin A2 and hepatitis C virus (HCV) 68
Annexin A2 and influenza A virus (IAV) 69
Annexin A2 and measles virus (MV) 70
Other viruses and species with Annexin A2 associations 71
5.5 Patterns, potential, and future directions 74
x
PUBLICATIONS BY THE AUTHOR 75
REFERENCES 77
1
Chapter 1

An introduction to HPV, annexin A2, and the host-pathogen
interaction
1.1 HPV and disease
High-risk human papillomavirus (HPV) is one of the most common sexually transmitted
viruses and leads to a variety of human cancers such as cervical and throat cancer.
Importantly, the link between HPV and head and neck cancer (HNC) is much stronger
than once predicted, as HPV is involved in up to 25% of HNCs and accounts for at least
60% of oropharyngeal carcinomas
1–3
. Despite the availability of a fully effective
preventative vaccine, vaccine coverage in the United States remains low
4
. Many
obstacles stand in the way of herd immunity and HPV eradication: clinically, the vaccine
requires a doctor’s prescription and a 3-dose series over the course of 6 months;
logistically, the vaccine is costly and requires refrigeration; and socially, barriers
include conservative ideals about adolescent sexual activity, anti-vaccination
campaigns, and today’s common misconceptions about vaccine safety and the existence
of male HPV-associated diseases. Moreover, the World Health Organization reports that
more than 85% of cervical cancer deaths occur in developing countries, which have
particularly limited access to the vaccine. In addition to breaking these barriers, there is
clearly an urgent need for alternative therapeutic strategies to prevent HPV-associated
cancers.
Viruses exploit many normal cellular entry processes to gain access into their
host organisms, and understanding these host-pathogen connections can lead to novel
anti-viral targets and strategies. Our knowledge of how HPV infiltrates human host cells
however, remains incomplete. Furthermore, a review summarizing the current status
of early investigational pharmacotherapies against HPV-induced precancerous lesions
concluded that presently, these drugs are neither viable nor efficacious options for
2
clearing HPV infection and resultant dysplasia
5
. Thus, investigating how cells process
oncogenic HPV type 16 (HPV16) may reveal new avenues for prevention and treatment
of HPV. Because of the significant HPV-associated disease burden, the challenges to
vaccine uptake, limited therapeutic options for infected individuals, and because we
lack a comprehensive understanding of the host-pathogen interaction, HPV cellular
entry and infection remain areas of active investigation.
1.2 HPV entry and intracellular trafficking
The mechanism of HPV endocytosis into epithelial cells is independent of common
canonical entry pathways including clathrin-, caveolin-, cholesterol-, flotillin-, and
dynamin-dependent endocytosis
6,7
. Although HPV entry is not fully characterized, it is
understood that HPV must gain access into host cells to deliver its double-stranded DNA
to the nucleus in a process that appears to be receptor-mediated and related to
micropinocytosis with respect to actin dynamics
7,8
.
Trafficking of HPV from the cell surface to the nucleus can be broken down into
five key stages: cell surface binding (Fig. 1.1a), entry (Fig. 1.1b), viral vesicle trafficking
(Fig. 1.1c), capsid uncoating (Fig. 1.1d), and transportation of the viral DNA (vDNA)
through the trans-Golgi network (TGN) to the nucleus (Fig. 1.1e). HPV binds to the cell
surface through two distinct attachment events. First, HPV capsid proteins interact with
heparan sulfate proteoglycans (HSPGs) found on the plasma membrane of basal
keratinocytes or within the surrounding extracellular matrix
9–12
. The binding of HPV to
HSPGs induces conformational changes in both HPV capsid proteins L1 and L2
13–15
,
exposing the amino terminus of HPV L2 which contains a furin/proprotein convertase
cleavage site
16
. These conformational changes in the capsid reduce HSPG-affinity and
the virion is then transferred to the elusive secondary uptake receptor/receptor complex
located within tetraspanin enriched microdomains (TEMs)
6,17,18
. Candidate receptors to
3
date have included a6 integrin
19,20
, epidermal growth factor receptor (EGFR)
21,22
, and the
protein complex studied herein – the annexin A2/S100A10 heterotetramer (A2t)
23,24
.
After handoff to this secondary receptor/receptor complex, HPV is internalized
through a non-canonical endocytic mechanism and trafficked through the degradative
endosomal system. While it has been shown that in optimal conditions viral trafficking
may be rapid, bulk internalization is relatively slow and asynchronous due to the time it
takes for extracellular structural modifications of the capsid and a hypothesized limited
availability of the secondary receptor/receptor complex
25,26
. Intracellular trafficking is
dependent on endocytic mediators including but not limited to Rab GTPases, certain
components of the ESCRT machinery, sorting nexin 17, tetraspanins, and the
cytoskeletal adapter protein obscurin-like 1 protein (OBSL1)
7,27–30
. Through this process,
early HPV-containing endosomes are delivered to multivesicular endosomes (MVEs)
where the majority of capsid uncoating occurs through compartment acidification and
cyclophilin-mediated dissociation of the vDNA from capsomeres
31,32
. Delivery of HPV to
MVEs is dependent on CD63, a tetraspanin that has been shown to facilitate HPV
trafficking and directly interact with the viral capsid
33
. The vDNA in complex with
remaining L1 and L2 is then diverted away from the lysosome and to the TGN through
interaction with the retromer complex
34,35
. Remarkably, it is currently hypothesized that
the vDNA/L1/L2 complex is concealed within a vesicle throughout the entirety of this
process, hidden from innate host defense molecules yet able to interact with the cytosol
through a transmembrane portion of L2
36
. The vDNA-containing vesicle eventually
infiltrates the nucleus during the nuclear envelope breakdown step of mitosis, and
finally infects the host
37,38
. Although many membrane-associated molecules have been
shown to be required for HPV endocytosis, to date, a central mediator of this pathway is
yet to be described.

4
1.3 HPV and the annexin A2/S100A10 heterotetramer (A2t)
A2t is a protein complex composed of two annexin A2 (AnxA2) monomers non-
covalently bridged by an S100A10 dimer, and found on the surface of various cell types
including endothelial cells, epithelial cells, monocytes, and macrophages.
Intracellularly, AnxA2 is found in the nucleus and cytoplasm, vesicle-bound, and on the
inner and outer leaflet of the plasma membrane as A2t. The functions of AnxA2 and A2t
are diverse, including calcium and membrane binding, membrane domain
organization, membrane fusion, vesicle aggregation, cytoskeletal-membrane dynamics,
protein trafficking, and translational regulation through RNA binding
39–42
. Interestingly,
structural studies have shown that annexin family members, including AnxA2, can
generate negative membrane curvature and undergo conformational changes that
disrupt membranes at acidic pH. Because of its role in endocytosis, a process that most
viruses exploit to gain entry into host cells, our lab investigated A2t in the context of HPV
infection and found that HPV infection of epithelial cells is dependent on A2t
23
. An
independent group confirmed these findings and went on to suggest that AnxA2 and
S100A10 have separable roles during HPV entry and intracellular trafficking
24
. Together,
these data suggest a role for AnxA2 and A2t in the endocytosis and vesicular transport of
HPV, yet the function and necessity of A2t at the cell surface and the precise endocytic
steps mediated by the heterotetramer and its individual subunits are not completely
clear.
In the presented dissertation, small molecule inhibitors of A2t (A2ti) were first
tested for their ability to prevent HPV infection, in vitro. We found that A2ti could
completely block HPV infection at high concentrations without substantial cellular
toxicity (published in the Journal of Antimicrobial Therapy in 2015
43
). Further
investigation revealed a decrease in cell proliferation after A2ti treatment at these high
concentrations (Chapter 2). Together, these results demonstrate that targeting A2t may
be an effective strategy to prevent HPV16 infection and perhaps even malignant cell
growth.
5
Our laboratory previously found that HPV16 minor capsid protein L2 binds
directly to S100A10
23
. Dziduszko and Ozbun went on to suggest separate roles for the A2t
subunits (S100A10 and AnxA2) during HPV16 entry and intracellular trafficking
24
.
Notably, both of these studies used AnxA2-targeting strategies
23,24
. Because S100A10 is
rapidly ubiquitinated and degraded in the absence of AnxA2, this method effectively
down-regulates both monomeric AnxA2 and heterotetrameric A2t
44,45
, limiting the
ability to interpret the effects of monomeric AnxA2 versus A2t. Additionally, our
observed effect of A2ti treatment on cell growth indicates possible non-specific effects
of the drug, rendering A2ti suboptimal to further investigate the mechanism of A2t-
mediated HPV infection. Therefore, to create a research tool that would enable us to
independently address the role of AnxA2 versus S100A10 and A2t, we genetically
engineered S100A10 knock-out (KO) and full A2t KO HeLa cells using CRISPR/Cas9 gene
editing technology (Chapter 3). Infection of HPV16 was significantly reduced and cell
proliferation was not affected in both S100A10 KO and A2t KO HeLa cells.
The previously described studies strongly indicate a central role for A2t in HPV16
endocytosis and intracellular trafficking, but they do not clearly describe the
contribution of monomeric AnxA2 versus A2t to HPV cell surface binding and
intracellular trafficking, nor do they assess if infection by other high-risk oncogenic HPV
genotypes requires A2t. Therefore, using our KO cells we sought to explore these
unknowns and found that that heterotetrameric A2t mediates post-entry trafficking of
HPV to MVEs, protecting HPV16 from lysosomal degradation, that monomeric AnxA2 is
involved in HPV infection post-capsid uncoating, that multiple oncogenic HPV rely on
AnxA2, and that in the absence of S100A10 and A2t, HPV16 endocytosis remains clathrin-
independent (Chapter 4). Together, these data further characterize the role of S100A10
and AnxA2 in protein trafficking, and suggest that A2t-mediated endocytosis represents
a distinct and previously uncharacterized trafficking pathway.
By bridging virology, genome engineering, protein chemistry, molecular biology,
and microscopy, the results from this dissertation further describe the role of A2t in HPV
entry and intracellular trafficking and expand our understanding of A2t-mediated
6
endocytosis. More broadly, AnxA2/A2t are associated with 13 other viruses, implicated
in various endocytic events, and have multiple associations with immunological
functions and pathological conditions (discussed in Chapter 5). The presented work
ultimately demonstrates the utility of our newly developed KO cells as a research tool to
further investigate AnxA2 and A2t in and outside of HPV biology.

Figure 1.1. An overview of HPV intracellular trafficking in the presence (left) and
absence (right) of A2t. (a) HPV binds to the cell surface through two distinct attachment
events. Frist, through interaction with heparan sulfate proteoglycans (HSPGs). Then, the
virion is transferred to a secondary receptor complex at tetraspanin enriched
microdomains. In the absence of S100A10 and A2t, we found no difference in the amount
of HPV bound to the cell surface (right). (b) HPV is internalized into the cell through a
clathrin-independent mechanism. Without S100A10 and A2t, the amount of HPV
7
internalized into the cell is unchanged and the process appears to remain clathrin-
independent. (c) After internalization, HPV is trafficked to early Rab5-, EEA1-positive
early endosomes. In the absence of S100A10 and A2t, more HPV was found to be present
in early endosomes as measured through immunofluorescence colocalization of HPV
with EEA1. (d) HPV then progresses to multivesicular endosomes (MVEs) where capsid
uncoating is understood to occur, the viral DNA (vDNA) is released, and disassembled
capsids are degraded in the lysosome. (e) Finally, HPV vDNA is shuttled through the
trans-Golgi network (TGN) and then into the nucleus during mitotic nuclear envelope
breakdown. (f) In S100A10 and A2t knock-out (KO) cells, less HPV was present in MVEs,
capsid uncoating was dramatically inhibited, and HPV lysosomal degradation was
accelerated.

8
Chapter 2

Small molecule inhibitors of heterotetrameric annexin
A2/S100A10 prevent HPV16 infection, in vitro

Associated publication: Woodham AW*, Taylor JR*, et al. 2015. Small molecule
inhibitors of the annexin A2 heterotetramer prevent human papillomavirus type 16
infection. Journal of Antimicrobial Chemotherapy, 70:1686–1690.
*Authors contributed equally to this work
2.1 Introduction
High-risk human papillomavirus (HPV) infection leads to the development of several
infection-related cancers including cervical, anogenital, and head and neck cancers that
are a significant health burden worldwide
3,46–49
. HPV type 16 (HPV16), the most common
of the high-risk oncogenic HPV genotypes, is an obligatory intracellular non-enveloped
virus that must gain entry into host basal cells of the epithelium to deliver its double-
stranded DNA (dsDNA) to the nucleus, and the HPV16 capsid proteins play a vital role in
these steps
50
. We have previously reported that the annexin A2/S100A10 heterotetramer
(A2t) facilitates infection of HPV16 in cervical epithelial cells through a direct protein-
protein interaction between the S100A10 subunit of A2t and the HPV16 minor capsid
protein L2
23
. Protein-protein interactions are increasingly being explored for small
molecule drug discovery, and the identification of A2t as central mediator of HPV16
infection makes it a promising target for inhibition. Annexin A2 (AnxA2) is found
cytoplasmically as a monomer, or at the cell surface as a heterotetramer consisting of
two AnxA2 monomers bridged non-covalently to an S100A10 dimer
51
. The dimeric
S100A10 structure yields two binding pockets that accommodate the N-terminus of
AnxA2
52,53
, and it is this interaction that preliminary drug discovery studies have targeted
54
. Reddy et al. used pharmacophore modeling techniques to identify several generations
of triazole compounds that moderately inhibit the interaction of the N-terminus of
9
AnxA2 with S100A10
55,56
, but these A2t inhibitors (A2ti) have not been explored in the
context of HPV infection. Here, we investigated the ability of first-generation A2ti
55

(A2ti-1) and second-generation A2ti
56
(A2ti-2) to inhibit HPV16 infection of epithelial
cells in vitro.
2.2 Results
A2ti bind to A2t and are non-toxic to HeLa cells
In the current study we examined the ability of two A2ti to block HPV16 infection in
vitro. The first of these inhibitors (Fig. 2.1a) is an earlier generation molecule (A2ti-1)
with a reported half maximal inhibitory concentration (IC 50) of 24µM
55
, and it was
hypothesized that A2ti-1 would block HPV16 pseudovirion (PsV) infection within a
reasonable range of this value. The group that developed these molecules published a
follow-up study in which they further optimized a subset of A2ti. The second generation
A2ti used in this study (A2ti-2; Fig. 2.1b) has a reported IC 50 of 10µM
56
, and was therefore
predicted to block HPV16 PsV infection at lower concentrations than A2ti-1. Before
performing infection assays, we confirmed that A2ti targeted the S100A10 dimer subunit
of A2t with isothermal titration calorimetry (ITC). We found that A2ti bind to S100A10
and observed an energy change that possibly indicates S100A10 dimer dissociation as the
action of A2ti on A2t (Fig. 2.2). Next, we analyzed if either A2ti or the DMSO carrier were
cytotoxic to HeLa cells and found no effect on cell viability after 48h treatment (Fig. 2.3).

10

Figure 2.1. Chemical structures for (a) A2ti-1 with a reported IC 50 value of 24µM, and (b)
A2ti-2 with a reported IC 50 value of 10µM.

11
Figure 2.2. Isothermal titration calorimetry (ITC) measurement of A2ti binding
interactions with the S100A10 dimer subunit of A2t. The top panel is the binding curve
shown as the heat in μcal/sec per injectant. The bottom panel shows the enthalpy
(KCal/Mole) as a function of the A2ti-S100A10 dimer molar ratio with the default fit line.
All titrations were performed at 37°C. The inset shows the stoichiometry (N), binding
affinity (K) and thermodynamic parameters (ΔH°, enthalpy; TΔS°, entropy) for each
interaction derived from a one site model fit of the binding isotherms. Integration of the
endothermic heat pulses as seen above is indicative of potential S100A10 dimer
dissociation as the action of A2ti on A2t.

Figure 2.3. A2ti are not cytotoxic to HeLa cells. Cells were left untreated or treated with
A2ti-1 (50µM), A2ti-2 (50µM), or 0.1% DMSO as a vehicle control. After 48h, cells were
collected, stained with propidium iodide (PI), and analyzed for viability (%PI-negative)
via flow cytometry. Results represent N=3 ± s.d. and are representative of at least 3
independent experiments. Statistics: 1-way ANOVA with Dunnett’s multiple comparison
test. No significant difference was found between all groups.
12
A2ti block HPV16 infection in HeLa cells
Next, we sought to determine the effect of A2ti on HPV16 PsV infection of HeLa cells in
a dose-dependent manner. As expected, we found that the higher affinity A2ti-2 (Fig.
2.4b) reduced HPV16 PsV infection to a greater extent than A2ti-1 (Fig. 2.4a), with
maximum inhibition of infection observed at 100µM (approximately 70% for A2ti-1 and
100% for A2ti-2; Fig 2.4a and 2.4b, respectively).

Figure 2.4. A2ti-1 and A2ti-2 block HPV16 PsV infection in HeLa cells. (a and b) Wild
type (WT) HeLa cells were treated with increasing concentrations of A2ti-1 (a), A2ti-2 (b),
or DMSO vehicle control. After 4h pre-treatment, cells were infected with HPV16 PsVs
carrying a GFP reporter plasmid. Infection (GFP gene transduction) was measured 48h
post-infection via flow cytometry as %GFP-positive cells. Background from mock
infected cells was subtracted from all groups and results show the mean %GFP-positive
cells ± s.d. (N=3; Infection in the absence of A2ti, was set to 100%). Statistics: 1-way
ANOVA with Dunnett’s multiple comparison test. **** P<0.0001.
13
A2ti treatment reduces HeLa cell proliferation
To determine the specificity of A2ti inhibition of HPV16 infection, we investigated
potential effects of A2ti on cell proliferation. Because successful HPV16 infection is
dependent on mitotic envelope breakdown, reduced cell proliferation would result in a
non-specific reduction in HPV16 infection. Using the [3H]-thymidine incorporation
assay, we observed a dose-dependent decrease in cell proliferation following treatment
with both A2ti-1 (Fig. 2.5a) and A2ti-2 (Fig. 2.5b) after 48h incubation, although the
decrease in infection was less significant than that following A2ti-1 treatment compared
to A2ti-2 (Fig. 2.4a and 2.4b, respectively).

Figure 2.5. A2ti treatment reduces cell proliferation. HeLa cells were seeded in 96-well
plates, treated with (a) A2ti-1, (b) A2ti-2, or DMSO and pulsed with [3H]-thymidine for
48h. Cells were then harvested using an automatic cell harvester and analyzed for
radioactivity indicating thymidine incorporation using a scintillation beta-counter
(Packard). Results represent N=12 ± s.d. for (a) and N=6 ± s.d. for (b) and are
14
representative of at least 3 independent experiments. Statistics: 1-way ANOVA with
Dunnett’s multiple comparison test. ns = not significant, * P<0.05, *** P<0.001, ****
P<0.0001.

