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WAVE1 is a novel mediator of HPV trafficking to the Golgi apparatus

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WAVE1 is a novel mediator of HPV trafficking to the Golgi apparatus

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WAVE1 is a Novel Mediator of HPV
Trafficking to the Golgi Apparatus

By
Stephanie Cheng

A Thesis Presented to
THE FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the
Requirements for the Degree

MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

August 2021

ii

ACKNOWLEDGEMENTS

This dissertation would not have been possible without the support of several individuals who
contributed to the completion of this research project.

I would like to express my appreciation to both Dr. W Martin Kast and Dr. Diane Da Silva
for their advice and continuous guidance. I am also thankful for my committee members Dr.
Keigo Machida and Dr. Weiming Yuan for their support and constructive comments.

Additionally, I would also like to extend a heartfelt thank you to all my lab mates, Ruben
Prins, Kim Lühen, and DJ Fernandez in particular, for useful technical suggestions and
consistent encouragement.

Last but most certainly not least I would like to thank my family and friends in supporting me
through this master’s program amid a global pandemic.

iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ....................................................................................................................... ii
LIST OF FIGURES ................................................................................................................................... iv
LIST OF ABBREVIATIONS .................................................................................................................... v
ABSTRACT ............................................................................................................................................... vii
CHAPTER 1 INTRODUCTION ............................................................................................................... 1
1.1 Human Papillomaviruses and Cancer ................................................................................................. 1
1.2 HPV16 Binding and Entry .................................................................................................................... 2
1.3 HPV16 Intracellular Trafficking ........................................................................................................... 3
1.4 Macropinocytosis Viral Entry Pathway ............................................................................................... 5
1.5 WAVE1 and WASP Family Proteins ..................................................................................................... 6
CHAPTER 2 MATERIALS AND METHODS ........................................................................................ 8
2.1 Cell Culture, CRISPR Gene Editing, Lentivirus Transfection ................................................................. 8
2.2 Cell Proliferation Assay ....................................................................................................................... 8
2.3 CyQuant Proliferation Assay ............................................................................................................... 8
2.4 Pseudovirus, Virus-Like Particle Production ........................................................................................ 9
2.5 Antibodies ........................................................................................................................................... 9
2.6 PsV Infection Assay ........................................................................................................................... 10
2.7 VLP Internalization Assay .................................................................................................................. 10
2.8 HPV Cell Surface Binding Assay ......................................................................................................... 10
2.9 Immunofluorescence Microscopy ..................................................................................................... 11
CHAPTER 3 RESULTS ........................................................................................................................... 12
3.1 WAVE1 KO and Overexpression cell line characterization ................................................................ 12
3.2 WAVE1 is required for efficient HPV16 infection .............................................................................. 14
3.3 WAVE1 is not necessary for HPV16 binding and early internalization ............................................. 15
3.4 WAVE1 KO and overexpression decreases HPV16 trafficking to acidic compartments .................... 17
3.5 WAVE1 is essential for HPV16 trafficking at Golgi Apparatus .......................................................... 19
CHAPTER 4 DISCUSSION ..................................................................................................................... 23
SUPPLEMENTAL DATA ....................................................................................................................... 26
BIBLIOGRAPHY ..................................................................................................................................... 27

iv

LIST OF FIGURES

Figure 1. HPV16 Viral Entry ............................................................................................. 3
Figure 2. HPV16 Intracellular Trafficking ........................................................................ 5
Figure 3. Successful KO and overexpression of WT in HeLa does not affect cell viability
and proliferation. .............................................................................................................. 13

Figure 4. WAVE1 is required for efficient HPV16 infection .......................................... 14
Figure 5. WAVE1 is not necessary for HPV16 binding. ................................................. 15
Figure 6. WAVE1 KO does not affect early HPV16 internalization. .............................. 17
Figure 7. WAVE1 KO and overexpression decreases HPV16 trafficking to acidic
compartments. .................................................................................................................. 18

Figure 8. WAVE1 is required for HPV trafficking to Golgi apparatus and not to
endosomes or lysosomes. ................................................................................................. 22

Figure 9. Speculative model of WASH involvement in HPV trafficking to Golgi
apparatus .......................................................................................................................... 25

Supplemental Figure 1. WAVE1 KO, Overexpression Western Blot. ............................ 26
Supplemental Figure 2. WAVE1 is required for HPV trafficking at golgi apparatus. .... 26

v

LIST OF ABBREVIATIONS
A2t Annexin A2 Heterotetramer
Arp Actin Related Protein
ATP Adenosine Triphosphate
BME Beta-Mercaptoethanol
CD Cluster of Differentiation
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
DNA Deoxyribonucleic Acid
EDTA EthyleneDiamineteTraacetic Acid
EGFP Enhanced Green Fluorescent Protein
EGFR Epidermal Growth Factor Receptor
ELISA Enzyme-Linked Immunosorbent Assay
ER Endoplasmic Reticulum
ESCRT Endosomal Sorting Complexes Required for Transport
FBS Fetal Bovine Serum
GTP Guanosine Triphosphate
HPV Human Papillomavirus
HSPG Heparan Sulfate Proteoglycans
IMDM Iscove's Modified Dulbecco's Medium
JMY Junction-Mediating Regulatory Protein
KO Knock Out
OBSCL1 Obscurin-Like 1
vi