2.3 Discussion
Our group previously identified A2t as an HPV16 associated-host factor using a
combination of cellular, molecular, and biochemical techniques including small hairpin
RNA (shRNA) knockdown, antibody neutralization, and electron paramagnetic
resonance
23
. Specifically, we demonstrated that the S100A10 subunit of A2t binds to
amino acids 108-126 of HPV16 L2; that A2t co-immunoprecipitates with HPV16 at the cell
surface; that A2t mediates HPV16 entry and infection in an L2-dependent manner; and
that a previously identified natural A2t ligand, secretory leukocyte protease inhibitor
(SLPI)
57
, reduced HPV16 PsV infection of epithelial cells
23
. Dziduszko and Ozbun
independently confirmed the role of A2t in HPV16 entry and infection by showing that
HPV16 binding results in the translocation of A2t to the extra-cellular surface; A2t
colocalizes with HPV16 at the cell surface; anti-AnxA2 antibodies alter the entry kinetics
of HPV16; and anti-S100A10 antibodies affect HPV16 PsV intracellular localization
24
.
In the current study, we show a significant decrease in HPV16 infection in
epithelial cells treated with a high-affinity A2ti that target the interaction between the
AnxA2 N-terminus and the S100A10 subunit of A2t. These results highlight the
importance of A2t in HPV16 infection and demonstrate the potential for targeting A2t to
block HPV16 infection. We also found that treatment with A2ti caused a decrease in cell
proliferation, yet it cannot be concluded that the reduction in proliferation completely
accounts for the reduction in HPV16 infection and further investigation is required.
15
Independent reports indicate that HPV entry may be dependent on A2t
24,43
, supporting
the notion that A2t is involved in events downstream of initial HPV16 binding and
internalization, but upstream of nuclear infiltration. Although ITC revealed an energy
change in S100A10 when treated with A2ti, co-immunoprecipitation experiments using
A2ti-1 and HeLa cell lysates showed no reduction in A2t complexes (not shown). Taken
together, these results suggest that A2ti might induce a conformational change that does
not completely dissociate the S100A10 dimer subunits. Alternatively, our proliferation
results may indicate that A2ti induce off-target effects that contribute to the reduction in
HPV16 infection. Although A2ti may be attractive as a therapeutic agent for blocking
HPV16 infection or for inhibiting cell growth, alternative A2t-targeting strategies are
required to specifically investigate the role of A2t and its individual subunits in HPV
infection.
2.4 Methods
Cell culture (HeLa), reagents, and pseudovirion (PsV) production
HeLa cells (CCL-2, ATCC) derived from cervical carcinoma were maintained in Iscove’s
Modified Dulbecco’s Medium (IMDM) with 10% FBS, 1x BME, and 1x gentamycin at 37°C
with 5% CO 2. A2ti-1 (2-[4-(2-Ethylphenyl)-5-o-tolyloxymethyl-4H-[1,2,4]triazol-3-
ylsulfanyl]acetamide) was purchased from Asinex (Moscow, Russia). A2ti-2 (2-[4-Allyl-5-
(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-N-(4-
isopropyl-phenyl)-acetamide) was a generous gift from Lodewijk V. Dekker (University
of Nottingham, United Kingdom). Both inhibitors were reconstituted in dimethyl
sulfoxide (DMSO). The ability of A2ti to disrupt A2t was verified via ITC following
standard procedures
58,59
. HPV16 PsVs were prepared as previously described
60
. Briefly,
293TT cells were co-transfected with codon-optimized plasmids for HPV16 L1 and L2
(p16sheLL; a kind gift from John T. Schiller (National Cancer Institute, USA)) and
16
pCIneoGFP reporter plasmid. Infectious titer was determined by flow cytometric
analysis of GFP+ 293TT cells 48h post-treatment and calculated as infectious units/mL.
Cytotoxicity assay
HeLa cells were seeded at 2E4 cells/well in 24-well plates and allowed to adhere
overnight. Cells were treated with increasing concentrations of A2ti-1 or A2ti-2, with
0.1% DMSO as vehicle control, or left untreated for 48h. Cells were collected via
trypsinization, stained with propidium iodide (PI), and viability was assessed via flow
cytometry (%PI-negative).
HPV16 pseudovirus infection assay
2E4 cells were seeded in 24-well plates and allowed to adhere overnight. The following
day, cells were pretreated with A2ti-1, A2ti-2, or 0.1% DMSO for 4h at 37°C. Cells were
then infected with HPV16 PsV at a multiplicity of infection (MOI) of 25 for an additional
48h in the presence of inhibitor. Infection was measured as %GFP-positive cells 48h
post-virus addition via flow cytometry (FC500, Beckman Coulter). In this study, infection
is defined as gene transduction of the GFP reporter plasmid.
Cell proliferation assay
1E4 cells were seeded in 96-well plates were treated with A2ti-1, A2ti-2, or DMSO for 48h
and proliferation during this time was measured using the [3H]-thymidine incorporation
assay. Briefly, cells were pulsed with [3H]-thymidine for 48h, harvested using an
automatic cell harvester, and radioactivity indicating thymidine incorporation into
mitotic cell DNA was measured using a scintillation beta-counter (Packard Top Count
NXT Microplate Scintillation and Luminescence Counter).

17
Chapter 3

Genetically engineering cells to further investigate A2t in HPV
infection
3.1 Introduction
The annexin A2/S100A10 heterotetramer (A2t) is a protein complex composed of two
annexin A2 (AnxA2) monomers and an S100A10 dimer
39,40
, and it is found on the surface
of various cell types and expressed in many tissues. A wide range of functions have been
suggested for A2t including anionic phospholipid binding, regulation of plasmin
activity, calcium binding, F-actin binding, exocytosis, and endocytosis (reviewed in
42,61
).
Because of its role in endocytosis, a process that most viruses exploit to infiltrate host
cells, A2t-mediated cellular infection has been investigated in the context of several viral
infections. For example, it was shown that secretory leukocyte protease inhibitor (SLPI),
a natural ligand of A2t, has anti-HIV properties
62
, and inhibits HIV infection of
macrophages by binding to and blocking A2t
57
. Furthermore, HSV infection causes a
sustained downregulation of SLPI thereby promoting viral entry through A2t
63
. Because
of the historic link between HSV and cervical cancer, and the sustained downregulation
of the A2t inhibitory protein SLPI in HSV infection, our lab began investigating the role
of A2t in high-risk human papillomavirus (HPV) type 16 (HPV16) infection, as HPV is the
known etiologic agent of cervical cancer. Our results demonstrated that HPV16 infection
of cervical epithelial cells is dependent on A2t, and that the S100A10 subunit of A2t
directly interacts with HPV minor capsid protein L2
23
. An independent group also
investigated A2t in the context of HPV16 infection and found an increase in A2t
translocation to the cell surface upon HPV binding, colocalization and co-
immunoprecipitation of AnxA2 and HPV at the cell surface, reduced HPV entry kinetics
upon anti-AnxA2 antibody treatment, and altered intracellular distribution of HPV after
anti-S100A10 antibody treatment
24
. Taken together, these data suggest that AnxA2 and
18
S100A10 may have separable roles in HPV infection. A2t plays a clear role in HPV
infection, however, how A2t and its individual subunits mediate intracellular trafficking
is not yet understood.
We previously investigated inhibitors of A2t (A2ti) in HPV infection and results
suggested promising anti-HPV blocking capability in vitro
43
, but potential non-A2t-
specific effects limit the use of A2ti as an experimental tool (summarized in Chapter 2).
Additionally, many studies investigating AnxA2 utilize knock-down strategies. Because
S100A10 is rapidly ubiquitinated and degraded in the absence of AnxA2
44,45
, it is not
possible to distinguish effects of monomeric AnxA2 versus A2t using this approach. In
some reports, this has led to ambiguous conclusions about the functions of AnxA2 versus
A2t. The AnxA2 monomer and A2t however, have independent functions and should
therefore be considered biochemically distinct and investigated as such. To study the
individual contribution of these proteins to HPV entry and intracellular trafficking, we
have generated two modified HeLa cell lines using CRISPR/Cas9 gene editing: (1) an
S100A10 knock-out (KO) expressing monomeric AnxA2 and (2) an AnxA2 KO, effectively
removing full A2t complexes (A2t KO). Moreover, the development of these cells creates
a research tool that will enable the larger scientific community to more rigorously
investigate AnxA2 and A2t biology.
3.2 Results
CRISPR/Cas9 gene editing effectively knocks out S100A10
and AnxA2 protein expression
Traditional Cas9 nucleases result in double-strand DNA breaks. Because optimal guide
sequences are relatively short (~30bp), non-specific, off-target genome editing can pose
an issue. For this reason, we employed the use of a mutant Cas9 nickase that results in
single-strand DNA breaks. Using two targeting guide RNA (gRNA) sequences per
19
breakage point, one on the plus strand and one on the minus strand that are 8-20
nucleotides apart, results in two single-stranded breaks that are recognized by the cell
as the desired double-strand DNA break. In this way, the likelihood of nonspecific
genome editing is greatly reduced
64
. Using this method, multiple clonal populations of
S100A10 KO and AnxA2 KO HeLa cells were generated. Because AnxA2 KO results in
S100A10 degradation, AnxA2 KO cells are referred to as A2t KO cells hereafter. S100A10
KO and A2t KO cell populations were expanded from a single cell, observed for
morphology and growth characteristics compared to wild type (WT) HeLa cells, and
analyzed for S100A10 and AnxA2 protein expression via western blot. Figure 3.1 shows
two representative western blots from protein expression screens in ten S100A10 KO
clonal populations (Fig. 3.1a) and ten AnxA2 KO clonal populations (Fig. 3.1b). 149 total
clones were screened and of these, ~20% of 78 A2t KO clones were found to be
successfully edited, and ~70% of 71 S100A10 KO clones were successfully edited.

20

Figure 3.1. Western blot analysis of S100A10 and AnxA2 protein expression in
CRISPR/Cas9 edited cells. Clonal cell populations were generated using guide RNA
(gRNA) targeting (a) the S100A10 gene and (b) the ANXA2 gene. After expansion, cells
were collected, lysed, and analyzed via western blot for expression of AnxA2 and
S100A10.
AnxA2 and A2t are essential for the infection of HPV16 in HeLa cells
We previously showed that downregulating A2t via shRNA or by targeting the AnxA2-
S100A10 binding interface using small molecule inhibitors (A2ti) significantly inhibits
HPV16 infection of epithelial cells
23,43
. However, results from A2ti experiments indicated
potential off-target effects (Chapter 2). Therefore, S100A10 and A2t KO clones were
generated to more precisely investigate AnxA2 and A2t. To confirm the requirement of
A2t in HPV16 infection and to investigate the effect of removing S100A10 alone, we
performed an infection assay (Fig. 3.2) using at least three clones of each protein KO
treated with HPV16 PsVs – HPV L1/L2 capsids with a GFP reporter plasmid. Clones were
selected based on growth characteristics and morphology that most closely resembled
that of WT HeLa cells during expansion. Results showed a modest yet significant
reduction in HPV16 infection in S100A10 KO cells and a dramatic reduction in A2t KO
cell lines compared to WT HeLa, indicating an essential role for monomeric AnxA2 in
HPV16 infection (Fig. 3.2).
S100A10 and AnxA2 are not required for efficient cell proliferation
Due to our results indicating decreased cell proliferation after A2ti treatment (Fig. 2.5),
it was essential to evaluate the effect of S100A10 KO and AnxA2 KO on cell proliferation
and exclude reduced mitosis as a confounding variable in HPV infection analysis. Here,
we selected two clones for in-depth proliferation analysis: P3-6-B2 and A1-6-B1 (referred
21
to hereafter as S100A10 KO and A2t KO, respectively). First, we examined proliferation
via cell counting 48h post-seeding, and did not find a significant difference in the
number of viable cells (Fig. 3.3a). Cytokinesis is the terminal step in cell division during
which the cytoplasm divides and the cell splits into two daughter cells, and AnxA2 has
been implicated in this process
65
. Because a cytokinetic defect would result in the
doubling of genomic material without doubling cell number, we quantified DNA in
S100A10 KO and A2t KO cells compared to WT HeLa cells 48h post-seeding and found no
change in the quantity of genomic DNA within these populations of cells (Fig 3.3b).
Finally, we utilized the CFDA-SE (5[6]-carboxyfluorescein diacetate succinimidyl ester)
cell proliferation assay to observe proliferation over time. CFDA-SE is a cell-permeable
reagent that is cleaved to fluorescent CFSE within cells and maintained as a stable,
covalent bond. As the cells divide, the fluorescence intensity is successively halved,
allowing analysis of each cell generation. Using this method, we found no significant
change in cell proliferation over the course of 72h when comparing WT cells to S100A10
A2t KO cells every 24h within this time frame (Fig. 3.3c).

Figure 3.2. S100A10 and AnxA2 KO (resulting in loss of full A2t) inhibit HPV16 infection
in HeLa cells. WT, S100A10 KO cells (P3-8-A3, P3-11-A1, P3-6-B2), and A2t KO cells (A1-6-
22
C2, A1-6-B1, A1-6-A1) were treated with HPV PsVs (TCID 50) carrying a GFP reporter
plasmid. Infection (GFP gene transduction) was measured 48h post-infection via flow
cytometry. Neutralizing anti-HPV L1 antibody H16.V5 was used as a positive control for
infection inhibition, and background from mock infected cells was subtracted. Results
show the mean %GFP-positive cells N=3 ± s.d. (relative to WT). Statistics: 1-way ANOVA
with Dunnett’s multiple comparisons test – * P< 0.05, ** P<0.01, ****P < 0.0001.

Figure 3.3. S100A10 and AnxA2 are not required for cell proliferation. (a and b) WT,
S100A10 KO, and A2t KO HeLa cells were seeded with equal cell number, grown for 48h,
23
and then analyzed for differences in cell proliferation via (a) assessing cell number and
viability via trypan blue exclusion test, and (b) quantification of nucleic acid content via
CyQUANT Cell Proliferation Assay Kit (Thermo Fisher) in S100A10 KO and A2t KO cells,
relative to WT HeLa cells. (c) HeLa cells were labeled with CFDA-SE, seeded at equal
densities, and then allowed to expand for 72h. At 24, 48, and 72h post-seeding, cells were
collected and mean fluorescence intensity (MFI) was measured via flow cytometry.
Statistics: (a and b) 1-way ANOVA with Dunnett’s multiple comparisons, ns = not
significant. (c) 2-way ANOVA with Tukey’s multiple comparisons test found no
significant difference between cell groups at each time point.
3.3 Discussion
Earlier work demonstrated a role for heterotetrameric A2t and potentially separable
roles for A2t subunits AnxA2 and S100A10 in HPV infection
23,24,43
. The entry and
intracellular trafficking of HPV leading to infection is poorly characterized, and data
supports the notion that HPV utilizes a novel endocytic mechanism
7,66
. Furthermore,
none of the identified HPV entry-associated molecules are capable of inducing
membrane curvature. While annexin family members, including AnxA2, are able to
generate the negative membrane curvature that is required for HPV endocytosis
67,68
.
Moreover, independent studies have shown that annexins undergo conformational
changes and disrupt membranes at acidic pH
69,70
, suggesting that A2t may promote
HPV16 endosomal escape or increased vesicle permeabilization that may be required
for infection. In a separate study, antibodies against the S100A10 subunit of A2t caused
an increase in colocalization between HPV16 and late endosome/lysosome marker
LAMP1, further supporting a role for A2t in late HPV16 trafficking
24
. A2t has also been
shown to link actin to endosomal membranes and promote endosome biogenesis
71,72
,
implicating a potential role for A2t in HPV16 intracellular trafficking. AnxA2 also
facilitates the endocytic trafficking of antisense oligonucleotides and the phagocytosis
24
of yeast
73,74
. Taken together, these data support a role for A2/A2t in HPV16 endocytosis
and intracellular trafficking, but it is not clear how this dynamic protein functions in
HPV infection.
Unfortunately, the current systems in place for studying AnxA2 do not allow for
differentiation between effects of monomeric AnxA2 and heterotetrameric A2t. For
example, small molecule inhibitors may have off-target effects, antibody blocking
strategies may trigger antibody-mediated endocytosis, and AnxA2 knock-down
strategies simultaneously remove AnxA2 and A2t. Because these experimental
challenges obfuscate interpretations, we have developed cell lines completely devoid of
either S100A10 or AnxA2 (resulting in the loss of full A2t). As expected, HPV16 infection
in these cell lines was significantly reduced. Although an earlier report claims that
AnxA2 functions as a necessary component in cytokinesis
65
, we did not observe any
effect of knocking out AnxA2 on this process. This study used transient knock-down
strategies and cytokinetic defects were assessed shortly thereafter. Although cytokinetic
defects were observed in significant proportions of the studied cell populations, these
effects were generally seen in approximately 50% of cell populations and assayed
immediately after knock-down. The authors conclude that AnxA2 likely participates in
spindle-equatorial plasma membrane communication, and although this may be
possible, we propose that redundancies in cytokinetic mechanisms overcome the loss of
AnxA2 during cell division. AnxA2 belongs to a large family of structurally similar
annexin proteins, supporting the hypothesis that compensatory mechanisms could
overcome an AnxA2 requirement during cytokinesis. Taken together, we hypothesize
that although there may be a defined role for AnxA2 in cytokinesis, this function is not
essential for overall cell division and survival, and any AnxA2 requirement has been
overcome in our cells after stable passage. In conclusion, the cells developed in this
study can be used to study the effects of long-term AnxA2 downregulation and to clearly
differentiate between the functions of monomeric AnxA2 and heterotetrameric A2t.
25
3.4 Methods
Cell culture
HeLa cells (CCL-2, ATCC) derived from cervical carcinoma were maintained in Iscove’s
Modified Dulbecco’s Medium (IMDM) with 10% FBS, 1x BME, and 1x gentamycin at 37°C
with 5% CO 2. During expansion, single cell clones were frequently microscopically
observed for morphology and growth characteristics.
Antibodies
Anti-AnxA2 (610069) and anti-S100A10 (610071) antibodies were purchased from BD
Biosciences. Anti-b actin (4970) was purchased Cell Signaling Technology. Goat-anti-
mouse IRDye 800CW (925-322) and goat-anti-rabbit (H+L) Alexa Fluor 680 (A27042)
secondary antibodies used for infrared western blot imaging were purchased from Li-
Cor and Invitrogen, respectively. Previously described anti-HPV16 L1-specific
neutralizing antibody H16.V5 was a generous gift from Neil Christensen
75
.
CRISPR/Cas9 gene editing
Addgene plasmid pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (#42335; a generous gift
from Ling Shao, USC) was used for CRISPR/Cas9-targeted ANXA2 and S100A10 gene
disruption via mutant Cas9 nickase (D10A variant). At least 3 distinct genomic targeting
regions per either ANXA2 or S100A10 gene were screened and the following target
sequences successfully edited HeLa cells: 5’-CAGGAAGCACGAACATCAGC-3’ and 5’-
ACAGGGGCTGGGAACCGACG-3’ for ANXA2; and 5’-AATGGTGAGGCCCGCAATTA-3’ and
5’-GTAGTACACATGAAGCAGAA-3’ for S100A10. Guide RNA (gRNA) were cloned using
Feng Zhang Laboratory protocols
76
. Non-target plasmid was used as negative control
and pMSCVpuro vector (a gift from Ling Shao) was used for selection. HeLa cells co-
26
transfected with Lipofectamine2000 (Thermo Fisher) were selected with puromycin for
72h, and clonal populations were generated through a limited dilution series. Clonal
populations were analyzed for protein expression via western blot.
Pseudovirus infection and infection rescue assay
2E4 cells were seeded in 24-well plates, allowed to adhere overnight, and infected with a
50% tissue culture infective dose (TCID 50) the following day. TCID 50 was determined by
titrating the multiplicity of infection (MOI) to result in approximately 50% infected cells,
48h post-infection. Infection is defined in this manuscript as gene transduction of the
GFP reporter plasmid. In infection assays, %GFP-positive cells were analyzed 48h post-
virus addition via flow cytometry (FC500, Beckman Coulter).
Cell proliferation analyses
WT and KO HeLa clones (A1-6-B1 and P3-6-B2) were analyzed for differences in cell
proliferation using three independent methods. 1) Cell counting via trypan blue
exclusion method – cells were seeded at equal densities, cultured for 48h, collected with
trypsin-EDTA, diluted 1:1 with trypan blue stain, and analyzed for viability. 2) DNA
quantification via CyQUANT Cell Proliferation Assay Kit (Invitrogen) – cells were seeded
at equal densities, grown for 48h, frozen at -80°C overnight, thawed at room
temperature, and lysed using CyQUANT cell lysis buffer with RNAse and GR dye
according to manufacturer protocol. Fluorescence of GR dye-bound DNA was measured
using a SpectraMax M5 plate reader (Molecular Devices) and DNA was quantified by
comparison to DNA standard curve. And 3) CFDA-SE Proliferation Assay – cells were
seeded at equal densities with DMSO as a vehicle control of 5µM CDK 1 inhibitor (RO-
3306, Merck) to reversibly synchronize the cells in G2/M phase of the cell cycle in early
mitosis, overnight. Cells were collected with trypsin-EDTA and each cell population was
labeled with Vibrant CFDA-SE cell tracer according to the manufactures protocol
27
(Invitrogen). Briefly, cells were re-suspended in PBS with 0.5% BSA. CFDA-SE was added
to the cell suspensions for a final concentration of 25µM and cells were incubated for 10
minutes at 37°C. The reaction was quenched with ice cold 1640 Medium (10% FBS;
Corning). The cells were pelleted and washed using HeLa media, counted, and seeded
at a density of 2.5E4 in 12-well plates. Baseline florescence at the time of seeding 24, 48,
and 72h post-seeding was measured via flow cytometry (Beckman Coulter Cytomics FC
500).

28
Chapter 4

Heterotetrameric annexin A2/S100A10 (A2t) is essential for
oncogenic human papillomavirus trafficking and capsid
disassembly, and protects virions from lysosomal degradation

Associated publication: Taylor JR, et al. 2018. Heterotetrameric annexin A2/S100A10
(A2t) is essential for oncogenic human papillomavirus trafficking and capsid
disassembly, and protects virions from lysosomal degradation. Scientific Reports,
8:11642.
4.1 Introduction

Persistent infection with mucosal-tropic high-risk human papillomavirus (HPV) causes
cervical, vaginal, anal, penile, and oropharyngeal cancers
2,49,77
. HPV-associated cancers
inflict a significant disease burden on the global population, yet there remain
unanswered questions about HPV cellular entry. As such, initial establishment of HPV
infection remains an active area of investigation. HPV is a non-enveloped double-
stranded DNA virus composed of major capsid protein L1 (HPV L1) and minor capsid
protein L2 (HPV L2)
78
. Although structurally simple, HPV infection depends on the
exploitation of complex host cell machinery and endocytic processes. HPV type 16
(HPV16) is the most common oncogenic genotype and is widely used to study the
infectious lifecycle of HPV. Since 1995, HPV entry has been thought to be receptor-
mediated; nevertheless, a consensus HPV receptor is still not identified
8
. Although
many HPV entry-associated molecules and co-factors have been recognized in what is
shaping up to be an incredibly complex and unique endocytic pathway (recently
reviewed in
79
), a central mediator has yet to be described.
The endocytosis of HPV16 into host basal epithelial cells is independent of
canonical clathrin-, caveolin-, flotillin-, lipid raft-, cholesterol-, and dynamin-mediated
endocytosis
6,7,80
. Trafficking of HPV from the cell surface to the nucleus can be broken
29
down into five key stages: cell surface binding, entry, viral vesicle trafficking, capsid
uncoating, and transporting of the viral genome (vDNA) through the trans-Golgi network
(TGN) to the nucleus. HPV binds to the cell surface through two distinct attachment
events. First, HPV capsid proteins interact with heparan sulfate proteoglycans (HSPGs)
found on the plasma membrane of basal keratinocytes or within the surrounding
extracellular matrix
9–12
. The binding of HPV to HSPGs induces conformational changes
in both HPV L1 and L2
13–15
, exposing the amino terminus of HPV L2 which contains a
furin/proprotein convertase cleavage site
16
. These conformational changes in the capsid
reduce HSPG-affinity and the virion is then transferred to the elusive secondary uptake
receptor/receptor complex located within tetraspanin enriched microdomains (TEMs)
6,17,18
. Candidate receptors to date have included a6 integrin
19,20
, epidermal growth factor
receptor (EGFR)
21,22
, and the protein complex studied herein – the annexin A2
heterotetramer (A2t)
23,24
. After handoff to this secondary receptor/receptor complex,
HPV is internalized through a non-canonical endocytic mechanism and trafficked
through the degradative endosomal system. While in optimal conditions viral trafficking
may be rapid, bulk internalization is relatively slow and asynchronous due to the time it
takes for extracellular structural modifications of the capsid and a hypothesized limited
availability of the secondary receptor/receptor complex
25,26
.
Internal trafficking of HPV is dependent on endocytic mediators including, but
not limited to Rab GTPases, certain components of the ESCRT machinery, sorting nexin
17, and the cytoskeletal adapter protein obscurin-like 1 protein (OBSL1)
7,27–30
. Through
this process, early HPV-containing endosomes are delivered to multivesicular
endosomes (MVEs) where the majority of capsid uncoating occurs through
compartment acidification and cyclophilin-mediated dissociation of the viral genome
(vDNA) and capsomeres
31,32
. Delivery of HPV to MVEs is dependent on CD63, a
tetraspanin that has been shown to facilitate HPV trafficking and directly interact with
the viral capsid
33
. The vDNA, concealed within a vesicle, then escapes lysosomal
degradation by transport to the TGN via interaction of cytosolically exposed HPV L2 with
the retromer complex
34,35,81
. The vDNA-containing vesicle eventually infiltrates the
30
nucleus during the nuclear envelope breakdown step of mitosis, completing
intracellular trafficking and establishing infection
37,38
.
Previous evidence supports a role for A2t at the cell surface and in the
intracellular trafficking of HPV
23,24
. However, the function and necessity of A2t at the
cell surface and the precise endocytic steps mediated by the heterotetramer and its
individual subunits in HPV infection are not well understood. A2t is a multifunctional
membrane-associated protein complex composed of two annexin A2 (AnxA2)
monomers bridged by an S100A10 homodimer
39–41
. AnxA2 and S100A10 are expressed in
many tissues, have been studied in the context of diverse cellular processes, and are
linked to multiple aspects of human health and disease
61,82,83
. The AnxA2 monomer and
A2t, however, have independent functions and should therefore be considered
biochemically distinct. In the mucosa, A2t is expressed in the basal layer of the
epithelium
84,85
, which is the initial site of HPV infection. On a cellular level, A2t is
localized to the inner and outer leaflets of the plasma membrane, while AnxA2 can be
found in the cytosol or membrane-bound. More specifically, these proteins function in
membrane domain organization, membrane trafficking events, membrane-cytoskeletal
connection through F-actin binding, MVE biogenesis, and membrane curvature
42,68,72,86–
88
. S100A10 is a small dimeric helix-loop-helix protein found in close association with
AnxA2 (complexed together to form A2t) and implicated in the trafficking of a variety of
membrane-resident proteins
40
.
S100A10 is rapidly ubiquitinated and degraded in the absence of AnxA2
44,45
. A
large proportion of studies investigating AnxA2 utilize anti-AnxA2 knock-down
strategies, effectively removing both monomeric AnxA2 and heterotetrameric A2t. In
the past, researchers have reported findings about either moiety without truly
distinguishing between the two. Therefore, to resolve the ambiguity between AnxA2 and
A2t in the context of HPV endocytosis, this study employs complete protein knock-out
of either S100A10 or AxnA2/S100A10 (A2t) in epithelial cells (HeLa) to distinguish the
specific contributions of these host factors in HPV entry and intracellular trafficking.
Here, we provide evidence supporting the role of A2t in HPV intracellular trafficking,
31
and further characterize the involvement of S100A10 in protein trafficking. Our data
show that AnxA2 and A2t are not required for cell surface attachment nor non-infectious
internalization of HPV. Instead, these data reveal a role for A2t in post-entry intracellular
trafficking and show that AnxA2 interacts with CD63, a known mediator of HPV
trafficking. We show that in the absence of complete A2t or S100A10 alone, cellular entry
of HPV is not reduced, however, progression of early HPV-containing endosomes to
MVEs is significantly inhibited. Consequently, HPV capsid disassembly is repressed, and
the virus is redirected to the lysosome for degradation. Taken together, these findings
suggest that A2t is required for the infectious post-entry intracellular trafficking of HPV.
Additionally, we reveal a previously underappreciated role for S100A10 in endosomal
transport.
4.2 Results
Annexin A2 (AnxA2) and heterotetrameric AnxA2/S100A10 (A2t) are essential for
the infection of high-risk oncogenic HPV in HeLa cells
We have previously shown that targeting A2t via siRNA knockdown of AnxA2, or
targeting the AnxA2-S100A10 binding interface via small molecule inhibition
significantly reduces HPV16 infection of cervical epithelial cells (HeLa and HaCaT)
23,43
.
It has also been suggested that the A2t subunits, AnxA2 and S100A10, may play separable
roles in HPV16 entry and intracellular trafficking
24
. Therefore, we sought to decipher
the specific contributions of AnxA2 and A2t in HPV infection and endocytosis. To
remove A2t, we knocked out either S100A10 or the full A2t complex via CRISPR/Cas9
gene editing and generated clonal HeLa cell populations. Given that S100A10 is rapidly
degraded in the absence of AnxA2
44,45
, the AnxA2 knock-out (KO) effectively serves as a
full A2t subunit KO. Western blot analysis confirmed the absence of protein expression
in the KO clones (Fig. 4.1a).
32
Because AnxA2 has been implicated in cytokinesis
65
, differences in cell
proliferation between clonal populations and WT cells were analyzed to exclude altered
proliferation rates as a potential confounding factor for the reduction in HPV infection
that we observed. Cell counts and proliferation were measured via trypan blue exclusion
and DNA-based quantification respectively (Fig. 4.1b and c), and no significant
difference in proliferation was observed.
HPV gene transduction is indicative of successful viral entry, intracellular
trafficking, and infiltration of the nucleus during mitosis; therefore, in vitro infection is
defined as reporter gene transduction. Using HPV16 pseudovirions (PsVs) containing a
GFP reporter plasmid, we then screened at least two S100A10 KO and two A2t KO clones
and found consistent inhibition of infection (Fig. 4.1d), confirming previously reported
findings
23,24
. To extend these findings and determine whether these proteins are used by
other HPV genotypes, we tested additional high-risk oncogenic HPVs as previously A2t
has only been implicated in HPV16 infection. Similar to HPV16, we found that infection
by HPV18, -31, and -45 was also significantly inhibited in both KO cell lines (Fig. 4.1d),
implicating this receptor complex in infection by multiple oncogenic HPV.

33

Figure 4.1. S100A10 knock-out (KO) and AnxA2 KO (resulting in loss of full A2t) does not
affect cell proliferation and inhibits multiple high-risk HPV infections in HeLa cells. (a)
*Because S100A10 is rapidly degraded in the absence of AnxA2, targeting AnxA2 results
in full A2t KO. Here, either dimeric S100A10 or heterotetrameric A2t (S100A10 + AnxA2)
was knocked out in wild type (WT) HeLa cells via CRISPR/Cas9 and confirmed by
western blot. (b and c) WT, S100A10 KO, and A2t KO HeLa cells were seeded with equal
cell number, grown for 48h, and then analyzed for differences in cell proliferation via
(b) analyzing cell number and viability via trypan blue exclusion test, and (c)
34
quantification of nucleic acid content via CyQUANT Cell Proliferation Assay Kit (Thermo
Fisher) and then analyzed relative to WT HeLa cells. Results (b and c) represent N=8 ±
s.d. and are representative of at least 3 independent experiments. Statistics: 1-way
ANOVA with Dunnett’s multiple comparison test. ns = not significant. (d) WT, S100A10
KO, and A2t KO HeLa cells were treated with HPV PsVs (TCID 50) carrying a GFP reporter
plasmid. Infection (GFP gene transduction) was measured 48h p.i. via flow cytometry.
Neutralizing anti-HPV L1 antibody H16.V5 (neut. Ab) was used as a positive control for
infection inhibition, and background from mock infected cells was subtracted. At least
3 independent S100A10 KO and 3 independent A2t KO clones were screened for
consistent inhibition of HPV16 infection. Results are representative of at least 3
independent experiments and show the mean %GFP-positive cells ± s.d. (n=3,
normalized to WT). Statistics: 1-way ANOVA with Dunnett’s multiple comparisons test –
ns = not significant, *P< 0.05, ***P < 0.001, ****P < 0.0001.
Restoration of A2t through AnxA2 knock-in restores HPV16 infectivity
To ensure that the observed inhibition of infection was specifically due to the S100A10
and AnxA2 gene editing, we restored AnxA2 expression in the A2t KO cells by plasmid
transfection which stabilized S100A10 protein expression and thus reintroduced
heterotetrameric A2t (Fig. 4.2a). Lane 3 in Figure 4.2a represents mCherry-tagged AnxA2
(mCherry-AnxA2) expression rescue in a total cell population of A2t KO cells, where
transfection efficiency was optimized to ~50% at 24h post-transfection. Live cells were
gated on forward and side scatter (Fig. 4.2b), and transfection efficiency was determined
by transfecting cells and measuring %mCherry-positive cells 24h post-transfection (Fig.
4.2c). In order to analyze infection rescue in the mCherry-AnxA2-transfected cells, the
labeled population was gated on mCherry (Fig. 4.2d) and analyzed for %GFP-positive
cells (Fig. 4.2e). The data show that upon AnxA2 re-expression and A2t re-stabilization,
infection was restored to WT levels (Fig. 4.2f), confirming that reduction in HPV16
infection in the A2t KO cells was not due to off-target effects.
35
Figure 4.2. HPV16 infectivity is rescued by
restoring A2t expression. (a) WT HeLa
cells were transiently transfected with a
control plasmid and A2t KO cells were
transfected with either an mCherry-
tagged AnxA2 expression plasmid or a
non-target control plasmid. AnxA2
expression was restored 24h post-
transfection, stabilizing S100A10 and
restoring heterotetrameric A2t (western
blot, lane 3). Lysates were prepared with
total populations of transfected cells, and
mCherry-AnxA2 transfection efficiency
was approximately 50%. (b-e) Gating
strategy for analysis of infection rescue.
Infection rescue was measured 48h post-
infection (p.i.) via flow cytometry by
gating on mCherry and analyzing the
%GFP-positive cells within that
population. Live cells were gated on
forward and side scatter in all plots. (b)
Control cell population: 50,000 total cells
analyzed, 42,350 live cells. (c) A2t knock-
out (KO) cells, 24h post-AnxA2-mCherry
transfection: 50,000 total cells analyzed,
37,502 live cells, and 19,173 live mCherry-
positive cells. (d) A2t KO cells 72h post-
AnxA2-mCherry transfection, 48h p.i.:
50,000 total cells analyzed, 43,861 live
36
cells, 3,701 live mCherry-positive cells. (e) A2t KO cells 72h post-AnxA2-mCherry
transfection, 48h post-infection: 50,000 total cells analyzed, 43,861 live cells, 3,701 live
mCherry-positive cells, 1,633 live mCherry-positive GFP-positive cells. (f) 24h post-
transfection, WT and A2t KO cells + non-targeting plasmid control and A2t KO cells + an
mCherry-AnxA2 expression plasmid were treated with HPV pseudovirions (TCID 50)
carrying a GFP reporter plasmid. Infection was measured 48h p.i. via flow cytometry,
and infection rescue was measured by gating on mCherry and analyzing the %GFP-
positive cells within that population. Results show the mean %GFP-positive cells ± s.d.
(n=3, normalized to WT), and are representative of at least 3 independent experiments.
Statistics: 1-way ANOVA with Dunnett’s multiple comparisons test – ****P < 0.0001.
A2t is not required for HPV cell surface attachment
HPV16 binds to the cell surface through two attachment events. First, through
interaction with HSPGs
9–12
and next, through a secondary receptor or receptor complex
at tetraspanin enriched microdomains
6
. Because HPV16 and A2t have been shown to
colocalize at the cell surface
24
, we investigated if this interaction is required for either
cell surface binding event. To first confirm cell surface AnxA2 localization, we
immunolabeled non-permeabilized cells for AnxA2 and analyzed surface expression via
immunofluorescent microscopy (Fig 4.3a). We observed ubiquitous cell surface
expression of AnxA2 in WT and S100A10 KO cells and no expression in A2t KO cells. To
examine HPV16 bound to the secondary receptor/receptor complex, HSPGs were
removed using heparinase. Treatment with heparinase I removes HSPGs from the cell
surface and results in a 50-80% reduction in cell surface binding of HPV. Here,
heparinase was titrated to reach a maximum reduction in binding (Fig. 4.3b). Consistent
with the reduction reported in HPV-HSPG binding studies, total cell surface-bound
HPV16 was reduced by about 70% in all cells treated with heparinase. Cells treated with
or without heparinase were incubated with HPV16 at 4°C to prevent internalization, and
the amount of cell surface-bound HPV was quantitated by flow cytometry. The left panel
37
of Figure 4.3c shows the amount of HPV16 bound to the cell surface in the presence of
HSPGs, and there is no statistical difference between WT, S100A10 KO, and A2t KO cells.
When comparing heparinase-treated KO cells to WT, there is also no observable
difference in surface-bound HPV16 (Fig. 4.3c, right panel). It is important to note that in
vitro capsid modifications in lab-produced pseudovirion and virus-like particle systems
have been shown to occur at the cell surface after interaction with HSPGs
89,90
. However,
furin cleavage is relevant in late-stage intracellular trafficking and is not required for
cell surface binding nor internalization
91,92
.
38

Figure 4.3. S100A10 and A2t are not required for cell surface binding of HPV16. HPV
binding occurs through binding heparan sulfate proteoglycans (HSPG) and a receptor
39
complex. (a) Wild type (WT) HeLa and S100A10 KO cells ubiquitously express AnxA2 at
the cell surface. Here, cells were grown in chamber slides, fixed with 2% PFA and
immunostained for AnxA2 under non-permeabilized conditions (no Triton-X). Nuclei
were stained with DAPI (blue). A single confocal slice is shown in each representative
image. Scale bar = 50µm. (b) WT HeLa cells were grown to 80% confluence, washed with
heparinase buffer, and incubated with either buffer or increasing concentrations of
heparinase I for 1h at 37°C. Cells were transferred to ice, washed with ice-cold PBS, and
then saturated with HPV16 PsVs (10µg/1E6 cells) in serum-free conditions for 1h at 4°C.
Cells were collected via scraping on ice, and the amount of surface-bound HPV was
analyzed by flow cytometry. (c) WT, S100A10 KO, and A2t KO HeLa cells were grown to
80% confluence and treated as described in (b) with 2U/mL Heparinase. Briefly, cells
were washed with heparinase buffer, and incubated with either buffer (vehicle ctrl; left
panel) or 2U/mL heparinase I (right panel) for 1h at 37°C, and then saturated with HPV16
PsV (10µg/1E6 cells) in serum-free conditions for 1h at 4°C and the amount of surface-
bound HPV was analyzed by flow cytometry. Results show the mean fluorescence
intensity (MFI) ± s.d., relative to WT HeLa, for three independent replicates (N=9).
Statistics: 2-way ANOVA with Sidak’s multiple comparisons test. ns = not significant.
Bulk HPV internalization is not affected by S100A10 KO and A2t KO, and
lysosomal degradation is increased
We next asked if the observed decrease in infection (Fig. 4.1b) could be explained by a
reduction in HPV internalization in the absence of S100A10 and A2t. By analyzing the
abundance of remaining surface-bound virions post-early endocytosis (4h), we found no
difference in HPV16 internalization into WT, S100A10 KO, and A2t KO cells as measured
by flow cytometry (Fig. 4.4a). To determine if virions are trafficked into degradative
endosomal compartments, we utilized pHrodo-labeled HPV16 (pHrodo-HPV16) virus-
like particles (VLP) to measure virus internalization. pHrodo is a pH-dependent
rhodamine dye that fluoresces in low pH environments, such as degradative endosomes.
40
The results show a significant increase in pHrodo-HPV16 signal intensity in both of the
KO cell lines compared to WT at 4 hours post-entry (Fig. 4.4b). Taken together, these
data suggest that virions are equally internalized between WT and KO cells, however,
HPV16 is preferentially transferred to more acidic pH environments in both S100A10
and A2t KO cells.
Given that the pH of lysosomes is the lowest of the intracellular compartments,
we investigated lysosomal degradation of HPV16. When HPV L1 is degraded via
proteolytic enzymes in the lysosome, cleaved HPV L1 is visible via western blot as double
bands that appear around 20 kDa using the CAMVIR-1 anti-L1 antibody (Fig. 4.4c, lanes
1-3). Treatment with chloroquine, a lysosome acidification inhibitor, results in complete
disappearance of the cleaved HPV L1 bands (Fig. 4.4c, lanes 4-6). By quantifying the band
intensities of the HPV L1 cleavage products normalized to actin in three independent
experiments, we confirmed that lysosome cleavage of HPV16 capsids is increased in the
absence of S100A10 and A2t (Fig. 4.4d).