PBS Phosphate Buffered Saline
PDGFR Platelet-Derived Growth Factor Receptor
PFA Paraformaldehyde
PI(3)K Phosphoinositide 3-Kinase
PKC Protein Kinase C
PsV Pseudovirus
Rab Ras-Related-Protein
RFP Red Fluorescent Protein
RNA Ribonucleic Acid
RTK Receptor Tyrosine Kinase
s.d. Standard Deviation
SNX Sorting Nexin
TGN Trans Golgi Network
VSP Voltage sensitive phosphatase
WASH WASP and SCAR Homologue
WASP Wiskott–Aldrich Syndrome Protein
WAVE WASP-family Verprolinhomologous Protein
WCA WH2 domain, central region, acidic region
WHAMM WASP Homolog associated with Actin, Membranes and
Microtubules

vii

ABSTRACT
Human Papilloma Virus (HPV) infection begins with viral entry into host cell, which has
been suggested to be a novel process independent of clathrin, caveolin, dynamin, cholesterol,
lipid raft, or flotillin. The cell entry pathway bears many similarities to macropinocytosis, which
is a bulk, nonspecific fluid uptake process. Actin polymerization is key for both HPV
endocytosis and macropinocytosis, thus, actin polymerization regulators could be important for
HPV entry. WAVE/WASP family proteins are responsible for actin nucleation and organization
at the cell membrane. Out of this protein family, WAVE1 in particular is critical for the
formation of circular dorsal ruffles, a type of membrane protrusion associated with
macropinocytosis. This implicates a potential role for WAVE1 in HPV endocytosis. Here, we
investigated the role of WAVE1 in binding, internalization, and infection of HPV16, a high-risk
variant subtype of HPV. WAVE1 knock out (KO) and overexpression cell lines were created
from wildtype (WT) HeLa cells. WAVE1 KO significantly reduced HPV16 infection while
overexpression significantly increased the rate of HPV16 infection. The absence of WAVE1 did
not affect HPV binding or early internalization, but it did play a role in HPV intracellular
trafficking. Utilizing immunofluorescence microscopy, we demonstrated that WAVE1is crucial
for HPV trafficking to the Golgi apparatus, and not for HPV endocytosis as we initially
hypothesized.

1

CHAPTER 1 INTRODUCTION

1.1 Human Papillomaviruses and Cancer
Human Papillomavirus (HPV) infection can lead to genital warts, precancerous lesions,
and many types of cancer, including cervical, anogenital, and head and neck cancers. Out of the
more than 200 genotypes of HPV, 12 are high risk HPV subtypes – HPVs 16, 18, 31, 33, 35, 39,
45, 51, 52, 56, 58, and 59. Among these subtypes, HPV 16 and 18 have the highest prevalence in
cancer and are responsible for 70% of cervical cancer and 95% of HPV-positive oropharyngeal
cancers (Serrano et al. 2018). In 2012, HPV attributed cancers accounted for 4.5% of all cancers
globally (630,000 new cases per year), with 8.6% in women and 0.8% in men. Cervical cancer
(530,000 new cases per year) consisted of the vast majority of HPV attributed cancers, as HPV
infections are seen as the primary drivers of cervical cancer development (de Martel et al. 2017).
Cervical cancer is the second most prevalent cancer in women and fourth leading cause of cancer
deaths globally, with 266,000 new deaths in 2012. Many of these new cervical cancer cases are
in developing countries, as detection methods (pap smear) and HPV vaccines are not as readily
available in these areas. In addition to cervical cancers, oropharyngeal cancers are on a steady
rise in United States and Western Europe, as 70-80% of these cancer cases are connected to HPV
infections (Berman 2017). In United States, oropharynx has replaced cervix as the most common
site of HPV related cancer, with 14,000 oropharynx cancer cases per year compared to 11,000
cervical cancer cases. HPV is also responsible for 60% of penile cancers, 70% of vaginal and
vulvar cancers, and 90% of anal cancers (CDC, 2016).

2

1.2 HPV16 Binding and Entry
HPV16 is a 55nm in diameter naked virus with an icosahedral capsid and 8kb of circular
double stranded DNA. The capsid is comprised of 72 pentameters of L1 proteins and between 12
and 72 molecules of L2 proteins. Both L1 and L2 proteins are required for infection, as L1 is
responsible for early binding and entry and L2 is involved in many viral trafficking processes in
the cytoplasm, endosome, nucleus (Raff et al. 2013). HPV enters through a micro wound in the
epithelium and infects mitotically active basal keratinocytes. The virus first binds to the
basement membrane, then interacts with heparan sulfate proteoglycans (HSPG) and induces L1
protein cleavage (Figure 1). Then, cyclophilin B mediates conformational changes in the viral
capsid, which exposes the L2 N terminus cleavage site and allows furin cleavage to occur. These
changes in the viral capsid lead to a decreased affinity towards HSPG and directs the virus
towards endocytosis. The viral uptake process involves integrins, tetraspanins CD63 and CD151,
EGFR, adaptor proteins (eg. OBSCL1, syntenin-1), and annexin A2 heterotetramer (A2t) (Aksoy
et al. 2017, Day and Schelhaas 2014, Raff et al). The viral endocytosis is described as a
macropinocytosis-like pathway that is actin polymerization dependent, and clathrin-, caveolin-,
dynamin-, cholesterol-, flotillin-, and lipid raft-independent (Schelhaas et al. 2012).

3

Figure 1. HPV16 Viral Entry (Aksoy et al. 2017) HPV16 detaches from the extracellular
matrix/basement membrane and binds to HSPG on the cell surface. The virus undergoes multiple
conformational changes in L1 and L2 proteins and is primed for endocytosis. Virus uptake is a
macropinocytosis-like pathway that involves integrins, tetraspanins CD63 and CD151, EGFR,
and A2t.