41

Figure 4.4 S100A10 and A2t are involved post-HPV entry into HeLa cells, and in the
absence of S100A10 and A2t, HPV is redirected to the lysosome and degraded. (a) WT,
S100A10 KO, and A2t KO cells were treated with HPV16 pseudovirions (PsVs) (5µg/1E6
cells) for 1h at 4°C and measured via flow cytometry. Cells were then incubated for 4h at
37°C to promote early internalization, and remaining cell surface-bound HPV was
measured via flow cytometry. The difference between the amount of surface-bound HPV
at 0h and 4h serves as a measure of internalization. Mean fluorescence intensity (MFI)
42
of WT was set to 100% and results show the mean ± s.d. for 3 independent replicates
(N=9). (b) pHrodo

is a pH-dependent fluorophore that fluoresces in low pH endosomes.
Cells were treated with pHrodo-labeled HPV16 (pHrodo-HPV16) VLPs (5µg/1E6 cells) for
4h at 37°C and then measured via flow cytometry. Results show the mean MFI ± s.d.,
relative to WT HeLa (N=3), and are representative of at least 3 independent replicates.
(c) WT, S100A10 KO, and A2t KO cells were pre-treated with either vehicle control or
chloroquine, a lysosome acidification inhibitor for 16h at 37°C. Cells were then washed
on ice and treated with HPV16 pseudovirions (PsVs) (5µg/1E6 cells) for 1h at 4°C, washed,
and incubated for 4h at 37°C to promote early internalization. Cells were harvested via
in-plate lysis and image shows western blot for HPV16 L1 capsid protein (CAMVIR-1 Ab).
Full length HPV L1 is visible as a single band (55kDa), and lysosome-cleaved HPV L1
(~20kDa) appears as a double band. Western blot is representative of 3 independent
experiments. (d) Quantification of western blot band intensity from 3 independent
replicates measured in Image Studio
TM
Lite. Results show mean band intensity ± s.d.
(N=3, normalized actin and relative to WT). Statistics: 1-way ANOVA with Dunnett’s
multiple comparison test – ns = not significant, *P< 0.05, ****P < 0.0001.
HPV endocytosis remains clathrin-independent in S100A10 KO and A2t KO cells
Because AnxA2 and S100A10 are involved in multiple membrane trafficking events
93
, we
asked if knocking out S100A10 or A2t would have general effects on endocytosis using
model endocytic cargos. We first examined the effect of S100A10 KO and A2t KO on
pinocytic mechanisms because HPV endocytosis is pinocytosis-like
7
. Fluorescein
isothiocyanate-conjugated bovine serum albumin (FITC-BSA) was used as model cargo
for pinocytosis
67
, and uptake was analyzed over time. We observed a significant
decrease in FITC-BSA uptake in both the S100A10 KO and A2t KO cells compared to WT
(Fig. 4.5a). To verify that clathrin-mediated endocytosis remained intact in the KO cells,
we used Alexa Fluor® 488-labeled transferrin (transferrin-AF488) as model cargo for
clathrin-mediated endocytosis
94,95
, and analyzed uptake over time. Consistent with the
43
literature, transferrin-AF488 uptake was rapid in WT HeLa cells, and when compared to
S100A10 and A2t KO cells, transferrin-AF488 uptake was not affected (Fig. 4.5b). Finally,
to verify that HPV uptake does not get shifted towards clathrin-mediated endocytosis,
we used confocal microscopy to analyze the presence of the virus in clathrin-containing
compartments (Fig. 4.5c). Visual and quantitative analysis showed that HPV16 and
clathrin do not colocalize in WT, S100A10 KO, and A2t KO cells, as measured by Mander’s
coefficient (Fig. 4.5d).

44
Figure 4.5. Pinocytosis is reduced, clathrin-mediated endocytosis is unaffected, and
HPV endocytosis remains clathrin-independent in S100A10 KO and A2t KO cells. (a)
Bovine serum albumin (BSA) is taken up via pinocytosis. Here, 10µg/mL of fluorescein
isothiocyanate-conjugated BSA (FITC-BSA) was added to pre-cooled WT, S100A10 KO,
and A2t KO cells at 4°C, transferred to 37°C, and analyzed via flow cytometry at 0.5, 1.5,
and 3h. Results show mean fluorescence intensity (MFI) ± s.d. (N=3) relative to WT HeLa.
(b) Transferrin is taken up via clathrin-mediated endocytosis. 10µg/mL of Alexa Flour®
488-conjugated transferrin (transferrin-AF488) was added to cells and analyzed as
described in (a). Results show MFI ± s.d. (N=3) relative to WT HeLa. (c) HPV16
endocytosis is shown to be clathrin-independent. HPV16 pseudovirions (0.5µg/1E6 cells)
were added to pre-cooled cells grown on chamber slides, bound for 1h at 4°C, washed,
and incubated for 1h at 37°C. Cells were then fixed with 4% PFA and immunostained for
clathrin (red) and HPV16 (green), and nuclei were stained with DAPI (blue). A single
confocal slice is shown in each representative image. Scale bar = 10µm. (d)
Quantification of extent of clathrin-HPV16 colocalization was measured as Mander’s
coefficient using the JACoP plugin for Fiji. Representative results are shown as the mean
± s.d. (N=11 images). Statistics (a) and (b): repeated measures ANOVA (rANOVA) with
Dunnett’s multiple comparisons test. Statistics (d): 1-way ANOVA w/ Dunnett’s multiple
comparisons test. ns = not significant.
A2t controls the progression of HPV-containing early endosomes to late
multivesicular endosomes (MVEs), inhibits capsid disassembly, and AnxA2 forms
a complex with tetraspanin CD63 during HPV trafficking
A previous study demonstrated that in vitro anti-S100A10 antibody treatment of
epithelial cells caused increased colocalization of HPV16 with LAMP1 – a marker of late
endosomes/lysosomes, and the authors suggested a role for S100A10 in HPV16
intracellular trafficking
24
. After binding to the cell surface, HPV is endocytosed into
early endosomes and then to MVEs in a CD63-dependent fashion
7,33
. We therefore
45
investigated these intracellular trafficking steps in S100A10 KO and A2t KO cells
compared to WT. Immunofluorescence microscopy revealed an increase in HPV16
colocalization with early endosome marker EEA1 in KO cells compared to WT (Fig. 4.6a).
Confirming our visual observations, quantification of the extent of red and green overlap
showed a significant increase in the extent of HPV16-EEA1 colocalization in the KO cells
compared to WT (Fig. 4.6b). Tetraspanin CD63 is a marker for MVEs, and because CD63
and HPV directly interact, we analyzed the amount of HPV-CD63 colocalization via
immunofluorescence microscopy. Visual and quantitative analysis revealed a
significant decrease in HPV16-CD63 colocalization (Fig. 4.6c and d), demonstrating that
in the absence of A2t, less HPV16 is shuttled to MVEs when compared to WT cells. In
either instance, both KO cells exhibited similar effects. These data indicate that A2t is
responsible for HPV trafficking to the MVE as opposed to monomeric AnxA2.

46

Figure 4.6. S100A10 knock-out (KO) and A2t KO inhibit the progression of HPV16 from
early endosomes to multivesicular endosomes (MVEs). After binding to the cell surface,
HPV is endocytosed into early endosomes. (a) Here, HPV16 pseudovirions (PsVs)
(0.5µg/1E6 cells) were added to pre-cooled cells grown on chamber slides, bound for 1h
at 4°C, washed, and incubated for 1h at 37°C. Cells were then fixed with 4% PFA and
immunostained for EEA1 (red) to mark early endosomes, HPV16 (green), and nuclei
47
were stained with DAPI (blue). A single confocal slice is shown in each representative
image. Scale bar = 10µm. (b) Quantification of extent of EEA1-HPV16 colocalization was
measured as Mander’s coefficient M2 using the JACoP plugin for Fiji. Representative
results are shown as the mean ± s.d. (N=11 images, ³2 cells/image). (c) Post-entry, HPV
travels from early endosomes to MVEs, and directly interacts with CD63, a marker for
MVEs. Cells were treated as described in (a), incubated for 7h at 37°C, fixed with 4% PFA,
and immunostained for CD63 (red) and HPV16 (green), and nuclei were stained with
DAPI (blue). A single confocal slice is shown in each representative image. Scale bar =
10µm. (d) Quantification of extent of CD63-HPV16 colocalization was measured as
Mander’s coefficient using the JACoP plugin for Fiji. Representative results are shown
as the mean ± s.d. (N=10 images). Statistics (b) and (d): 1-way ANOVA with Dunnett’s
multiple comparisons test – *P< 0.05, **P < 0.01, ****P < 0.0001.

HPV capsid disassembly occurs MVEs, and given that HPV16 abundance in MVEs
is significantly decreased in the absence of S100A10 and A2t (Fig. 4.6c and d), we
investigated if capsid disassembly was affected. Using an antibody that specifically
recognizes an internal epitope of the HPV capsid (L1-7 Ab), we stained HPV16-treated
cells and saw a striking signal reduction in the S100A10 KO and A2t KO cells compared
to WT (Fig. 4.7a), confirmed by quantitative analysis of fluorescent signal (Fig. 4.7b).

48

Figure 4.7. HPV16 capsid disassembly is dramatically inhibited in the absence of
S100A10 and A2t. (a) WT, S100A10 KO, and A2t KO cells grown on chamber slides were
treated with HPV16 pseudovirions (PsVs) (0.5µg/1E6 cells) for 1h at 4°C to promote bulk
binding. Cells were then washed and incubated for 7h at 37°C to promote endocytosis
and intracellular trafficking. Cells were fixed with 4% PFA and immunostained with the
anti-HPV 33L1-7 antibody (L1-7 Ab) that recognizes an internal epitope on the HPV
capsid (green), marking disassembled capsid. Nuclei were stained with DAPI (blue) and
a single confocal slice is shown in each representative image. Scale bar = 10µm. (b)
Quantification of L1-7 Ab signal intensity was measured as mean tonal intensity using
Fiji. Results are shown as the mean ± s.d. (N=15 images). Statistics: 1-way ANOVA with
Dunnett’s multiple comparisons test – ****P < 0.0001.

Taken together, these data indicate that failure of infection in the knock-outs is
due to a blockage of HPV progression from early endosomes to MVEs and subsequent
redirection to lysosomes before capsid disassembly can occur. Similar to our results, a
study investigating the CD63-syntenin-1 complex in HPV infection found that post-entry
49
delivery of HPV to MVEs and consequent capsid disassembly are dependent on the CD63
complex
33
. Therefore, we hypothesized that AnxA2 and CD63 interact in the context of
HPV infection. Immunofluorescence microscopy revealed a significant increase in
AnxA2 colocalization with CD63 at 4h and 7h post-HPV infection in WT HeLa cells (Fig.
4.8a and b). The formation of an AnxA2-CD63 complex in HPV infection warrants further
investigation, however, these data support a functional role for AnxA2 in HPV
intracellular trafficking. In fact, given our HPV trafficking results showing equivalent
effects in S100A10 KO cells compared to A2t KO cells, we can speculate that the
functional form of AnxA2 is AnxA2 in its heterotetrameric form, A2t.

Figure 4.8. HPV16 infection stimulates CD63-AnxA2 complex formation. (a) WT cells
grown on chamber slides were treated with HPV16 PsVs (0.5µg/1E6 cells) for 4h and 7h
at 37°C (4h shown). At 4h and 7h post-infection (p.i.) cells were then fixed with 4% PFA
and immunostained with the anti-CD63 (green) and anti-AnxA2 (red) antibodies. Nuclei
were stained with DAPI (blue) and a single confocal slice is shown in each representative
image. Scale bar = 10µm. (b) Quantification of AnxA2-CD63 colocalization was measured
as Mander’s coefficient M2 using the JACoP plugin for Fiji. Representative results are
50
shown as the mean ± s.d. (N=15 images) Statistics: 2-way ANOVA with Sidak’s multiple
comparisons test – ****P < 0.0001.
4.3 Discussion
Earlier reports have shown that A2t is required for HPV16 infection of epithelial cells.
Our data expand these findings to HPV18, -31, and -45, a subset of genotypes with a high
risk for oncogenic transformation
96
, supporting a role for A2t in multiple high-risk HPV
infections. Because interpreting the role of AnxA2 versus A2t is unclear in many study
designs, we independently knocked out S100A10 and A2t to define the roles of A2t and
its subunits in HPV endocytosis. Despite previous evidence demonstrating a direct
interaction between S100A10 and HPV L2
23
, inhibition of infection in the S100A10 KO
was reproducibly less pronounced compared to the full A2t KO for all genotypes tested.
These data strongly suggest that monomeric AnxA2 and heterotetrameric A2t play
separable roles in HPV infection, although both KOs showed similar trafficking
phenotypes in other experiments. For the additional high-risk genotypes, the reduction
in infection in the KO cells is less pronounced compared to HPV16, indicating that
although A2t plays an important role, its significance for HPV16 infection cannot be
understated. The differences between infection in S100A10 KO versus A2t KO cells could
be explained by the fact that S100 family members S100A4, S100A6, and S100A11 are also
able to complex with AnxA2 and are expressed in cervical epithelium
97–100
. If other S100-
AnxA2 complexes behave similarly to A2t, these alternative binding modes could
compensate for the loss of S100A10 and result in the dampened inhibition of HPV
infection. Accordingly, AnxA2 re-expression was sufficient to restore HPV16 infection
in our rescue experiments, although S100A10 protein levels were not comparable to WT.
Alternatively, monomeric AnxA2 may serve an independent function post-endocytic
trafficking.
51
Two primary models for the involvement of A2t in HPV infection have been
previously described
23,24
. These models are not mutually exclusive, but A2t’s
involvement was studied from two distinct perspectives. The first is based on previous
work from our laboratory and emphasizes a direct interaction between the S100A10
subunit of A2t and HPV16 capsid protein L2 (HPV L2), and further posits that A2t is an
HPV L2-specific cell surface receptor for HPV16
23,43,50
. The second, based on detailed
studies from Ozbun’s laboratory, emphasizes an interaction between AnxA2 and HPV16
capsid protein L1 (HPV L1) at the cell surface, and suggests that S100A10 is involved in
post-entry intracellular trafficking
24
. In the absence of S100A10 and A2t, we were
surprised to find no difference in the quantity of HPV bound to the cell surface. Given
that HPV exposure increases AnxA2 translocation to the cell surface, and because A2t
and HPV colocalize at the cell surface
24
, it is reasonable to hypothesize that A2t functions
as an HPV receptor. However, we show quantitative evidence that A2t is not a sole
requirement for the binding of HPV to the host cell surface. These data do not negate the
involvement of A2t at the plasma membrane, but rather indicate that a direct HPV-A2t
interaction is not required for cell surface binding. On a larger scale, these data are in
agreement with the emerging consensus that HPV cell surface binding involves multi-
protein complexes and warrants further work defining an HPV-entry associated
membrane platform
101
.
Following HPV binding to the cell surface, there is no significant difference in
HPV internalization into S100A10 KO and A2t KO cells compared to WT HeLa cells. These
results are inconsistent with previous findings that negative regulation of AnxA2 results
in delayed HPV16 internalization
24
. Dziduszko and Ozbun interrogated this pathway
using an anti-AnxA2 antibody targeting the N-terminus to inhibit interactions at the
surface, and it is difficult to discern whether the observed kinetic effect is due to
functional silencing of AnxA2 or antibody-triggered endocytosis. Nonetheless, work
from the same study showed an increased colocalization of HPV16 with late
endosome/lysosome marker LAMP1 following anti-S100A10 antibody treatment. These
data suggested a role for A2t in HPV intracellular trafficking but fell short of giving a
52
complete picture of the discrete post-endocytic steps that A2t and/or its subunits may be
regulating. To directly interrogate these differences, we used our KO cells to investigate
at which points S100A10 and A2t are required in HPV intracellular trafficking. Our
results revealed the same phenotype in the S100A10 and A2t KO cells with respect to HPV
binding, endocytosis, and intracellular trafficking events. These findings indicate that
the AnxA2 monomer alone is not capable of shuttling the virus to the MVE.
An important feature of this study was analyzing the effect that knocking out
S100A10 and A2t has on non-HPV endocytic mechanisms. HPV endocytosis has been
shown to be pinocytosis-like, but independent of canonical endocytic pathways such as
clathrin-mediated endocytosis, and suggested to be novel
7
. Conversely, some
investigators have hypothesized that association of HPV with various cell factors may be
indicative of the utilization of multiple entry routes depending on receptor engagement
24
. Our data show that targeting AnxA2 and S100A10 does not lead to aberrant HPV
internalization through alternative endocytic pathways, and support the previous
hypothesis that HPV employs a specific and potentially novel endocytic pathway.
Furthermore, our data directly demonstrate that both the AnxA2 monomer and A2t are
involved in pinocytosis but not receptor-mediated, clathrin-dependent endocytosis.
By tracking HPV via confocal microscopy, we found that A2t is required for the
progression of HPV from early endosomes to CD63+ MVEs (the late endosome stage for
HPV
7,33
), and that monomeric AnxA2 alone is not sufficient to facilitate this process.
Subsequently, capsid disassembly was dramatically inhibited in the absence of S100A10
and A2t, confirming previous reports and corroborating our intracellular trafficking
results.
These results are strikingly similar to those published by Gräßel et al. in 2016. In
this study, knockdown and knock-in techniques were utilized to demonstrate the
requirement of the CD63-syntenin-1 complex in HPV infection and trafficking to MVEs
33
. Not surprisingly, we found that colocalization of AnxA2 and CD63 is increased upon
HPV infection. Consistent with observations in the literature, we also observed a notable
increase in AnxA2 intensity in HPV-treated cells
24
. Throughout the intracellular
53
trafficking of HPV, AnxA2 and CD63 appear to be members of a complex that likely
includes other members (e.g. syntenin-1 and S100A10) and is critical for efficient HPV
infection. Taken together, these data suggest that successful infiltration and unchecked
descent of HPV into the host nucleus requires A2t-dependent vesicle trafficking from
early endosomes to MVEs.
S100A10 facilitates the trafficking of a variety of membrane-resident proteins
(reviewed in
40
), but in contradiction to the data supporting these conclusions, another
group claims that S100A10 is dispensable for the endosomal transport functions of
AnxA2
71,102
. As a result of our study design, we were able to directly test if S100A10 is
involved in AnxA2-dependent vesicle trafficking. Utilizing both HPV as model cargo and
BSA as pathway-specific canonical cargo in our uptake experiments, we provide clear
evidence for a role for S100A10 in AnxA2-associated trafficking. Furthermore, a study
investigating MVE biogenesis did not discern between monomeric AnxA2 and A2t by
examining the role of S100A10
72
. Our findings support previous work defining a role for
AnxA2 and provide the first evidence that heterotetrameric A2t is functioning in MVE
biogenesis.
Annexin A2 and its binding partner S100A10 serve dynamic roles in and outside
of HPV intracellular trafficking
51,61,93
. The role that AnxA2 and A2t serve at the HPV-host
cell surface interface however, is poorly understood. AnxA2 and A2t are involved in
membrane trafficking events including microvesicle formation, vesicle aggregation,
phagocytosis, and nucleotide and protein trafficking
73,74,103–107
. Notably, these functions
are relevant for binding to and co-internalization of HPV with host cell surface receptors
and subsequent vesicle trafficking. Although not necessary for cell surface binding,
AnxA2 may be required at the cell surface to recruit and organize essential co-factors at
the HPV entry platform/receptor complex. In combination with the fact that HPV
exposure leads to an increase in cell surface-bound A2t, this is an area that should be
further investigated.
This study delineates the involvement of A2t in HPV trafficking from early
endosomes to MVEs in a CD63-dependent fashion, protection from lysosomal
54
degradation, and as a requirement for HPV capsid disassembly. Furthermore, using
HPV as a model we provide evidence that S100A10 is involved in AnxA2-mediated
endosomal transport and MVE biogenesis. By implementing methods that can
differentiate between AnxA2- and A2t-dependent effects in HPV intracellular
trafficking, we solidify existing knowledge about AnxA2-dependent endocytosis, and
provide a framework for future studies investigating the role of AnxA2 versus A2t in non-
HPV endocytosis through analysis of S100A10-associated functions.
4.4 Methods
Cell culture, CRISPR/Cas9 gene editing, and proliferation analysis
HeLa cells (CCL-2, ATCC) derived from cervical carcinoma were maintained in Iscove’s
Modified Dulbecco’s Medium (IMDM) with 10% FBS, 1x BME, and 1x gentamycin at 37°C
with 5% CO 2.
Addgene plasmid pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) (#42335; a gift
from Ling Shao) was used for CRISPR/Cas9-targeted ANXA2 and S100A10 gene
disruption via mutant Cas9 nickase (D10A variant). The following target sequences were
used: 5’-CAGGAAGCACGAACATCAGC-3’ and 5’-ACAGGGGCTGGGAACCGACG-3’ for
ANXA2; and 5’-AATGGTGAGGCCCGCAATTA-3’ and 5’-GTAGTACACATGAAGCAGAA-3’
for S100A10. Guide RNA (gRNA) were cloned using Feng Zhang Laboratory protocols
76
.
Non-target plasmid was used as negative control and pMSCVpuro vector (a gift from Ling
Shao) was used for selection. HeLa cells co-transfected with Lipofectamine2000 (Thermo
Fisher) were selected with puromycin for 72h, and clonal populations were generated
through a limited dilution series. Clonal populations were then sequenced to verify gene
disruption and analyzed for protein expression via western blot.
Knock-out clones were analyzed for differences in cell proliferation via cell
counting (trypan blue exclusion method) as well as DNA quantification using the
55
CyQUANT Cell Proliferation Assay Kit (Invitrogen). Briefly, WT, S100A10 KO, and A2t KO
cells were grown for 48h, collected with trypsin-EDTA, diluted 1:1 with trypan blue stain
(Invitrogen), and viable cells were counted. To quantify DNA via CyQUANT kit, cells
were cultured for 48h, frozen at -80°C overnight, thawed at room temperature, and lysed
using CyQUANT cell lysis buffer with RNAse and GR dye according to manufacturer
protocol. Fluorescence of GR dye-bound DNA was measured using a SpectraMax M5
plate reader (Molecular Devices) and DNA was quantified by comparison to DNA
standard curve.
Pseudovirion and virus-like particle production
HPV16, -18, -31, and -45 PsVs were prepared as previously described
60

(http://home.ccr.cancer.gov/LCO/ripcord.htm). Briefly, 293TT cells were co-transfected
with codon-optimized L1 and L2 plasmids for HPV16, -18, -31, and -45, and pCIneoGFP
reporter plasmid. For bulk PsV preparations, the self-packaging p16L1L2 plasmid was
utilized. HPV L1 and L2 expression vectors used were p16L1L2 (bulk), p16sheLL,
p18sheLL, p31sheLL, and p45sheLL (all kind gifts from J. Schiller). Infectious titer was
determined by flow cytometric analysis of GFP+ 293TT cells 48h post-treatment and
calculated as infectious units/mL. Bulk PsV preps were quantified via coomassie blue
staining utilizing known BSA concentrations.
HPV16 virus-like particles (VLPs) were produced using a recombinant
baculovirus expression system in insect cells as previous described
108
. Western blot
analysis confirmed the presence of HPV L1 and L2, while a neutralizing antibody ELISA
confirmed the presence of intact particles. Coomassie Blue staining was performed to
determine protein purity and standardize the concentration of HPV L1 content of the
VLP preparations.
Antibodies
56
Previously described anti-HPV16 L1-specific neutralizing antibody H16.V5 and internal
epitope-recognizing 33L1-7 (L1-7 Ab) were gifts from Neil Christensen and Martin Sapp,
respectively
75,109
. Anti-HPV16 L1 (H16.56E) used in immunofluorescence experiments
was a gift from Martin Sapp
110
. Anti-AnxA2 (610069), anti-S100A10 (610071), and anti-
HPV16 L1 (550840) were purchased from BD Biosciences. Mouse IgG isotype control
(ab37355), rabbit IgG isotype control (ab37415), anti-CD63 (ab118307), and goat-anti-
rabbit TRITC (ab6718) were purchased from Abcam. Anti-b actin (4970), anti-clathrin
(4796), and anti-EEA1 (3288) were purchased from Cell Signaling Technology. Anti-
mCherry (677702) and goat-anti-mouse DyLight 488 (405310) were purchased from
BioLegend. Antibodies used in AnxA2-CD63 colocalization immunofluorescence
microscopy were anti-AnxA2 (11256-1-AP) and anti-CD63 (ab8219) from Proteintech and
Abcam, respectively. Goat-anti-mouse IRDye 800CW (925-322) and goat-anti-rabbit
(H+L) Alexa Fluor 680 (A27042) secondary antibodies used for infrared western blot
imaging were purchased from Li-Cor and Invitrogen, respectively. For
immunofluorescence microscopy controls: isotype controls and secondary antibody-
only controls yielded minimal background (Fig. 4.9).

57

Figure 4.9. Immunofluorescence control antibody experiments show minimal
background. and WT cells grown on chamber slides were fixed with 4% PFA and
immunostained with either mouse (Ms) or rabbit (Rb) isotype control primary
antibodies (top) or no primary antibodies (bottom). All slides were stained with either
goat-anti-Ms DyLight 488 (left) which is fluorescent in the FITC channel or goat-anti-Rb
TRITC (right) secondary antibodies. Nuclei were stained with DAPI (blue) and a single
confocal slice is shown in each representative image.
Pseudovirus infection and infection rescue assay
2E4 cells were seeded in 24-well plates and infected with a 50% tissue culture infective
dose (TCID 50) the following day. TCID 50 was determined by titrating the multiplicity of
infection (MOI) for each genotype to result in approximately 50% infected cells, 48h
post-infection. Infection is defined in this manuscript as gene transduction of the GFP
reporter plasmid. In infection assays, %GFP-positive cells were analyzed 48h post-virus
addition via flow cytometry (FC500, Beckman Coulter). For rescue experiments, cells
were seeded in 6-well plates, grown to 80% confluency, and transfected with pEGFP-
N3_ANXA2-mCherry (a gift from Volker Gerke) or an empty vector using Lipofectamine
2000 (Thermo Fisher). Utilizing HPV16, cells were subjected to the HPV-infectivity assay
as described above and infection rescue was analyzed by first gating mCherry-positive
cells and then measuring %GFP-positive cells within that population using CXP software
(version 2.2).
Cell surface binding assay
Concentration of heparinase I was determined via titration and analyzing surface-bound
HPV before and after treatment. Reductions in binding were then compared to reported
decreases in HPV binding upon HSPG removal following heparinase I treatment. Briefly,
58
WT HeLa cells were grown to 80% confluence, washed with heparinase buffer, and
incubated with either buffer or increasing concentrations of heparinase I for 1h at 37°C.
Cells were transferred to ice, washed with ice-cold PBS, and saturated with HPV16
pseudovirions (10µg/1E6 cells) in serum-free conditions for 1h at 4°C. Cells were
collected via scraping on ice, and the amount of surface-bound HPV was analyzed by
flow cytometry.
For binding assay, 2.5E5 cells were seeded in 6-well plates and grown overnight.
Cells were washed with ice cold buffer (20mM Tris-HCl, 50mM NaCl, 4mM CaCl 2, and
0.01% BSA) and then treated with either buffer (vehicle control) or 2U/mL heparinase I
(Sigma-Aldrich) for 1h at 37°C to remove HSPGs. Plates were transferred to ice and
washed thoroughly with ice cold PBS+CaCl 2. PBS supplemented with 1mM CaCl 2 was
used for all wash steps as AnxA2 binds membranes in a calcium-dependent manner.
Cells were then treated with 10µg/1E6 cells HPV16 in cold serum-free media for 1h at 4°C
to saturate binding. Cells were collected on ice via scraping, stained with H16.V5 (1:100)
for 30min at 4°C, and fixed with 2% PFA in order to ensure measurement of particles
that might internalize at room temperature during analysis. Mean fluorescence intensity
(MFI) was measured via flow cytometry.
Virus internalization assays
2.5E5 cells were seeded in 6-well plates and grown overnight. Plates were cooled to 4°C
for 30min and 5µg/1E6 cells of HPV16 PsVs were added for 1hr at 4°C. Cells were
collected and surface-bound HPV was measured as described in cell surface binding
assay. Unbound virus was washed away with PBS+CaCl 2 and cells were incubated for an
additional 4h at 37°C to promote internalization of surface-bound HPV. After 4h,
remaining surface-bound HPV was again analyzed via flow cytometry. The amount of
internalized PsV was determined by subtracting MFI at 4h (remaining surface HPV)
59
from MFI at 0h (initial surface HPV). MFI of WT HeLa cells was set to 100% and KO cells
were normalized to that value (Fig. 4.10).

Figure 4.10. Internalization assay study design. Cells pre-cooled to 4°C (30min) were
treated with HPV16 L1L2 PsV for 1h at 4°C. Cells were washed and then incubated for 4h
at 37°C to promote surface-bound virus internalization. MFI was measured at 0hr (1h
post-binding) and after 4h incubation at 37°C. Amount internalized was determined by
subtracting MFI at 4h from MFI at 0h.

For pHrodo-HPV16 uptake assays, HPV16 VLPs were labeled with pH-dependent
rhodamine fluorophore (pHrodo iFL Red STP, Life Technologies) in a 10:1 (dye:HPV L1
protein) ratio, purified with 2% agarose beads (sized 50-150µm) (Gold Biotechnology),
and treated as described above. Briefly, after 30min at 4°C cells were treated with HPV16
PsVs for 4h at 37°C and MFI of pHrodo-HPV16 was measured via flow cytometry. MFI
was measured from 0-12h and trends remained consistent.

60
Lysosome degradation assay via Western blot
2.5E5 cells were seeded in 6-well plates, allowed to adhere, and then treated overnight
(16h) with 50µM chloroquine diphosphate crystalline (Sigma-Aldrich) or vehicle control.
Plates were cooled to 4°C for 30min, treated with 5µg/1E6 cells HPV16 PsVs for 1hr at
4°C, thoroughly washed with PBS+CaCl 2, and then incubated for 4h at 37°C to promote
internalization. Cells were collected by in-plate lysis using M-PER Mammalian Protein
Extraction Reagent (Thermo Fisher) and lysates were analyzed via western blot for
cleaved HPV L1 (CAMVIR-1 antibody) and b-actin using Odyssey Infrared blot imager
(Li-Cor).
Endocytic uptake assays
2.5E5 cells were seeded in 6-well plates and grown overnight. Plates were cooled to 4°C
for 30min and either Alexa Fluor 488-conjugated transferrin from human serum
(Transferrin-AF488, Thermo Fisher) or fluorescein isothiocyanate (FITC)-conjugated
bovine serum albumin (BSA) (FITC-BSA, Sigma-Aldrich) were added, and cells were
transferred to 37°C. Cells were collected at 0.5, 1.5, and 3h after the temperature shift,
and MFI was measured via flow cytometry.
Immunofluorescence microscopy
For HPV colocalization with EEA1 and CD63, and for L1-7 Ab staining experiments, cells
were seeded on 8-well chamber slides (1E4 cell/well) and grown overnight. Slides were
cooled to 4°C for 30min prior to treatment with 0.5µg/1E6 cells of HPV16 PsVs for 1h or
7h at 37°C. At 1h, cells were stained for HPV16 (H16.56E, 1:100) and either EEA1 (1:100)
or clathrin (1:50). At 7h, cells were stained for HPV16 (H16.56E) and either CD63 (1:100),
or L1-7 Ab (1:10). For AnxA2-CD63 colocalization, cells were seeded on 3-well chamber
slides (2.5E4 cells/well) and grown overnight. Slides were treated with HPV16 PsVs at
61
0.5µg/1E6 cells for 4h or 7h at 37°C. Cells were stained for AnxA2 (1:100) and CD63 (1:100).
For imaging in non-permeabilized conditions, Triton X-100 was omitted from the
immunostaining procedure. All slides were fixed with 2-4% PFA prior to staining and
mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher). Images
were visualized on a Nikon Eclipse Ti-E laser scanning confocal microscope. For
colocalization analysis, 10-20 images were analyzed with Fiji (a distribution of ImageJ,
NIH)
111,112
. Using the JACoP plugin
113
, thresholds were automatically set, and the extent
of colocalization was measured using Mander’s colocalization coefficient. Batch image
analysis was achieved through an application-specific script in Fiji macro language.
Statistics
Background from control groups was subtracted in all experiments and KO cells were
normalized to WT cells for comparison. Statistical analyses were performed using
GraphPad Prism (version 6) and detailed statistical tests for each figure are provided in
legends.

62
Chapter 5

Discussion

Associated publication: Taylor JR, et al. 2018. Annexin A2 in virus infection. Frontiers in
Microbiology, 9:2954.
5.1 Introduction
To successfully replicate, viruses must hijack and reprogram host cells to produce viral
progeny. The life cycle of a virus consists of three main phases intimately reliant on host
cell proteins and mechanisms. The first is cellular attachment and penetration.
Attachment and penetration can occur through receptor-mediated endocytosis or
through direct membrane fusion. Second, the viral genome is released for replication
and protein expression. In this phase, the virus can rely on host enzymes to facilitate
capsid uncoating or host machinery to replicate the viral genome. Finally, assembly and
maturation yield newly constructed viral particles poised for release. In this stage, viral
proteins can require post-translational modification by host factors, or intracellular
transport systems for proper localization. During egress, virions are released by taking
advantage of apoptosis, exocytosis, cell lysis, and by appropriating host membranes to
bud directly from the cell. In this study, we shed light on a host factor that has been
repeatedly exploited for the benefit of viral infection: the annexin A2/S100A10
heterotetramer (A2t) and monomeric annexin A2 (AnxA2). AnxA2 is a membrane-
associated protein implicated in a number of human, animal, and zoonotic infections.
In the presented work we specifically show that cell surface A2t is not required
for HPV attachment, and that in the absence of A2t, virion internalization remains
clathrin-independent. These results moderately contrast the previously hypothesized
HPV entry model, which positioned A2t as the primary cell surface binding receptor for
HPV
50
. This model was built from the fact that A2t is predominantly located at the cell
63
surface, HPV L2 and S100A10 interact, and HPV and A2t colocalize and co-
immunoprecipitate at the cell surface, albeit through an indirect interaction
24
.
Furthermore, this study found that anti-AnxA2 antibodies did not alter the amount of
HPV bound to the cell surface, which directly support our findings demonstrating no
difference in the amount of HPV bound to the cell surface in the absence of S100A10 and
A2t. These findings are also in agreement with the current consensus that HPV entry
requires a multiprotein complex as opposed to a single receptor. Although A2t is not
required for cell surface binding, it is likely that A2t serves an important function at the
cell surface, in organizing the proposed HPV entry complex or in a signaling capacity,
for example. More broadly, we show that successful infection by multiple oncogenic
HPV genotypes is dependent on A2t, although the observed reduction in infection is less
significant in the absence of S100A10 alone compared to full A2t. These findings support
an independent role for monomeric AnxA2 post-viral uncoating, and suggest that
S100A10 and A2t are broad mediators of high-risk HPV intracellular trafficking post-
entry, pre-viral uncoating.
In the absence of both S100A10 and A2t, we found that viral progression from
early endosomes to multivesicular endosomes is significantly decreased, strongly
indicating that heterotetrameric A2t controls trafficking from the early endosome to the
MVE. Without A2t, lysosomal degradation of HPV is accelerated, demonstrating that in
the presence of A2t, HPV is able to avoid trafficking to the lysosome. These results
suggest that A2t may inhibit viral recognition, however a more detail investigation would
be required to accurately describe this interaction. Additionally, AnxA2 has
immunomodulatory functions
61,114,115
, and it is therefore possible that A2t expression
promotes a permissive environment for infection through modulation of innate immune
responses. Ultimately, future studies into AnxA2 in the host-pathogen interaction should
investigate interactions not only between the host cell and the pathogen, but between
the host cell and its own immunological environment in the context of the pathogen.
In wild type cells, we found that AnxA2 forms a complex with CD63, a known
mediator of HPV intracellular trafficking that co-localizes with HPV at the cell surface
64
33
. These results support the hypothesis that HPV entry occurs at large multi-protein
complexes and that A2t is a major component of this functional complex. Lastly, by
demonstrating a clear role for S100A10 in HPV and BSA trafficking, our findings support
a previously contested role for S100A10 in vesicle trafficking
40,71,102
, exemplifying the
importance of differentiating functions of AnxA2 versus A2t. Finally, by experimentally
addressing the involvement of AnxA2 and A2t in HPV endocytosis, these studies have
advanced our understanding of HPV endocytosis and further characterized the role of
S100A10 in AnxA2-dependent protein trafficking. By further discussing the involvement
of these proteins in the life cycle of multiple viruses, we propose that A2t is an emerging
viral host factor.
5.2 Broader implications
Annexin A2 as an emerging viral host factor
AnxA2 is a multifunctional calcium- and lipid-binding protein that is expressed in nearly
all human tissues and cell types. AnxA2 exists as a monomer localized to the cytoplasm,
vesicle-bound, or as a heterotetrameric complex termed A2t consisting of two AnxA2
monomers bridged by an S100A10 dimer found on the inner and outer leaflet of the
plasma membrane. Both AnxA2 and A2t have been implicated in a wide range of
intracellular processes including membrane domain organization, membrane fusion,
vesicle aggregation, cytoskeletal-membrane dynamics, epithelial cell polarity,
exocytosis, endocytosis, phagocytosis, and transcriptional regulation through binding of
AnxA2 to RNA (reviewed in
41,42,51,61,83,93
).
More broadly, AnxA2 has been implicated in immune function, multiple human
diseases, and viral infection
61,83,116,117
. AnxA2 expression in some cancers can promote
metastasis and function as a prognostic marker of recurrence and survival
104,118,119
. This
involvement of AnxA2 in human health and disease has prompted the development of
65
pharmacological inhibitors of AnxA2 and A2t
54–56,120
, and these compounds are being
explored in a growing number of therapeutic contexts. One class of inhibitors, for
example, has been shown to block human papillomavirus (HPV) type 16 (HPV16)
infection in cervical epithelial cells
43
. It is presumed that these A2t inhibitors disrupt the
function of A2t during viral infection, though this specific mechanism still needs to be
verified. Importantly, HPV is just one virus in a list of at least 13 viruses with known
AnxA2 associations during binding, endocytosis, and egress (Table 5.1). By addressing
the involvement of AnxA2 in the context of multiple viruses and viral life cycle stages,
we offer a broad perspective on an emerging host-pathogen interaction and highlight
the complexities in AnxA2 biology.
The annexin superfamily is highly conserved across eukaryotic phyla, from
unicellular organisms to complex plants and animals
121–123
, and annexins have been
associated with both human and non-human viral pathogens (summarized in Fig. 1).
This discussion focuses on seven viruses with direct links to AnxA2 during their lifecycle.
To more confidently cross-compare cellular functions, we specifically examine the
viruses that target human epithelial cells. AnxA2 is utilized by HPV, enterovirus 71
(EV71), respiratory syncytial virus (RSV), and cytomegalovirus (CMV) during cell
attachment and penetration
23,24,43,124–130
, by hepatitis C virus (HCV) and influenza A virus
(IAV) during replication
131–135
, and by measles virus (MV) during assembly and
maturation
136
. In some cases, AnxA2 has been implicated in multiple life cycle steps of
the aforementioned viruses, underscoring the importance of a more complete approach
to understanding the role of AnxA2 in viral infection.
Virus attachment and entry into target cells occurs through host receptor-
mediated endocytic mechanisms, or less frequently through direct fusion between virus
envelope and the plasma membrane. The first steps of infection are attractive antiviral
targets and are therefore studied extensively in a vast array of viral infections (selected
reviews include:
137–140
). During the first phase of infection, cell attachment and
penetration, AnxA2 is utilized by HPV, enterovirus 71 (EV71), respiratory syncytial virus
(RSV), and cytomegalovirus (CMV)
23,24,43,124–130
.
66