1.3 HPV16 Intracellular Trafficking
After HPV internalization, the viral particles are localized to early endosomes with the
help of A2t, tetraspanin CD63, synthenin-1 and ESCRT proteins. In addition, Rab5 is key for
endosomal biogenesis and recruitment of ATP-dependent ion channels to lower the pH in the
endosome, which promotes viral capsid dissociation (Young et al. 2019) (Figure 2 step 3). HPV
particles remain in the vesicle during the endosome maturation. Rab7a is responsible for the
conversion from early endosome to late endosome (Figure 2 step5ii). During this process, host
4

cell cyclophillins are involved with dissociating most L1 proteins from the L2/viral DNA
complex. Some of the L1 proteins are then targeted for degradation and diverted to the lysosome,
while some L1 remains with the L2/viral DNA complex (DiGiuseppe et al. 2016). From the late
endosome, HPV particles are trafficked by Rab7b and Rab9a to TGN (Figure 2 step7ii). The C-
terminus of the L2 protein protrudes out to the cytosol and acts as a cell-penetrating peptide
(CPP) to bind to cytosolic retromer. L2 also interacts with SNX27, SNX17, VSP26 as a part of
the retrograde transport machinery (Xie et al. 2020). The L2/viral DNA complex remains in
TGN until the nuclear membrane breaks down during prophase of mitosis. Then, microtubule
organizing center (MOTC) transports vesicles containing L2/viral DNA to mitotic spindle where
L2 binds to the chromatin (Figure 2 step8). This process is facilitated by a novel protein
complex of Ran-binding protein 10, karyopherin alpha2, and dynein light chain DYNLT3 (Lai et
al. 2021). Then, early viral protein E1 and E2 transcription begins with the help of promyelocytic
leukemia (PML) and nuclear domain (ND) 10. This viral gene transcription signifies successful
HPV16 infection (Young et al. 2019).
5

Figure 2. HPV16 Intracellular Trafficking (Young et al. 2019) After viral uptake, HPV16 is
trafficked by a network of Rab-GTPases to early endosome, late endosome, golgi apparatus and
enters the nucleus.

1.4 Macropinocytosis Viral Entry Pathway
Macropinocytosis is an actin-driven endocytic process that leads to a bulk, non-specific
uptake of fluid in a large vacuole (up to 10 micrometers in diameter). The process is typically
stimulated by growth factors (ie. EGFR, PDGFR), which activate RTKs and induces actin
polymerization at the cell surface. These actin filaments generate transient ruffles that can be
planar or circular in shape. Most ruffles extend out of the cell and merge back to the cell
membrane, but some fold back and undergo membrane fission to form endocytic vacuoles called
macropinosomes (Mercer and Helenius 2009, Kerr and Teasdale 2009). Macropinocytosis was
first observed in amoebas nearly a century ago, and it is also found in human macrophages and
ras-activated tumor cells. There are viruses and other pathogens that take advantage of the
6

cellular macropinocytosis, such as vaccina virus and herpes simplex 1 (Lim and Gleeson 2011).
Viruses are able to trigger macropinocytosis by first attaching to the cell using either specific
receptors or extracellular matrix components and activate RTKs. There are five stages in viral
macropinocytosis: binding, activation of signaling, protrusion, vacuole closure/formation,
trafficking. Mercer and Helenius proposed a set of cellular factors that is important for each of
these stages which can be used to categorize a viral entry pathway as macropinocytosis. PI(3)K,
protein kinase C (PKC), Rho GTPases (ie. Rac1, cdc42) are key regulators in stages from
membrane protrusion to macropinosome trafficking. Actin, Na
+
/H
+
exchangers, p21-activated
kinase 1 (Pak1) are essential for membrane protrusion step. (Mercer and Helenius 2012, Lim and
Gleeson 2011). HPV16 endocytosis shares many of these hallmarks of macropinocytosis but it
does not depend on any Rho GTPases (Schelhaas et al. 2012). However, its similarities with
macropinocytosis could help shed light on other factors that are involved in HPV16 uptake.
1.5 WAVE1 and WASP Family Proteins
As previously discussed, actin polymerization is crucial for both HPV16 endocytosis and
macropinocytosis. In order to form membrane protrusions like ruffles, actin monomers (G-actin)
need to assemble into filamentous actin (F-actin) and then be organized into different branching
structures. This actin reorganization is tightly regulated by actin branching initiator Arp2/3
complex and nucleation promoting factors in the WASP family. Arp2/3 on its own is relatively
poor at actin nucleation in vivo and requires WASP family proteins to activate actin branching.
(Rotty et al. 2013). The WASP family includes WASP, WAVE1-3, WHAMM, WASH, JMY,
and each protein regulates actin remodeling in different parts of the cell (Alekhina et al. 2017).
Both WASP and WAVE1-3 are present at the cell membrane. WASP is involved in
phagocytosis, filopodium and podosome formation WAVE1-3 are tissue specific and have some
7