Table 5.1. Annexin A2-associated viruses

Virus Family Envelope Genome Primary
host
tropism
Reference(s)
Human
papillomavirus
(HPV)
Papillomaviridae – dsDNA Human Woodham et al.,
2012*; Dziduszko
and Ozbun, 2013*;
Taylor et al., 2018
Enterovirus 71
(EV71)
Picornaviridae – ssRNA Human Yang et al., 2011
Respiratory
syncytial virus
(RSV)
Paramyxoviridae ＋ ssRNA Human Malhotra et al.,
2003
Cytomegalovirus
(CMV)
Herpesviridae

＋ dsDNA Human Wright et al., 1994,
1995; Raynor et al.,
1999*; Derry et al.,
2007*
Hepatitis C virus
(HCV)
Flaviviridae

＋ ssRNA Human Lai et al., 2008;
Saxena et al., 2012;
Backes et al., 2010*
Influenza A virus
(IAV) H1N1
Orthomyxoviridae

＋ ssRNA Human Lebouder et al.,
2008*
Avian influenza A
virus (IAV) H5N1
Orthomyxoviridae

＋ ssRNA Avian/human Ma et al., 2017
Measles virus
(MV)
Paramyxoviridae

＋ ssRNA Human Koga et al., 2018*
Human
immunodeficiency
virus type 1 (HIV-
1)
Retroviridae ＋ ssRNA Human Ma et al., 2004;
Ryzhova et al.,
2006; Rai et al.,
2010*; Woodham et
al., 2016*
Rabbit vesivirus
(RaV)
Caliciviridae – ssRNA Rabbit Gonzàlez-Reyes et
al., 2009
Infectious
bronchitis virus
(IBV)
Coronaviridae ＋ ssRNA Avian Kwak et al., 2011
Classical swine
fever virus (CSFV)
Flaviviridae

＋ ssRNA Porcine Sheng et al., 2015;
Yang et al., 2015
Bluetongue virus
(BTV)
Reoviridae

– dsRNA Livestock Beaton et al.,
2002*; Celma and
Roy, 2011*
Porcine
reproductive and
respiratory
syndrome virus
(PRRSV)
Arteriviridae

＋ ssRNA Porcine Li et al., 2014;
Chang et al., 2018
* = study addressed monomeric AnxA2 and heterotetrameric AnxA2+S100A10 (as A2t)

67
Annexin A2 and human papillomavirus (HPV)
Persistent infection with HPV can lead to the development of a variety of anogenital and
oropharyngeal cancers causing significant morbidity worldwide
2,49,77
. HPV is a non-
enveloped double stranded DNA (dsDNA) virus that enters basal keratinocytes through
a non-canonical endocytic pathway while interacting with a number of host molecules
6,7,50,79,141
. In the search to identify an HPV uptake receptor, AnxA2 and A2t were
discovered as central mediators of HPV entry and intracellular trafficking. Interestingly,
it has been suggested AnxA2 and A2t have independent functions in HPV attachment
and intracellular trafficking
24
. For example, it was shown that AnxA2 and A2t colocalize
with HPV at the cell surface and that antibodies against AnxA2 alter entry kinetics
24
, but
antibodies against the S100A10 subunit
24
and targeted knock-out via CRISPR/Cas9
130

does not affect cellular entry in vitro. Furthermore, when the full A2t complex is
knocked-out HPV infection is significantly reduced as measured by reporter gene
transduction. However, when S100A10 alone is knocked-out, only a moderate reduction
in infection is observed
130
, emphasizing the importance of delineating the roles of
monomeric AnxA2 versus heterotetrameric A2t.
Annexin A2 and enterovirus 71 (EV71)
EV71 is a causative agent of hand, foot, and mouth disease (HFMD), a common infection
in infants and children that can sometimes lead to severe illness and long term
neurological conditions
142
. EV71 is a non-enveloped single-stranded RNA (ssRNA) virus
that enters cells through an unknown dynamin-independent pathway
143
. In an effort to
understand initial host-virus interactions, AnxA2 was identified as a cell surface
attachment factor through anti-EV71 immunoprecipitation and mass spectrometric
analysis of infected cells in vitro
124
. Using immunofluorescence microscopy, these
authors also demonstrated that AnxA2 and EV71 colocalize at the cell surface, and that
pretreatment with recombinant AnxA2 (rAnxA2) or antibodies against AnxA2 yields
68
reduced infectivity. Results from this work showed that AnxA2 and EV71 colocalize at
the cell surface, but they did not address if the reduction in infectivity was due to reduced
binding, entry, or replication. Furthermore, their yeast two-hybrid experiments showed
that EV71 capsid protein VP1 interacted with the C-terminus of AnxA2, which may also
implicate A2t as serving a functional role. Future studies investigating AnxA2 or A2t in
EV71 endocytosis could yield interesting results given the varied implications of AnxA2
in this process.
Annexin A2 and respiratory syncytial virus (RSV)
Infants and elderly can develop severe lower respiratory disease from infection by RSV
144
– an enveloped ssRNA virus. The mechanism of RSV cellular entry is disputed, with
independent reports suggesting plasma membrane fusion, clathrin-dependent
endocytosis, and macropinocytic mechanisms
145–148
. Fucoidan is a polysaccharide that
inhibits RSV infection in vitro and in vivo. Because it is assumed that fucoidan works by
binding to RSV receptors, Malhotra et al. employed solid-phase-immobilized fucoidan
as an affinity matrix to isolate potential RSV-binding partners on epithelial cells and
identified AnxA2 using mass spectrometry
125
. The authors show that treatment with
rAnxA2 reduced RSV infection as measured by fluorescent focus assay 24h post-
infection, but similar to Yang et al., they did not investigate the mechanism of infection
reduction beyond cell surface interactions. An independent study did however
demonstrate that AnxA2 is not involved in virus assembly
149
. As was the case with EV71,
a more detailed analysis of RSV endocytosis has the potential advance our understanding
of AnxA2-mediated endocytosis.
Annexin A2 and cytomegalovirus (CMV)
CMV infection in immunocompromised individuals or through congenital transmission
can lead to serious diseases including pneumonia and hearing loss
150
. CMV is an
69
enveloped dsDNA virus that is able to establish life-long persistence, and multiple CMV
entry mechanisms have been described. Interestingly, it has been hypothesized that the
viral entry route may actually influence the outcome of infection
151
. Early work first
discovered AnxA2 on the surface of CMV particles isolated from human fibroblasts, and
found that rabbit antiserum against AnxA2 inhibited CMV infection in vitro
126,127
. Using
synthetic membrane systems and rAnxA2, Raynor et al demonstrated enhanced binding
of CMV if rAnxA2 was present and attributed fusion events to A2t
128
. Follow up studies
elucidated that AnxA2 is not essential for CMV entry
152,153
, however viral gene expression
and completion of viral life cycle are dependent on AnxA2 and A2t
129
and progeny virions
have been shown to contain both forms of AnxA2 on viral envelopes
126
. These findings
together suggest multiple roles of both AnxA2 and A2t for CMV trafficking and progeny
egress.
The ultimate goal of a virus is to produce and release progeny virions. In order to
make new infectious particles, viruses must transcribe and replicate their genomes in
either the cytoplasm or the nucleus of a host cell. To accomplish this, the virus
orchestrates cellular factors to form replication complexes: organelle-like structures
that form in the nucleus, the cytoplasm, endoplasmic reticulum (ER), or at the plasma
membrane and shield cytoplasmic genome replication from host defenses
154,155
. Post-
replication, virus particles must reassemble and traffic to the plasma membrane for
release.
Annexin A2 and hepatitis C virus (HCV)
Chronic infection with HCV can lead to the development of liver cirrhosis and
hepatocellular carcinoma. HCV is an enveloped ssRNA virus that enters the host cell
through endocytosis, replicates in ER-replication complexes, and exits via exocytosis
156–
158
. Many viruses express non-structural (NS) proteins to aid in efficient and successful
infection; accordingly, NS proteins often function within viral replication complexes. In
2008, Lai et al. investigated host factors that might interact with a specific NS protein
70
complex of HCV (NS3/NS4A) known to interact with actin filaments in kidney epithelial
cells. NS3/NS4A expression and co-immunoprecipitation followed by mass spectrometry
identified AnxA2 as an interacting host factor
159
. Given that lipid rafts were
demonstrated to be involved in the formations of HCV RC complexes and because AnxA2
is associated with both lipid rafts and interacted with NS4A
160
, the authors published a
follow-up study asking if AnxA2 aids in the formation of HCV replication complexes
132
.
Their report details the localization of AnxA2 at HCV replication complexes via
immunofluorescence and immuno-electron microscopy, a reduction in the number of
these structures following AnxA2 siRNA silencing, and a reduction in HCV RNA
synthesis. The reduction in RNA synthesis, however, was measured via HCV replicase
activity although there was no observed change in relative mRNA levels. An independent
report conclusively demonstrated that although monomeric AnxA2 colocalizes with
HCV NS proteins, AnxA2 silencing has no direct effect on HCV RNA replication but
causes a significant reduction in intra- and extracellular virus titers
131
. Based on these
findings, the authors conclude that AnxA2 is involved in viral assembly as opposed to
replication. Interestingly, overexpression of AnxA2 led to an enrichment of HCV NS
proteins at replication complex sites
132
, a mechanism that may in fact promote virus
assembly and support the claim of these authors.
Annexin A2 and influenza A virus (IAV)
Of the four types of influenza viruses, A, B, C, and D, influenza A viruses (IAV) and
influenza B viruses cause epidemics of seasonal disease and respiratory infections. IAV
type H1N1 and zoonotic avian IAV type H5N1 have both been associated with AnxA2
134,135
. IAV is an enveloped ssRNA virus that enters host cells via endocytosis, replicates
in the nucleus, and buds from the plasma membrane for release
161
. It has been shown
that AnxA2 and A2t are present on IAV H1N1 viral envelopes and that A2t, a plasminogen
receptor, is responsible for the conversion of plasminogen to plasmin, a process
involved in IAV replication
134
. The authors demonstrated reduced viral titer after
71
inhibiting plasminogen activation but did not tease out the precise involvement of
AnxA2. An independent report investigated the role of AnxA2 in IAV H5N1 replication
and found that silencing AnxA2 via siRNA inhibited viral protein expression and reduced
progeny titer and proposed a mechanism by which AnxA2 bridged the gap between NS1
and p53, extending the amount of time cells could produce new virions
135
. These data
support the hypothesis that AnxA2 is involved in viral replication or assembly but do not
preclude AnxA2 involvement during the preceding steps.
Annexin A2 and measles virus (MV)
Measles is a highly contagious respiratory infection that is caused by MV – an enveloped
ssRNA virus. After MV fuses with the host cell membrane, genome replication occurs in
the cytoplasm and the virus is released by budding at the plasma membrane
162
.
Knockdown of AnxA2 via shRNA in cervical epithelial cells (HeLa) caused reduced MV
progeny virus generation 24h post-infection, but did not affect MV entry and RNA
replication
136
. Normally, MV matrix protein (M protein) aids in connecting the viral
capsid to the viral envelope and localizes to the plasma membrane where MV particles
will form. Koga et al. went on to reveal that in the absence of AnxA2, M protein
expression is decreased and mis-localized from the plasma membrane to the
perinuclear space. Finally, the authors found that the observed M trafficking effect was
due to monomeric AnxA2 versus A2t
136
.
Other viruses and species with Annexin A2 associations
The viruses discussed above represent a subpopulation of epithelial-targeting viruses
that cause disease in the human population and have all been shown to utilize AnxA2 or
A2t in some capacity during their viral lifecycle. Outside of humans AnxA2, A2t, or a
combination of the two have been implicated in the attachment and entry of rabbit
vesivirus (RaV)
163
, the replication of porcine reproductive and respiratory syndrome
72
virus (PRRSV; pig) and avian infectious bronchitis virus (IBV; chicken)
164–166
, and in the
assembly and release stages of classical swine fever virus (CSFV; pig) and bluetongue
virus (BTV; livestock)
167–170
. Additionally, there have been multiple reports of AnxA2-
viral associations occurring within cell types beyond epithelial cells (ex.
Alphaherpesviruses with neuronal cells
171
and human immunodeficiency virus with
macrophages
57,172–174
), however addressing viral tropisms for non-epithelial cells and
how different cell types utilize AnxA2/A2t does not fall within the scope of this
dissertation. Alas, a deeper understanding of how AnxA2 biology is manipulated during
the course of viral infections may uncover novel treatment routes or expand our
understanding of cellular biology in general.
Figure 5.1 summarizes where and how different viruses exploit AnxA2 and A2t
and serves as a visual representation of the complexity of this subject. It should be noted
that nearly half of these studies fail to address whether or not the observed AnxA2-
associated effects are due to monomeric AnxA2 or heterotetrameric A2t (indicated in
Table 1). Synthesizing our current understanding of AnxA2 in viral infections will reveal
similarities between pathogens, highlight deficiencies in our experimental approaches,
and help us better understand the diversity in AnxA2 functionality.

73

Figure 1. Schematic diagram of annexin A2 (AnxA2) and heterotetrameric
AnxA2/S100A10 (A2t) in viral infections. Placements of AnxA2 depict where either AnxA2
or A2t have been implicated in a viral infection. (A) Viruses with evidence for AnxA2
involvement in attachment and entry, which can occur via endocytosis or by fusion of
the viral envelope with the plasma membrane. (B) Viruses shown to utilize AnxA2 during
genome replication and expression, a process that can occur in the cytoplasm or
nucleus. (C) Viruses that have been shown to involve AnxA2 during assembly,
maturation, and egress. Assembly can occur in the nucleus or in the cytoplasm, and
release is achieved via cell lysis, apoptosis, exocytosis, or direct budding from the
plasma membrane. * = evidence for AnxA2 involvement in more than one phase of the
viral life cycle.
74
5.5 Patterns, potential, and future directions
Given the vast diversity in A2t functionality, it is probable that AnxA2 plays a highly
complex and dynamic role in virus infection. As mentioned above, AnxA2 also has
immunomodulatory effects
61,114,115
, and it therefore possible that AnxA2 expression
promotes a permissive environment for infection through modulation of innate immune
responses. Additionally, AnxA2 may play a role in initiating adaptive immune responses
against infections. For example, exogenous addition of A2t to the antigen-presenting
cells of the epithelium, Langerhans cells (LC), induced suppression of immune
activation and reduced Th-1 cytokine production in vitro, suggesting that A2t may
function as an immune modulator in the epithelium. The small molecule inhibitor
against A2t that was used in the HPV16 studies was also able to reverse HPV-induced
immune suppression of LC populations, further supporting the notion that a deeper
understanding of AnxA2 biology may reveal novel avenues for treatment options
114
.
In presenting our original data detailing the roles of AnxA2 and A2t in HPV
infection (Chapters 2
43
, 4
130
, and 5), along with a general overview of the different ways
AnxA2 is utilized by viruses (summarized in Table 5.1 and Fig. 5.1), we shed light on
broad mechanistic patterns and identify potential avenues for future research. We
conclude that AnxA2-mediated endocytosis represents a distinct trafficking pathway
utilized by multiple viruses. Future studies should implement a holistic research
approach that investigates the interactions between the host cell, the pathogen, and the
immunological environment in which the viral lifecycle takes place. Ultimately, multi-
modal research approaches may provide a more comprehensive understanding: we can
learn more about AnxA2 endocytosis by studying different viruses, and we can use
AnxA2 endocytosis as a model to better understand viral infection. In this dissertation,
we implemented methods that can differentiate between AnxA2- and A2t-dependent
effects, solidifying existing knowledge about-AnxA2-dependent endocytosis and A2t in
HPV infection, while providing a framework for future studies investigating AnxA2-
versus A2t-mediated functions.
75
Publications by the author

1. Taylor JR, Skeate JG, Kast WM. 2018. Annexin A2 in virus infection. Frontiers in
Microbiology, 9:2954.

Included in Chapter 5.

2. Taylor JR, Fernandez DJ, Thornton SM, Skeate JG, Lühen KP, Da Silva DM,
Langen R, Kast WM. 2018. Heterotetrameric annexin A2/S100A10 (A2t) is
essential for oncogenic human papillomavirus trafficking and capsid
disassembly, and protects virions from lysosomal degradation. Scientific
Reports, 8:11642.
Included in Chapter 4.

3. Woodham AW, Sanna AM, Taylor JR, Skeate JG, Da Silva DM, Dekker L V, Kast
WM. 2016. Annexin A2 antibodies but not inhibitors of the annexin A2
heterotetramer impair productive HIV-1 infection of macrophages in vitro.
Virology Journal, 13:1–5.

4. Woodham AW, Skeate JG, Sanna AM, Taylor JR, Da Silva DM, Cannon PM, Kast
WM. 2016. Human Immunodeficiency Virus Immune Cell Receptors,
Coreceptors, and Cofactors: Implications for Prevention and Treatment. AIDS
Patient Care and STDs, 30:291–306.