overlapping functions in cell membrane protrusions, but WAVE1 is essential for dorsal ruffle
formation, WAVE2 is required for lamellipodia formation (Takenawa and Suetsugu 2007).
WHAMM regulates vesicle transport at ER and Golgi immediate compartment. JMY is present
at leading edge of the cell as well as the nucleus during DNA damage response. WASH localizes
to the early endosome and is involved in endosomal trafficking to the endolysosome. (Alekhina
et al. 2017, Burianek and Soderling 2013). All WASP family proteins share a common WCA
domain in the carboxy terminus that activates Arp2/3 complex, and each protein has a unique
amino terminus for regulating actin assembly in their specific function. For example, WAVE
proteins contain a N-terminal WAVE homology domain (WHD), which allows WAVE proteins
to form the WAVE Regulatory Complex (WRC), which consists of Sra1, Nap1, HSPC300, and
Abi1.
In the context of HPV endocytosis, WAVE1 is a protein of interest as it regulates circular
dorsal ruffles that are involved in macropinocytosis. Therefore, the hypothesis of this thesis is
that HPV endocytosis is mediated by WAVE1. To test this hypothesis, WAVE1 knockout
(KO) and overexpression cell lines were created from HeLa cells, and HPV binding,
internalization, and infection were characterized in these cell lines. Our findings suggest that
WAVE1 plays a role in HPV intracellular trafficking and not endocytosis as we initially
anticipated.

8

CHAPTER 2 MATERIALS AND METHODS
2.1 Cell Culture, CRISPR Gene Editing, Lentivirus Transfection
HeLa cells (CCL-2, ATCC) derived from cervical carcinoma were maintained in Iscove’s
Modified Dulbecco’s Medium (IMDM) (Corning, Corning NY) with 10% FBS, 1x BME, and 1x
gentamycin at 37 °C with 5% CO 2.
WAVE1 knockout (KO) clones were generated from HeLa cells, using Lipofectamine
CRISPRMAX Transfection reagent (Thermo Fisher Scientific, Waltham, MA) and guide RNA
sequence 5’-ACATCGTACGTCTCCTGTAA-3. Single clone dilution was performed 48hr post
transfection. The clones were then screened by DNA sequencing at the cut site and Western Blot
to ensure no WAVE1 protein is present.
WAVE1 overexpression clones were generated from HeLa cells transfected with
lentivirus containing EGFP linked WAVE1 and puromycin selection factor. Single clone dilution
was performed after 1 week of puromycin selection. The clone with the highest fluorescent
intensity (determined by flow cytometry) was selected, as this indicates the highest expression of
EGFP-WAVE1.
2.2 Cell Proliferation Assay
20,000 Cells were per well in a 24-well plate with 0.5ml IMDM with 10% FBS. After 48-
hour incubation at 37°C with 5% CO2, the cells were harvested using 0.05% Trypsin-EDTA
(Gibco, Waltham, MA). The cell suspension was then diluted with trypan blue stain (Invitrogen,
Carlsbad, CA) at 1:1 ratio and counted using Countess automated cell counter (Invitrogen).
2.3 CyQuant Proliferation Assay
1500 Cells were seeded per well in a 96-well plate with 150µl IMDM with 10% FBS and
incubated for 48 hours at 37°C with 5% CO2. The wells were then washed with PBS twice and
9

after the final aspiration step, the plate was frozen at -80°C overnight. The plate was thawed at
room temperature and the CyQuant assay (Thermo Fisher Scientific) was performed according to
manufacture protocol. The fluorescent intensity at 595nm was read using a Clario Star Plate
Reader (BMG Lab Tech, Ortenberg, Germany).
2.4 Pseudovirus, Virus-Like Particle Production
HPV16 Pseudovirion (PsV) was produced from 293TT cells co transfected with self-
packaging p16L1L2 and RFP reporter (pRwB) plasmids using published methods (Buck and
Thompson 2007). The PsV infectious titer was determined by flow cytometry using a standard of
293TT cells treated with different dilutions of PsV vector stock. PsV protein concentration was
quantified using coomassie blue stain on SDS page gel with known BSA concentrations.
HPV16L1L2 virus-like particles (VLP) were produced using a recombinant baculovirus
expression system in insert cells as previously published (Kirnbauer et al. 1992). The presence of
HPV L1 and L2 proteins were confirmed with western blot and the presence of intact particles
were confirmed by a neutralizing antibody ELISA. The L1 protein concentration of VLP was
quantified with Coomassie blue staining.
2.5 Antibodies
The following antibodies were used in this study: HPV16.5A, HPV16.V5 mouse anti-
HPV16 L1(gifted by Neil Christensen, Penn State, Philadelphia, PA); rabbit anti WAVE1 (PA5-
78273, Invitrogen); goat anti-mouse IR Dye 800 (LiCor, Lincoln, NE); goat anti-rabbit Alexa
Fluor 680 (Invitrogen); goat anti-mouse Alexa Fluor 488 (Invitrogen); Texas Red goat anti-rabbit
(Invitrogen); goat anti-mouse Alexa Fluor 594 (Invitrogen). Rabbit anti EEA1 (2411), rabbit anti
Rab5 (2143), rabbit anti Rab7 (9367); rabbit anti Golgin97 (13192); rabbit anti GM130 (12480);
10

rabbit anti LAMP1 (9091), mouse anti beta-actin (3700) were purchased from Cell Signaling
(Danvers, MA).;
2.6 PsV Infection Assay
20,000 Cells/well were seeded with IMDM and 10% FBS in a 24 well plate and
incubated at 37C overnight with 5% CO2. HPV16 PsV with pRwB reporter gene was added to
cells at 35 multiplicity of infection (MOI) to achieve 30% infection in HeLa cells. After 48 hours
of incubation at 37°C, 5% CO2, the cells were harvested with 0.05% Trypsin-EDTA and then
fixed with 4% paraformaldehyde (PFA). %RFP-positive cells were determined with flow
cytometry (FC500, Beckman Coulter, Brea, CA).
2.7 VLP Internalization Assay
HPV16 VLPs were labeled with pH-dependent rhodamine fluorophore (pHrodo iFL Red
STP, Life Technologies, Grand Island, NY) at 10:1 (dye:HPV L1 protein) ratio. The labeled
VLPs were then filtered with 2% agarose beads size standards 50-150µm (Agarose Bead
Technologies, Tampa, FL) to remove unconjugated dye particles. The pHrodo labeled VLP
concentrations were determined with the same Coomassie blue staining method in Section 2.4.
20,000 Cells/well were seeded with IMDM and 10% FBS in a 24 well plate and
incubated at 37C overnight with 5% CO2. pHrodo labeled VLP in IMDM (no phenol red)
(Gibco) were added at 2µg/ml per 1 million cells. The plate was then placed in the 37°C
incubator and at every hour, the fluorescence intensity was measured using Clario Star plate
reader (BMG Lab Tech).
2.8 HPV Cell Surface Binding Assay
300,000 Cells were seeded with IMDM and 10% FBS in 6 well plates and incubated at
37°C overnight with 5% CO2. Cells were washed with ice cold DPBS and treated with HPV16
11

L1L2VLP at 1µg/ml per 1 million cells in cold IMDM for 1 hour at 4°C to saturate binding.
Cells were collected on ice via scraping, stained with H16.V5 (1:100) for 30 min at 4°C, Alexa
Fluor 594 Goat anti mouse secondary antibody (1:200), and fixed with 2% PFA in order to
ensure measurement of particles that might internalize at room temperature during analysis.
Mean fluorescent intensity (MFI) was measured via flow cytometry.
2.9 Immunofluorescence Microscopy
Cells were seeded on 8-well ibiTreat slides (Ibidi, Planegg, Germany) at 20,000 cell/well
in 200µl of IMDM and 10% FBS and incubated at 37°C overnight with 5% CO2. HPV16L1L2
PsV was then added at 0.5ug/ml per 1 million cells, and the slide was incubated for 1 hour at 4C.
The wells were washed 3 times with 200µl of PBS, then fresh media was added, and the slide
was incubated at 37°C. At each time point, the slide was washed 3 times with 200ul of PBS and
fixed with 4% PFA. Then, the cells were stained for HPV16 (HPV16.5A, 1:1000) along with
EEA1 (1:100), or Rab5 (1:150), or Rab7 (1:100), or Golgin97 (1:100), or GM130 (1:4000), or
LAMP1 (1:200). Alexa Fluor 488 goat anti mouse and Texas red goat anti rabbit were used as
secondary antibody staining. Images were visualized on a Nikon Eclipse Ti-E laser scanning
confocal microscope and analyzed with Nikon NIS-Elements software.

12

CHAPTER 3 RESULTS
3.1 WAVE1 KO and Overexpression cell line characterization
In order to interrogate the function of WAVE1 in HPV infection, WAVE1 was knocked
out via CRISPR/Cas9 gene editing and overexpressed using a lentivirus coding for linked EGFP-
WAVE1 protein in HeLa cell background. Figure 3a shows a western blot verifying the absence
of WAVE1 in the knockout and presence of EGFP-WAVE1 protein in the overexpression cell
line (full western blot in Supplemental Figure 1). Flow cytometry also confirms successful
EGFP-WAVE1 expression, with 97.7% GFP+ cells (Figure 3b). Cell proliferation of these two
cell lines were also compared to WT HeLa cells, as differences in growth rate could impact HPV
infection assays. Viable cells were counted using trypan blue exclusion and proliferation was
measured by DNA based quantification assay, and there were no significant differences in
viability and proliferation among these cell lines (Figure 3c, d).
A
WT
WAVE1
KO
WAVE1
Overexpression
EGFP-
WAVE1

WAVE1

Β-Actin

B
WT HeLa
WAVE1 Overexpression
0
50
100
150
%GFP+

13

C
WT HeLa
WAVE1 KO
WAVE1 Overexpression
0
20000
40000
60000
80000
100000
Total Viable Cells
ns
ns

D
WT HeLa
WAVE1 KO
WAVE1 Overexpression
0.0
0.5
1.0
1.5
Fold Change in Proliferation
ns
ns

Figure 3. Successful KO and overexpression of WT in HeLa does not affect cell viability
and proliferation. (a) Western blot image of WAVE1 KO and overexpression; protein input
was normalized to 25µg. (b) flow cytometry measurement of %GFP+ cells in WT and WAVE1
overexpression HeLa cell lines, N=9 biological repeats, error bars indicate s.d. (c, d) WT,
WAVE1 KO, overexpression cells were seeded at equal amounts and grown for 48hr, then cell
proliferation was measured by (c) counting cells via trypan blue exclusion test, and (d)
CyQUANT fluorescence based DNA quantification used to measure cell proliferation. Results
(c) N=3, (d) N=5, error bars indicated s.d. Statistics: 1-way ANOVA with Dunnett’s multiple
comparison test. ns = not significant.