5. Skeate JG, Porras TB, Woodham AW, Jang JK, Taylor JR, Brand HE, Kelly TJ, Jung
JU, Da Silva DM, Yuan W, Kast WM. 2016. Herpes simplex virus downregulation
of secretory leukocyte protease inhibitor enhances human papillomavirus type
16 infection. Journal of General Virology, 97:422–434.
6. Da Silva DM, Woodham AW, Skeate JG, Rijkee LK, Taylor JR, Brand HE,
Muderspach LI, Roman LD, Yessaian AA, Pham HQ, Matsuo K, Lin YG, McKee
GM, Salazar AM, Kast WM. 2015. Langerhans cells from women with cervical
76
precancerous lesions become functionally responsive against human
papillomavirus after activation with stabilized Poly-I:C. Clinical Immunology,
161:197–208.

7. Woodham AW*, Taylor JR*, Jimenez AI, Skeate JG, Schmidt T, Brand HE, Silva
DM Da, Kast WM. 2015. Small molecule inhibitors of the annexin A2
heterotetramer prevent human papillomavirus type 16 infection. Journal of
Antimicrobial Chemotherapy, 70:1686–1690.

Included in Chapter 2.

8. Da Silva DM*, Woodham AW*, Rijkee LK, Skeate JG, Taylor JR, Koopman ME,
Brand HE, Wong MK, McKee GM, Salazar AM, Kast WM. 2015. Human
papillomavirus-exposed Langerhans cells are activated by stabilized Poly-I: C.
Papillomavirus Research, 1:12–21.

*Authors contributed equally to this work

77
REFERENCES

1. Garbuglia, A. R. Human papillomavirus in head and neck cancer. Cancers
(Basel). 6, 1705–26 (2014).
2. Spence, T., Bruce, J., Yip, K. W. & Liu, F. F. HPV associated head and neck
cancer. Cancers (Basel). 8, 1–12 (2016).
3. Vokes, E. E., Agrawal, N. & Seiwert, T. Y. HPV-Associated Head and Neck
Cancer. Journal of the National Cancer Institute (2015). doi:10.1093/jnci/djv344
4. Walker, T. Y. et al. National, Regional, State, and Selected Local Area Vaccination
Coverage Among Adolescents Aged 13–17 Years — United States, 2016. MMWR.
Morb. Mortal. Wkly. Rep. (2017). doi:10.15585/mmwr.mm6633a2
5. Hampson, L., Martin-Hirsch, P. & Hampson, I. N. An overview of early
investigational drugs for the treatment of human papilloma virus infection and
associated dysplasia. Expert Opin. Investig. Drugs 24, 1529–1537 (2015).
6. Spoden, G. et al. Clathrin- and caveolin-independent entry of human
papillomavirus type 16 - Involvement of tetraspanin-enriched microdomains
(TEMs). PLoS One 3, (2008).
7. Schelhaas, M. et al. Entry of human papillomavirus type 16 by actin-dependent,
clathrin- and lipid raft-independent endocytosis. PLoS Pathog. 8, (2012).
8. Qi, Y. M. et al. Epithelial cells display separate receptors for papillomavirus VLPs
and for soluble L1 capsid protein. Virology 216, 35–45 (1996).
9. Giroglou, T., Florin, L., Schafer, F., Streeck, R. E. & Sapp, M. Human
Papillomavirus Infection Requires Cell Surface Heparan Sulfate. J. Virol. 75,
1565–1570 (2001).
10. Selinka, H.-C. et al. Inhibition of Transfer to Secondary Receptors by Heparan
Sulfate-Binding Drug or Antibody Induces Noninfectious Uptake of Human
Papillomavirus. J. Virol. 81, 10970–10980 (2007).
11. Johnson, K. M. et al. Role of Heparan Sulfate in Attachment to and Infection of
the Murine Female Genital Tract by Human Papillomavirus. J. Virol. 83, 2067–
2074 (2009).
12. Kines, R. C., Thompson, C. D., Lowy, D. R., Schiller, J. T. & Day, P. M. The initial
steps leading to papillomavirus infection occur on the basement membrane prior
to cell surface binding. Proc. Natl. Acad. Sci. 106, 20458–20463 (2009).
78
13. Selinka, H.-C., Giroglou, T., Nowak, T., Christensen, N. D. & Sapp, M. Further
evidence that papillomavirus capsids exist in two distinct conformations. J. Virol.
77, 12961–7 (2003).
14. Dasgupta, J. et al. Structural basis of oligosaccharide receptor recognition by
human papillomavirus. J. Biol. Chem. 286, 2617–2624 (2011).
15. Richards, K. F., Bienkowska-Haba, M., Dasgupta, J., Chen, X. S. & Sapp, M.
Multiple Heparan Sulfate Binding Site Engagements Are Required for the
Infectious Entry of Human Papillomavirus Type 16. J. Virol. 87, 11426–11437
(2013).
16. Bienkowska-Haba, M., Patel, H. D. & Sapp, M. Target cell cyclophilins facilitate
human papillomavirus type 16 infection. PLoS Pathog. 5, (2009).
17. Scheffer, K. D. et al. Tetraspanin CD151 Mediates Papillomavirus Type 16
Endocytosis. J. Virol. 87, 3435–3446 (2013).
18. Scheffer, K. D., Berditchevski, F. & Florin, L. The tetraspanin CD151 in
papillomavirus infection. Viruses 6, 893–908 (2014).
19. Evander, M. et al. Identification of the alpha6 integrin as a candidate receptor for
papillomaviruses. J. Virol. 71, 2449–56 (1997).
20. McMillan, N. A. J., Payne, E., Frazer, I. H. & Evander, M. Expression of the α6
integrin confers papillomavirus binding upon receptor-negative B-cells. Virology
261, 271–279 (1999).
21. Surviladze, Z., Dziduszko, A. & Ozbun, M. A. Essential roles for soluble virion-
associated heparan sulfonated proteoglycans and growth factors in human
papillomavirus infections. PLoS Pathog. 8, (2012).
22. Surviladze, Z., Sterk, R. T., DeHaro, S. A. & Ozbun, M. A. Cellular Entry of
Human Papillomavirus Type 16 Involves Activation of the Phosphatidylinositol 3-
Kinase/Akt/mTOR Pathway and Inhibition of Autophagy. J. Virol. 87, 2508–2517
(2013).
23. Woodham, A. W. et al. The S100A10 subunit of the annexin A2 heterotetramer
facilitates L2-mediated human papillomavirus infection. PLoS One 7, (2012).
24. Dziduszko, A. & Ozbun, M. A. Annexin A2 and S100A10 Regulate Human
Papillomavirus Type 16 Entry and Intracellular Trafficking in Human
Keratinocytes. J. Virol. 87, 7502–7515 (2013).
25. Broniarczyk, J., Massimi, P., Bergant, M. & Banks, L. Human Papillomavirus
79
Infectious Entry and Trafficking Is a Rapid Process. J. Virol. 89, 8727–8732 (2015).
26. Becker, M., Greune, L., Schmidt, M. A. & Schelhaas, M. Extracellular
conformational changes in the capsid of human papillomaviruses contribute to
asynchronous uptake into host cells. J. Virol. (2018). doi:10.1128/JVI.02106-17
27. Broniarczyk, J., Bergant, M., Goździcka-Józefiak, A. & Banks, L. Human
papillomavirus infection requires the TSG101 component of the ESCRT
machinery. Virology 460–461, 83–90 (2014).
28. Broniarczyk, J. et al. The VPS4 component of the ESCRT machinery plays an
essential role in HPV infectious entry and capsid disassembly. Sci. Rep. 7, 1–11
(2017).
29. Bergant, M. & Banks, L. SNX17 Facilitates Infection with Diverse Papillomavirus
Types. J. Virol. 87, 1270–1273 (2013).
30. Wüstenhagen, E. et al. The cytoskeletal adaptor obscurin-like 1 interacts with the
HPV16 capsid protein L2 and is required for HPV16 endocytosis. J. Virol. 185,
JVI.01222-16 (2016).
31. Selinka, H. C., Giroglou, T. & Sapp, M. Analysis of the infectious entry pathway of
human papillomavirus type 33 pseudovirions. Virology 299, 279–287 (2002).
32. Bienkowska-Haba, M., Williams, C., Kim, S. M., Garcea, R. L. & Sapp, M.
Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid
Protein L1 from the L2/DNA Complex following Virus Entry. J. Virol. 86, 9875–
9887 (2012).
33. Gräßel, L. et al. The CD63-Syntenin-1 Complex Controls Post-Endocytic
Trafficking of Oncogenic Human Papillomaviruses. Sci. Rep. 6, 1–8 (2016).
34. Day, P. M., Thompson, C. D., Schowalter, R. M., Lowy, D. R. & Schiller, J. T.
Identification of a Role for the trans-Golgi Network in Human Papillomavirus 16
Pseudovirus Infection. J. Virol. 87, 3862–3870 (2013).
35. Lipovsky, A. et al. Genome-wide siRNA screen identifies the retromer as a
cellular entry factor for human papillomavirus. Proc. Natl. Acad. Sci. 110, 7452–
7457 (2013).
36. DiGiuseppe, S. et al. Incoming human papillomavirus type 16 genome resides in
a vesicular compartment throughout mitosis. Proc. Natl. Acad. Sci. 113, 6289–
6294 (2016).
37. Pyeon, D., Pearce, S. M., Lank, S. M., Ahlquist, P. & Lambert, P. F. Establishment
80
of human papillomavirus infection requires cell cycle progression. PLoS Pathog.
5, (2009).
38. Aydin, I. et al. Large Scale RNAi Reveals the Requirement of Nuclear Envelope
Breakdown for Nuclear Import of Human Papillomaviruses. PLoS Pathog. 10,
(2014).
39. Waisman, D. M. Annexin II tetramer: structure and function. Mol. Cell. Biochem.
149–150, 301–322 (1995).
40. Rescher, U. & Gerke, V. S100A10/p11: Family, friends and functions. Pflugers
Arch. Eur. J. Physiol. 455, 575–582 (2008).
41. Hitchcock, J. K., Katz, A. A. & Schäfer, G. Dynamic reciprocity: The role of
annexin A2 in tissue integrity. J. Cell Commun. Signal. 8, 125–133 (2014).
42. Bharadwaj, A., Bydoun, M., Holloway, R. & Waisman, D. Annexin A2
heterotetramer: Structure and function. International Journal of Molecular
Sciences 14, (2013).
43. Woodham, A. W. et al. Small molecule inhibitors of the annexin A2
heterotetramer prevent human papillomavirus type 16 infection. J. Antimicrob.
Chemother. 1686–1690 (2015). doi:10.1093/jac/dkv045
44. Puisieux, A., Ji, J. & Ozturk, M. Annexin II up-regulates cellular levels of p11
protein by a post-translational mechanisms. Biochem J 313 ( Pt 1, 51–55 (1996).
45. He, K. L. et al. Endothelial cell annexin A2 regulates polyubiquitination and
degradation of its binding partner S100A10/p11. J. Biol. Chem. 283, 19192–19200
(2008).
46. Crosbie, E. J., Einstein, M. H., Franceschi, S. & Kitchener, H. C. Human
papillomavirus and cervical cancer. Lancet (2013). doi:10.1016/S0140-
6736(13)60022-7
47. Arbyn, M. et al. Worldwide burden of cervical cancer in 2008. Ann. Oncol. (2011).
doi:10.1093/annonc/mdr015
48. Radosevich, J. A. HPV and cancer. HPV and Cancer (2012). doi:10.1007/978-94-
007-5437-9
49. Forman, D. et al. Global Burden of Human Papillomavirus and Related Diseases.
Vaccine 30, F12–F23 (2012).
50. Raff, A. B. et al. The Evolving Field of Human Papillomavirus Receptor Research:
81
a Review of Binding and Entry. J. Virol. 87, 6062–6072 (2013).
51. Gerke, V. & Moss, S. E. Annexins: From Structure to Function. Physiol. Rev. 82,
331–371 (2002).
52. Johnsson, N., Marriott, G. & Weber, K. P36, the Major Cytoplasmic Substrate of
Src Tyrosine Protein Kinase, Binds To Its P11 Regulatory Subunit Via a Short
Amino-Terminal Amphiphatic Helix. EMBO J. (1988).
53. Becker, T., Weber, K. & Johnsson, N. Protein-protein recognition via short
amphiphilic helices; a mutational analysis of the binding site of annexin II for
p11. EMBO J. (1990). doi:10.1002/j.1460-2075.1990.tb07868.x
54. Reddy, T. R. K. et al. Design , Synthesis , and Structure - Activity Relationship
Exploration of 1-Substituted 4-Aroyl-3-hydroxy-5-phenyl-1 H -pyrrol-2 ( 5 H ) -one
Analogues as Inhibitors of the Annexin A2 - S100A10 Protein Interaction. 2, 2080–
2094 (2011).
55. Reddy, T. R. K., Li, C., Fischer, P. M. & Dekker, L. V. Three-Dimensional
Pharmacophore Design and Biochemical Screening Identifies Substituted 1,2,4-
Triazoles as Inhibitors of the AnnexinA2-S100A10 Protein Interaction.
ChemMedChem 7, 1435–1446 (2012).
56. Reddy, T. R. K., Li, C., Guo, X., Fischer, P. M. & Dekker, L. V. Bioorganic &
Medicinal Chemistry Design , synthesis and SAR exploration of tri-substituted 1 ,
2 , 4-triazoles as inhibitors of the annexin A2 – S100A10 protein interaction.
Bioorg. Med. Chem. 22, 5378–5391 (2014).
57. Ma, G. et al. Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a
Cofactor for Macrophage HIV-1 Infection. J. Exp. Med. 200, 1337–1346 (2004).
58. Situ, A. J., Schmidt, T., Mazumder, P. & Ulmer, T. S. Characterization of
membrane protein interactions by isothermal titration calorimetry. J. Mol. Biol.
(2014). doi:10.1016/j.jmb.2014.08.020
59. Velázquez-Campoy, A., Ohtaka, H., Nezami, A., Muzammil, S. & Freire, E.
Isothermal titration calorimetry. Curr. Protoc. Cell Biol. (2004).
doi:10.1021/ac00217a715
60. Buck, C. B. & Thompson, C. D. Production of Papillomavirus-Based Gene
Transfer Vectors. Curr. Protoc. Cell Biol. 1–19 (2007).
doi:10.1002/0471143030.cb2601s37
61. Hajjar, K. A. The Biology of Annexin A2: From Vascular Fibrinolysis to Innate
Immunity. Trans. Am. Clin. Climatol. Assoc. 126, 144–55 (2015).
82
62. McNeely, T. B. et al. Secretory leukocyte protease inhibitor: a human saliva
protein exhibiting anti-human immunodeficiency virus 1 activity in vitro. J. Clin.
Invest. (1995). doi:10.1172/JCI118056
63. Fakioglu, E. et al. Herpes simplex virus downregulates secretory leukocyte
protease inhibitor: a novel immune evasion mechanism. J Virol 82, 9337–9344
(2008).
64. Ran, F. A. et al. Double nicking by RNA-guided CRISPR cas9 for enhanced
genome editing specificity. Cell 154, 1380–1389 (2013).
65. Benaud, C. et al. Annexin A2 is required for the early steps of cytokinesis. EMBO
Rep. 16, 481–489 (2015).
66. Spoden, G. et al. Clathrin- and caveolin-independent entry of human
papillomavirus type 16--involvement of tetraspanin-enriched microdomains
(TEMs). PLoS One 3, e3313 (2008).
67. Kenisi, H. et al. Cell surface-expressed phosphatidylserine and annexin A5 open
a novel portal of cell entry. J. Biol. Chem. 279, 52623–52629 (2004).
68. Drücker, P., Pejic, M., Galla, H. J. & Gerke, V. Lipid segregation and membrane
budding induced by the peripheral membrane binding protein annexin A2. J.
Biol. Chem. 288, 24764–24776 (2013).
69. Isas, J. M. et al. Global structural changes in annexin 12: The roles of
phospholipid, Ca2+, and pH. J. Biol. Chem. 278, 30227–30234 (2003).
70. Kim, Y. E., Isas, J. M., Haigler, H. T. & Langen, R. A helical hairpin region of
soluble annexin B12 refolds and forms a continuous transmembrane helix at
mildly acidic pH. J. Biol. Chem. 280, 32398–32404 (2005).
71. Morel, E., Parton, R. G. & Gruenberg, J. Annexin A2-Dependent Polymerization
of Actin Mediates Endosome Biogenesis. Dev. Cell 16, 445–457 (2009).
72. Mayran, N., Parton, R. G. & Gruenberg, J. Annexin II regulates multivesicular
endosome biogenesis in the degradation pathway of animal cells. EMBO J. 22,
3242–3253 (2003).
73. Wang, S., Sun, H., Tanowitz, M., Liang, X. H. & Crooke, S. T. Annexin A2
facilitates endocytic trafficking of antisense oligonucleotides. Nucleic Acids Res.
44, 7314–7330 (2016).
74. Stukes, S. et al. The Membrane Phospholipid Binding Protein Annexin A2
Promotes Phagocytosis and Nonlytic Exocytosis of Cryptococcus neoformans and
83
Impacts Survival in Fungal Infection. J. Immunol. 197, 1252–1261 (2016).
75. Christensen, N. D. et al. Surface conformational and linear epitopes on HPV-16
and HPV-18 L1 virus-like particles as defined by monoclonal antibodies. Virology
223, 174–184 (1996).
76. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas9 Systems.
Science (80-. ). 339, 819–823 (2013).
77. Watson, M. et al. Using population-based cancer registry data to assess the
burden of human papillomavirus-associated cancers in the United States:
Overview of methods. Cancer 113, 2841–2854 (2008).
78. Zheng, Z.-M. & Baker, C. C. Papillomavirus genome structure, expression, and
post-transcriptional regulation. Front. Biosci. 11, 2286–302 (2006).
79. DiGiuseppe, S., Bienkowska-Haba, M., Guion, L. G. & Sapp, M. Cruising the
cellular highways: How human papillomavirus travels from the surface to the
nucleus. Virus Res. 231, 1–9 (2017).
80. Fausch, S. C., Da Silva, D. M. & Kast, W. M. Differential uptake and cross-
presentation of human papillomavirus virus-like particles by dendritic cells and
Langerhans cells. Cancer Res. 63, 3478–3482 (2003).
81. Bienkowska-Haba, M. et al. Incoming human papillomavirus 16 genome is lost in
PML protein-deficient HaCaT keratinocytes. Cell. Microbiol. 19, 1–15 (2017).
82. Hedhli, N. et al. The annexin A2/S100A10 system in health and disease: Emerging
paradigms. J. Biomed. Biotechnol. 2012, (2012).
83. Schloer, S., Pajonczyk, D. & Rescher, U. Annexins in Translational Research :
Hidden Treasures to Be Found. Int. J. Mol. Sci. 19, 1–17 (2018).
84. Pena-Alonso, E. et al. Annexin A2 localizes to the basal epithelial layer and is
down-regulated in dysplasia and head and neck squamous cell carcinoma.
Cancer Lett. 263, 89–98 (2008).
85. Munz, B., Gerke, V., Gillitzer, R. & Werner, S. Differential expression of the
calpactin I subunits annexin II and p11 in cultured keratinocytes and during
wound repair. J. Invest. Dermatol. 108, 307–312 (1997).
86. Menke, M., Gerke, V. & Steinem, C. Phosphatidylserine membrane domain
clustering induced by annexin A2/S100A10 heterotetramer. Biochemistry 44,
15296–15303 (2005).
84
87. Wayne, N. & Waismanj, M. Calcium-dependent by Lipocortin-85 ” Regulation of
Actin Filament Bundling. 265, 3392–3401 (1990).
88. Hayes, M. J., Shao, D., Bailly, M. & Moss, S. E. Regulation of actin dynamics by
annexin 2. EMBO J. 25, 1816–1826 (2006).
89. Buck, C. B. et al. Human alpha-defensins block papillomavirus infection. Proc.
Natl. Acad. Sci. U. S. A. 103, 1516–21 (2006).
90. Calton, C. M. et al. Translocation of the papillomavirus L2/vDNA complex across
the limiting membrane requires the onset of mitosis. PLoS Pathog. 13, 1–29
(2017).
91. Richards, R. M., Lowy, D. R., Schiller, J. T. & Day, P. M. Cleavage of the
papillomavirus minor capsid protein, L2, at a furin consensus site is necessary
for infection. Proc. Natl. Acad. Sci. U. S. A. 103, 1522–7 (2006).
92. Wiens, M. E. & Smith, J. G. Alpha-Defensin HD5 Inhibits Furin Cleavage of
Human Papillomavirus 16 L2 To Block Infection. J. Virol. 89, 2866–2874 (2015).
93. Rescher, U. Annexins - unique membrane binding proteins with diverse
functions. J. Cell Sci. 117, 2631–2639 (2004).
94. Conrad, M. E. & Umbreit, J. N. Iron absorption and transport-an update. Am. J.
Hematol. 64, 287–298 (2000).
95. Conner, S. D. & Schmid, S. L. Regulated portals of enrty into the cell. Nature 422,
37–43 (2003).
96. Ramanakumar, A. V. et al. Human papillomavirus (HPV) types 16, 18, 31, 45 DNA
loads and HPV-16 integration in persistent and transient infections in young
women. BMC Infect. Dis. 10, 326 (2010).
97. Semov, A. et al. Metastasis-associated protein S100A4 induces angiogenesis
through interaction with annexin II and accelerated plasmin formation. J. Biol.
Chem. 280, 20833–20841 (2005).
98. Nedjadi, T. et al. S100A6 binds to annexin 2 in pancreatic cancer cells and
promotes pancreatic cancer cell motility. Br. J. Cancer 101, 1145–1154 (2009).
99. Rintala-Dempsey, A. C., Santamaria-Kisiel, L., Liao, Y., Lajoie, G. & Shaw, G. S.
Insights into S100 target specificity examined by a new interaction between
S100A11 and annexin A2. Biochemistry 45, 14695–14705 (2006).
100. Rintala-Dempsey, A. C., Rezvanpour, A. & Shaw, G. S. S100-annexin complexes -
85
Structural insights. FEBS J. 275, 4956–4966 (2008).
101. Aksoy, P., Gottschalk, E. Y. & Meneses, P. I. HPV entry into cells. Mutat. Res. -
Rev. Mutat. Res. 772, 13–22 (2017).
102. Morel, E. & Gruenberg, J. The p11/S100A10 light chain of annexin A2 is
dispensable for annexin A2 association to endosomes and functions in
endosomal transport. PLoS One 2, (2007).
103. Grewal, T. & Enrich, C. Annexins - Modulators of EGF receptor signalling and
trafficking. Cell. Signal. 21, 847–858 (2009).
104. Zhang, W. et al. Annexin A2 Promotes the Migration and Invasion of Human
Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-
Harboring Microvesicles from Tumor Cells. PLoS One 8, (2013).
105. Tcatchoff, L. et al. Annexin A1 and A2: Roles in retrograde trafficking of Shiga
toxin. PLoS One 7, (2012).
106. Johnstone, S. A., Hubaishy, I. & Waisman, D. M. Phosphorylation of annexin II
tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.
J. Biol. Chem. 267, 25976–25981 (1992).
107. Dathe, C. et al. Annexin A2 mediates apical trafficking of renal Na+-K +-2Cl-
Cotransporter. J. Biol. Chem. 289, 9983–9997 (2014).
108. Kirnbauer, R., Booyt, F., Chengt, N., Lowy, D. R. & Schiller, J. T. Papillomavirus
Li major capsid protein self-assembles into virus-like particles that are highly
immunogenic. Med. Sci. 89, 12180–12184 (1992).
109. Sapp, M. et al. Analysis of type-restricted and cross-reactive epitopes on virus-
like particles of human papillomavirus type 33 and in infected tissues using
monoclonal antibodies to the major capsid protein. J. Gen. Virol. 75, 3375–3383
(1994).
110. Rommel, O. et al. Heparan sulfate proteoglycans interact exclusively with
conformationally intact HPV L1 assemblies: Basis for a virus-like particle ELISA.
J. Med. Virol. 75, 114–121 (2005).
111. Schindelin, J., Rueden, C. T., Hiner, M. C. & Eliceiri, K. W. The ImageJ
ecosystem: An open platform for biomedical image analysis. Mol. Reprod. Dev.
82, 518–529 (2015).
112. Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis.
Nat. Methods 9, 676–682 (2012).
86
113. Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalisation
analysis in light microscopy. J. Microsc. 224, 13–232 (2006).
114. Woodham, A. W. et al. Inhibition of Langerhans Cell Maturation by Human
Papillomavirus Type 16: A Novel Role for the Annexin A2 Heterotetramer in
Immune Suppression. J. Immunol. 192, 4748–4757 (2014).
115. Zhang, S. et al. Annexin A2 binds to endosomes and negatively regulates TLR4-
triggered inflammatory responses via the TRAM-TRIF pathway. Sci. Rep. 5, 1–15
(2015).
116. Bećarević, M. TNF-alpha and annexin A2: inflammation in thrombotic primary
antiphospholipid syndrome. Rheumatol. Int. 36, 1649–1656 (2016).
117. Tanida, S. et al. Advances in refractory ulcerative colitis treatment: A new
therapeutic target, Annexin A2. World J. Gastroenterol. 21, 8776–8786 (2015).
118. Lokman, N. A., Ween, M. P., Oehler, M. K. & Ricciardelli, C. The role of annexin
A2 in tumorigenesis and cancer progression. Cancer Microenviron. 4, 199–208
(2011).
119. Xu, X. H., Pan, W., Kang, L. H., Feng, H. & Song, Y. Q. Association of Annexin A2
with cancer development (review). Oncol. Rep. 33, 2121–2128 (2015).
120. Liu, Y., Myrvang, H. K. & Dekker, L. V. Annexin A2 complexes with S100
proteins: Structure, function and pharmacological manipulation. Br. J.
Pharmacol. 172, 1664–1676 (2015).
121. Moss, S. E. & Morgan, R. O. The annexins. Genome Biol. 5, 1–8 (2004).
122. Einarsson, E. et al. Comparative Cell Biology and Evolution of Annexins in
Diplomonads. Mol. Biol. Physiol. 1, e00032-15 (2016).
123. Jami, S. K., Clark, G. B., Ayele, B. T., Ashe, P. & Kirti, P. B. Genome-wide
Comparative Analysis of Annexin Superfamily in Plants. PLoS One 7, (2012).
124. Yang, S.-L., Chou, Y.-T., Wu, C.-N. & Ho, M.-S. Annexin II Binds to Capsid
Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity. J. Virol. 85, 11809–
11820 (2011).
125. Malhotra, R. et al. Isolation and characterisation of potential respiratory
syncytial virus receptor(s) on epithelial cells. Microbes Infect. 5, 123–133 (2003).
126. Wright, J. F., Kurosky, A. & Wasi, S. An endothelial cell-surface form of annexin
II binds human cytomegalovirus. Biochemical and Biophysical Research
87
Communications 198, 983–989 (1994).
127. Wright, J. F., Kurosky, A., Pryzdial, E. L. & Wasi, S. Host cellular annexin II is
associated with cytomegalovirus particles isolated from cultured human
fibroblasts. J. Virol. 69, 4784–91 (1995).
128. Raynor, C. M., Wright, J. F., Waisman, D. M. & Pryzdial, E. L. G. Annexin II
enhances cytomegalovirus binding and fusion to phospholipid membranes.
Biochemistry 38, 5089–5095 (1999).
129. Derry, M. C., Sutherland, M. R., Restall, C. M., Waisman, D. M. & Pryzdial, E. L.
G. Annexin 2-mediated enhancement of cytomegalovirus infection opposes
inhibition by annexin 1 or annexin 5. J. Gen. Virol. 88, 19–27 (2007).
130. Taylor, J. R. et al. Heterotetrameric annexin A2/S100A10 (A2t) is essential for
oncogenic human papillomavirus trafficking and capsid disassembly, and
protects virions from lysosomal degradation. Sci. Rep. 8, 11642 (2018).
131. Backes, P. et al. Role of Annexin A2 in the Production of Infectious Hepatitis C
Virus Particles. J. Virol. 84, 5775–5789 (2010).
132. Saxena, V., Lai, C.-K., Chao, T.-C., Jeng, K.-S. & Lai, M. M. C. Annexin A2 Is
Involved in the Formation of Hepatitis C Virus Replication Complex on the Lipid
Raft. J. Virol. 86, 4139–4150 (2012).
133. Solbak, S. M. Ø., Abdurakhmanov, E., Vedeler, A. & Danielson, U. H.
Characterization of interactions between hepatitis C virus NS5B polymerase,
annexin A2 and RNA - Effects on NS5B catalysis and allosteric inhibition. Virol. J.
14, 1–11 (2017).
134. LeBouder, F. et al. Annexin II Incorporated into Influenza Virus Particles
Supports Virus Replication by Converting Plasminogen into Plasmin. J. Virol. 82,
6820–6828 (2008).
135. Ma, Y. et al. Annexin A2 (ANXA2) interacts with nonstructural protein 1 and
promotes the replication of highly pathogenic H5N1 avian influenza virus. BMC
Microbiol. 17, 1–9 (2017).
136. Koga, R., Kubota, M., Hashiguchi, T., Yanagi, Y. & Ohno, S. Annexin A2 Mediates
the Localization of Measles Virus Matrix Protein at the Plasma Membrane. 92, 1–
11 (2018).
137. Mercer, J., Schelhaas, M. & Helenius, A. Virus Entry by Endocytosis. Annu. Rev.
Biochem. 79, 803–833 (2010).
88
138. Barrow, E., Nicola, A. V. & Liu, J. Multiscale perspectives of virus entry via
endocytosis. Virol. J. 10, 1 (2013).
139. Yamauchi, Y. & Helenius, A. Virus entry at a glance. J. Cell Sci. 126, 1289–1295
(2013).
140. Helenius, A. Virus Entry: Looking Back and Moving Forward. Journal of
Molecular Biology (2018). doi:10.1016/j.jmb.2018.03.034
141. Day, P. M. & Schelhaas, M. Concepts of papillomavirus entry into host cells.
Curr. Opin. Virol. 4, 24–31 (2014).
142. Chan, Y. F. & AbuBakar, S. Recombinant human enterovirus 71 in hand, foot and
mouth disease patients. Emerg. Infect. Dis. 10, 1468–1470 (2004).
143. Yuan, M. et al. Enhanced human enterovirus 71 infection by endocytosis
inhibitors reveals multiple entry pathways by enterovirus causing hand-foot-and-
mouth diseases. Virol. J. 15, 1–13 (2018).
144. Nair, H. et al. Global burden of acute lower respiratory infections due to
respiratory syncytial virus in young children: a systematic review and meta-
analysis. Lancet 375, 1545–1555 (2010).
145. Collins, P. L. & Graham, B. S. Viral and Host Factors in Human Respiratory
Syncytial Virus Pathogenesis. J. Virol. 82, 2040–2055 (2008).
146. Kolokoltsov, A. A. et al. Small Interfering RNA Profiling Reveals Key Role of
Clathrin-Mediated Endocytosis and Early Endosome Formation for Infection by
Respiratory Syncytial Virus. J. Virol. 81, 7786–7800 (2007).
147. Gutiérrez-Ortega, A., Sánchez-Hernández, C. & Gómez-García, B. Respiratory
syncytial virus glycoproteins uptake occurs through clathrin-mediated
endocytosis in a human epithelial cell line. Virol. J. 5, 1–4 (2008).
148. Krzyzaniak, M. A., Zumstein, M. T., Gerez, J. A., Picotti, P. & Helenius, A. Host
Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by
Proteolytic Activation of the F Protein. PLoS Pathog. 9, (2013).
149. Shaikh, F. Y. et al. Respiratory syncytial virus assembles into structured
filamentous virion particles independently of host cytoskeleton and related
proteins. PLoS One 7, (2012).
150. Fowler, K. B. & Boppana, S. B. Congenital cytomegalovirus infection. Semin.
Perinatol. 42, 149–154 (2018).
89
151. Murray, M., Peters, N. & Reeves, M. Navigating the Host Cell Response during
Entry into Sites of Latent Cytomegalovirus Infection. Pathogens 7, 30 (2018).
152. Pietropaolo, R. & Compton, T. Interference with annexin II has no effect on entry
of human cytomegalovirus into fibroblast cells. J. Gen. Virol. (1999).
doi:10.1099/0022-1317-80-7-1807
153. Esclatine, A. et al. Differentiation-dependent redistribution of heparan sulfate in
epithelial intestinal Caco-2 cells leads to basolateral entry of cytomegalovirus.
Virology 289, 23–33 (2001).
154. Den Boon, J. A., Diaz, A. & Ahlquist, P. Cytoplasmic viral replication complexes.
Cell Host Microbe 8, 77–85 (2010).
155. Schmid, M., Speiseder, T., Dobner, T. & Gonzalez, R. A. DNA Virus Replication
Compartments. J. Virol. 88, 1404–1420 (2014).
156. Farquhar, M. J. et al. Hepatitis C Virus Induces CD81 and Claudin-1 Endocytosis.
J. Virol. 86, 4305–4316 (2012).
157. Benedicto, I. et al. Clathrin Mediates Infectious Hepatitis C Virus Particle Egress.
J. Virol. 89, 4180–4190 (2015).
158. Lindenbach, B. D. & Rice, C. M. The ins and outs of hepatitis C virus antry and
assemplu. Nat. Rev. Microbiol. 11, 688–700 (2014).
159. Lai, C.-K., Jeng, K.-S., Machida, K. & Lai, M. M. C. Association of Hepatitis C
Virus Replication Complexes with Microtubules and Actin Filaments Is
Dependent on the Interaction of NS3 and NS5A. J. Virol. 82, 8838–8848 (2008).
160. Gokhale, N. A., Abraham, A., Digman, M. A., Gratton, E. & Cho, W.
Phosphoinositide specificity of and mechanism of lipid domain formation by
annexin A2-p11 heterotetramer. J. Biol. Chem. 280, 42831–42840 (2005).
161. Salomon, R. & Webster, R. G. The Influenza Virus Enigma. Cell 136, 402–410
(2009).
162. Jiang, Y., Qin, Y. & Chen, M. Host-pathogen interactions in measles virus
replication and anti-viral immunity. Viruses 8, (2016).
163. Gonzàlez-Reyes, S. et al. Role of annexin A2 in cellular entry of rabbit vesivirus. J.
Gen. Virol. 90, 2724–2730 (2009).
164. Li, J. et al. The interaction between host Annexin A2 and viral Nsp9 is beneficial
for replication of porcine reproductive and respiratory syndrome virus. Virus
90
Res. 189, 106–113 (2014).
165. Kwak, H., Park, M. W. & Jeong, S. Annexin A2 binds RNA and reduces the
frameshifting efficiency of Infectious Bronchitis Virus. PLoS One 6, (2011).
166. Chang, X. B. et al. Annexin A2 binds to vimentin and contributes to porcine
reproductive and respiratory syndrome virus multiplication. Vet. Res. 1–12
(2018). doi:10.1186/s13567-018-0571-5
167. Yang, Z. et al. Annexin 2 is a host protein binding to classical swine fever virus E2
glycoprotein and promoting viral growth in PK-15 cells. Virus Res. 201, 16–23
(2015).
168. Beaton, A. R., Rodriguez, J., Reddy, Y. K. & Roy, P. The membrane trafficking
protein calpactin forms a complex with bluetongue virus protein NS3 and
mediates virus release. Proc. Natl. Acad. Sci. U. S. A. 99, 13154–13159 (2002).
169. Celma, C. C. P. & Roy, P. Interaction of Calpactin Light Chain (S100A10/p11) and
a Viral NS Protein Is Essential for Intracellular Trafficking of Nonenveloped
Bluetongue Virus. J. Virol. 85, 4783–4791 (2011).
170. Sheng, C. et al. Annexin A2 is involved in the production of classical swine fever
virus infectious particles. J. Gen. Virol. 96, 1027–1032 (2015).
171. Koyuncu, O. O., Perlman, D. H. & Enquist, L. W. Efficient retrograde transport of
pseudorabies virus within neurons requires local protein synthesis in axons. Cell
Host Microbe (2013). doi:10.1016/j.chom.2012.10.021
172. Ryzhova, E. V, Harrist, A. V, Harvey, T. & Gonza, F. Annexin 2: a Novel Human
Immunodeficiency Virus Type 1 Gag Binding Protein Involved in Replication in
Monocyte-Derived Macrophages. Society 80, 2694–2704 (2006).
173. Rai, T., Mosoian, A. & Resh, M. D. Annexin 2 Is Not Required for Human
Immunodeficiency Virus Type 1 Particle Production but Plays a Cell Type-
Dependent Role in Regulating Infectivity. J. Virol. 84, 9783–9792 (2010).
174. Woodham, A. W. et al. Annexin A2 antibodies but not inhibitors of the annexin
A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.
Virol. J. 13, 1–5 (2016).