14

3.2 WAVE1 is required for efficient HPV16 infection
Differences in HPV16 infection among WT, KO, and overexpression of WAVE1 in
HeLa cells were determined with an in vitro infection assay using HPV16 pseudo virions (PsV)
carrying an RFP reporter plasmid. RFP expression, which indicates successful HPV infection,
was measured using flow cytometry. HPV16 infection was significantly reduced in WAVE1 KO
cells by roughly 50% (P<0.0001) and significantly increased in WAVE1 overexpression cells by
more than 50% (P<0.0001) (Figure 4). The remaining 50% infection in WAVE1 KO cells could
be due to some sort of compensatory mechanism, such as other WAVE/WASP family proteins
fulfilling some WAVE1 functions. These results show that WAVE1 is essential for HPV16
infection. The following experiments were performed to determine which step in the HPV16
infection pathway is WAVE1 implicated in.
WT HeLa
WAVE1 KO
WAVE1 Overexpression
0
50
100
150
200
250
%HPV Infection
(%RFP+, Relative to WT HeLa)
✱✱✱✱
✱✱✱✱

Figure 4. WAVE1 is required for efficient HPV16 infection WT, KO and overexpression of
WAVE1 in HeLa cells were treated with HPV16 PsV carrying the RFP reporter plasmid for 48h,
and infection (RFP positive cells) was measured by flow cytometry. Results are representative of
3 independent experiments with 3 biological replicates in each experiment, and show mean
%RFP positive cells ± s.d. Statistics: 1-way ANOVA with Dunnett’s multiple comparisons test,
****=P < 0.0001.
15

3.3 WAVE1 is not necessary for HPV16 binding and early internalization
WAVE1 influences cell membrane structures via actin polymerization on the cytosolic
side of the cell membrane and is therefore not expected to be directly involved in HPV binding at
the cell surface. To confirm that the decreased HPV infection in WAVE1 KO is not due to HPV
binding differences, cells were incubated with HPV at 4°C (to prevent internalization) and the
amount of surface-bound HPV was measured using flow cytometry. No statistical difference
could be observed in binding of HPV to the cell surface among WT, WAVE1 KO and
overexpression HeLa cells (Figure 5).
WT HeLa
WAVE1 KO
WAVE1 Overexpression
0
50
100
150
%Surface-Bound HPV
(MFI, Relative to WT Hela)
ns
ns

Figure 5. WAVE1 is not necessary for HPV16 binding. WT, WAVE1 KO, overexpression
HeLa cells were incubated with HPV16 L1L2 VLPs (1µg/ml/1E6 cells) at 4°C for 1h. The
amount of surface-bound HPV on collected cells was quantified by flow cytometry. The results
show mean fluorescent intensity (MFI) ± s.d., relative to WT HeLa, for six independent
replicates in each cell line. Statistics: ANOVA with Dunnett’s multiple comparisons test.
ns = not significant.

16

To understand if WAVE1 is potentially involved in HPV endocytosis, we next
investigated the role of WAVE1 in early HPV internalization. To set a baseline, the amount of
surface bound HPV was measured after cold binding and subsequently allowed to internalized
for 1 hour at 37°C. The difference between the two time points is considered as the amount of
HPV internalized. Results show that there is no significant difference in internalized HPV
between WT and WAVE1 KO HeLa cells, but a significant decrease between WT and WAVE1
overexpression HeLa cells (Figure 6). The similar amount of HPV internalization between WT
and WAVE1 KO HeLa cells is surprising, and this suggests that WAVE1 is functioning in a
different part of the HPV infection process than initially anticipated. As for the WAVE1
overexpression cells, the decrease in internalization could be explained by overexpressed
WAVE1 localizing to other areas of the cell. Previous studies have shown that WAVE1
overexpression causes displacement of Arp2/3 complex from the cell periphery and increases
Arp2/3 activity at the cytosol (Machesky and Insall 1998). The decreased actin polymerization at
the cell membrane could lead to decreased HPV endocytosis.
17

WT HeLa
WAVE1 KO
WAVE1 Overexpression
0
10
20
30
40
% Internalized HPV16
ns
✱✱✱✱

Figure 6. WAVE1 KO does not affect early HPV16 internalization. Using the same
procedure used for the HPV cell surface binding experiment, cells were treated with HPV16
L1L2 VLP at 1ug/ml/10E6 cells. The amount of surface bound HPV was measured with flow
cytometry at 1h of 4°C cold binding, and subsequently after 1hr incubation at 37°C to faciliate
internalization. HPV internalization was calculated as the difference between the amount of
surface bound HPV at 0h and 1hr. Results show mean MFI ± s.d from 6 independent replicates in
each cell line. Statistics: ANOVA with Dunnett’s multiple comparisons test. ns = not significant,
**** = P < 0.0001.

3.4 WAVE1 KO and overexpression decreases HPV16 trafficking to acidic
compartments
To investigate downstream HPV trafficking in endosomal compartments, we utilized
pHrodo labeled HPV16 VLP. pHrodo is a pH sensitive rhodamine dye which fluoresces in acidic
environments and shows little to no signal in neutral pH environments. pHrodo labeled HPV16
VLPs can be used to track HPV in the cell as it is trafficked to increasing acidic environments,
from early to late endosomes and lysosomes. WT, KO, and overexpression of WAVE1 HeLa
18

cells were treated with pHrodo VLPs and the RFU was measured at every hour from 0 to 5
hours. The results show both the absence and overexpression of WAVE1 decreases pHrodo
signal intensity over time (Figure 7). Despite WAVE1 KO and overexpression having different
effects on HPV infection, they both decrease HPV trafficking in the cell. As previously
mentioned, overexpressed WAVE1 protein could localized to different parts of the cell and it
could be altering HPV trafficking via different mechanism than the endogenous WAVE1 protein.
Also, the decreased HPV early internalization rate could contribute to decreased intracellular
trafficking. From the WAVE1 KO result, it is clear that WAVE1 plays a role in HPV
intracellular trafficking post entry.
0 2 4 6
0
1000
2000
3000
Time (hr)
RFU
WT HeLa
WAVE1 KO
WAVE1 Overexpression
WT HeLa No Virus Control

Figure 7. WAVE1 KO and overexpression decreases HPV16 trafficking to acidic
compartments. Cells were treated with pHrodo-labeled HPV16 VLPs at 2µg/mg per 1 million
cells and incubated at 37°C. At every hour from 0 to 5h, the fluorescent intensity was measured
using a plate reader. Results show 3 replicates per cell line and is representative of 3 independent
experiments.

19

3.5 WAVE1 is essential for HPV16 trafficking at Golgi Apparatus
In order to determine which step of the HPV intracellular trafficking pathway WAVE1 is
involved in, immunofluorescent microscopy was used to analyze colocalization of HPV16 and
cellular compartments in WT and WAVE1 KO HeLa cells post infection. Cells grown on an 8-
chamber slide were treated with HPV16 PsVs, which were bound to cells at 4°C for 1 hour,
washed with DPBS, and incubated at 37°C to allow internalization for different lengths of time.
Cells were then fixed and stained with HPV16 antibody, DAPI, and a marker for early endosome
late endosomes, golgi apparatus, or lysosome. The immunostaining was visualized using a
confocal microscope. Z-stacks, a series of images at different heights within a field of view, were
taken in order to capture all HPV particles from top to bottom of the cell. A maximum intensity
projection was generated from the z stack and the colocalization between HPV and cellular
compartment staining was measured using Mander’s overlap coefficient. Mander’s overlap
coefficient was used to determine the amount of fluorescent signal overlap between HPV and
cellular compartment staining without assuming a correlation of signal intensity in the two
channels. Figure 8a shows the Mander’s overlap coefficient over the course of 7 hours of HPV
internalization in WT and WAVE1 KO cells. There is little to no effect of WAVE1 KO on HPV
trafficking to the early and late endosome at any time points measured, but some increase can be
observed colocalization of HPV with the lysosome marker LAMP-1 at the 7hr time point.
However, there is visibly less HPV-Golgi apparatus colocalization in WAVE1 KO cells
compared to WT at the 7hr time point using both Golgin97 and GM130 markers (Figure 8b).
This result is also confirmed in a separate experiment with a 2, 4, 6, 8-hour HPV infection time
course (Supplemental Figure 2). This demonstrates that WAVE1 is involved in HPV trafficking
20

to the Golgi apparatus, and without WAVE1, this trafficking is stalled and leads to decreased
intracellular trafficking and infection.

21

A
0 2 4 6 8
0.0
0.5
1.0
1.5
EEA1
(Early Endosome)
Infection Time (hr)
Mander's Overlap Coefficient
0 2 4 6 8
0.0
0.5
1.0
1.5
Rab5
(Early Endosome)
Infection Time (hr)
Mander's Overlap Coefficient
0 2 4 6 8
0.0
0.5
1.0
1.5
GM130
(Golgi Apparatus)
Infection Time (hr)
Mander's Overlap Coefficient
0 2 4 6 8
0.0
0.5
1.0
1.5
Golgin97
(Golgi Apparatus)
Infection Time (hr)
Mander's Overlap Coefficient
WT HeLa WAVE1 KO
0 2 4 6 8
0.0
0.5
1.0
1.5
Rab7
(Late Endosome)
Infection Time (hr)
Mander's Overlap Coefficient
0 2 4 6 8
0.0
0.5
1.0
1.5
LAMP1
(Lysosome)
Infection Time (hr)
Mander's Overlap Coefficient

22

B
GM130 HPV16 Merge
WT
HeLa

WAVE1
KO

Figure 8. WAVE1 is required for HPV trafficking to Golgi apparatus and not to endosomes
or lysosomes. HPV16 PsV (0.5 μg per 1 million cells) were added to cells on 8 chamber slides,
bound for 4°C at 1 hour, washed with DPBS, and incubated at 37 °C. At each time point (2, 5, 7
hour), cells were fixed with and immunostained for the indicated cellular compartment marker,
HPV16 , and DAPI. A z-stack was taken using a confocal microscope and a maximum intensity
projection image was generated from the z-stack. (a) 2 z-stacks were taken for each cellular
compartment marker and time point. Colocalization of HPV16 and cellular compartment was
measured using Mander’s overlap coefficient (k2) using Nikon NIS-Elements software. (b)
Images from cells that were incubated with HPV16 PsV for 7 hours, stained with GM130
(green), HPV16 (red), DAPI (blue).