Abstract (if available)

Abstract High-risk human papillomavirus (HPV) is one of the most common sexually transmitted viruses and leads to a variety of human cancers such as cervical and throat cancer. HPV establishes infection independently of canonical cellular entry mechanisms such as clathrin- and caveolin-mediated endocytosis but is dependent on the annexin A2/S100A10 heterotetramer (A2t). This dissertation sought to block HPV infection by targeting A2t via small molecule inhibitors and protein knock-out (KO) via CRISPR/Cas9, while examining the contribution of monomeric annexin A2 (AnxA2) vs. A2t and further characterizing the broader role of these molecules in protein trafficking. The data presented herein show that inhibitors of A2t and A2t KO via CRISPR/Cas9 significantly inhibit HPV type 16 (HPV16) infection of epithelial cells. Specifically, we show that cell surface A2t is not required for HPV attachment, and in the absence of A2t, virion internalization remains clathrin-independent. Without A2t, viral progression from early endosomes to multivesicular endosomes is significantly inhibited, capsid uncoating is dramatically reduced, and lysosomal degradation of HPV is accelerated. Furthermore, we present evidence that AnxA2 forms a complex with CD63, a known mediator of HPV trafficking. Overall, the observed reduction in infection is less significant in the absence of S100A10 alone compared to full A2t, supporting an independent role for monomeric AnxA2. More broadly, we show that successful infection by multiple oncogenic HPV types is dependent on A2t. Finally, by discussing the emergent role of AnxA2 as a key host-virus interaction molecule throughout the lifecycle of multiple epithelial-cell targeting viruses, we highlight recurrent themes, identify discrepancies, and reveal potential avenues for future research that could have a broad impact on the field of virus-induced diseases.

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Asset Metadata

Creator Taylor, Julia Rose (author)

Core Title The annexin A2 heterotetramer as a host cell cofactor in human papillomavirus infection

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Medical Biology

Publication Date 05/06/2019

Defense Date 10/22/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag annexin A2,endocytosis,epithelial cells,host-pathogen interaction,HPV,human papillomavirus,OAI-PMH Harvest,virology

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Language English

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Advisor Ouellette, Andre J. (committee chair), Da Silva, Diane (committee member), Kast, W. Martin (committee member), Okamoto, Curtis (committee member)

Creator Email jrtaylor924@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-168082

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