23

CHAPTER 4 DISCUSSION
It is crucial to understand HPV endocytosis in order to develop new strategies in
preventing HPV induced cancers, which cause hundreds of thousands of deaths worldwide every
year (de Martel et al. 2017). HPV enters the cell in a novel macropinocytosis-like pathway;
therefore, elucidating the factors involved in this process could contribute to discoveries in
endocytosis of other viruses that utilize a macropinocytosis or macropinocytosis-like pathway,
such as ebola, vaccina, and influenza (Mercer and Helenius 2012). HPV endocytosis shares
many hallmarks with macropinocytosis, such as actin polymerization, PKC, PI3K, PAK1, and
Na
+
/H
+
exchanger (Schelhaas et al. 2012). Given that HPV entry is actin dependent, it would
seem possible for actin binding partners to be involved. WASP/WAVE family proteins regulate
actin nucleation and organization at the cell membrane as well as in organelles. Out of this
protein family, WAVE1 is a likely candidate to be involved in HPV endocytosis, as it is
responsible for circular dorsal ruffles which are membrane protrusions associated with
macropinocytosis.
WAVE1 KO and overexpression cells were generated from WT HeLa cell background to
determine differences in HPV16 overall infection, binding, and internalization. HPV16 infection
decreased by 50% in WAVE1 KO cells and increased by more than 50% in the overexpression
cells. This establishes the critical involvement of WAVE1 in the HPV infection pathway, and
additional experiments were done to narrow down which step of HPV infection is WAVE1
dependent. Unsurprisingly, WAVE 1 is not required for HPV binding, as it is not expressed on
the cell membrane. WAVE1 was initially expected to be important for HPV endocytosis, but the
1-hour HPV internalization assay revealed that WAVE1 is not necessary for early HPV entry.
This suggests that WAVE1 is involved in downstream HPV trafficking processes, which was
24

confirmed in the pHrodo labeled HPV16 internalization assay results. In order to determine at
which step of HPV trafficking is WAVE1 required for, WT and WAVE1 KO HeLa cells post
HPV infection were immunostained for HPV16 and early endosome, late endosome, Golgi
apparatus, and lysosome markers. The results show a decrease in HPV-Golgi apparatus
colocalization in WAVE1 KO cells compared to WT HeLa cells. This indicates that WAVE1 is
necessary for HPV trafficking to the Golgi apparatus. In addition to the experiments done in
HeLa cells presented in this thesis, it is important to validate the results in the future using a
relevant primary cell line, such as HaCaT cells.
Not much is known about the function of WAVE1 inside the cell, as most WAVE1
research is on its activity at the cell membrane. There are only two papers discussing WAVE1 at
activity at the Golgi apparatus. Rawe et al. examined WAVE1 localization throughout
mammalian spermatogenesis and found that it was located at Golgi apparatus during
spermatocyte and round spermatid stages. Ceglia et al. investigated the role of WAVE1 in
amyloid β production in the context of Alzheimer’s disease, and demonstrated WAVE1
localization at Golgi apparatus in neuroblastoma cells. WAVE1 was also shown to facilitate the
budding process of vesicles containing amyloid precursor protein (APP) at the Golgi apparatus.
This adds confidence to the conclusion of WAVE1 function in HPV transport at this
compartment.
While there is no published research on WAVE1 activity in viral intracellular trafficking,
WASH, another member of the WAVE/WASP family, has been shown be key in retromer
transport at endosomes in vaccina virus (Hsiao et al. 2015). Similar to HPV, vaccina virus is a
double stranded DNA virus and it belongs in the poxvirus family. Post cell entry, vaccina virus is
trafficked from early endosome to recycling endosomes and released into the cytoplasm. WASH
25

recruits Arp2/3 and regulates actin polymerization at the early endosome. WASH was found to
interact with FAM21 and recruits FAM21 associated retromer complex VPS26,29, 35. This
WASH/FAM21 retromer complex mediates the sorting of virus containing vesicles from early
endosomes to recycling endosomes. Since HPV is transported from endosome to Golgi apparatus
in a similar retromer complex with FAM21, VSP26, 29, 35, Bugnon Valdano et al. speculates
that WASH is also involved in this complex, though this has not yet been proven (Figure 9).
Given the data presented here which points to WAVE1 playing a role in HPV trafficking to the
Golgi apparatus, it is possible that WAVE1 is a part of this retromer machinery in a similar
fashion to WASH.

Figure 9. Speculative model of WASH involvement in HPV trafficking to Golgi apparatus
(Bugnon Valdano et al. 2019) WASH is likely to be recruited by FAM21 binding to VPS29
subunit, a core component of the retromer transport machinery.

26

SUPPLEMENTAL DATA

Supplemental Figure 1. WAVE1 KO, Overexpression Western Blot. Western blot image of
WAVE1 KO and overexpression; protein input was normalized to 25µg.

0 2 4 6 8 10
0.0
0.5
1.0
1.5
2.0
GM130
(Golgi Apparatus)
Infection Time (hr)
Mander's Overlap Coefficient
0 2 4 6 8 10
0.0
0.5
1.0
1.5
Golgin97
(Golgi Apparatus)
Infection Time (hr)
Mander's Overlap Coefficient
WT HeLa WAVE1 KO

Supplemental Figure 2. WAVE1 is required for HPV trafficking at golgi apparatus.
Immunostaining protocol as described in Figure 8 was performed with time course of 2,4,6,8
hours post infection. 3 z-stacks were taken at each time point. Graph shows Mander’s Overlap
Coefficient mean ± s.d.

27

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Abstract (if available)

Abstract Human Papilloma Virus (HPV) infection begins with viral entry into host cell, which has been suggested to be a novel process independent of clathrin, caveolin, dynamin, cholesterol, lipid raft, or flotillin. The cell entry pathway bears many similarities to macropinocytosis, which is a bulk, nonspecific fluid uptake process. Actin polymerization is key for both HPV endocytosis and macropinocytosis, thus, actin polymerization regulators could be important for HPV entry. WAVE/WASP family proteins are responsible for actin nucleation and organization at the cell membrane. Out of this protein family, WAVE1 in particular is critical for the formation of circular dorsal ruffles, a type of membrane protrusion associated with macropinocytosis. This implicates a potential role for WAVE1 in HPV endocytosis. Here, we investigated the role of WAVE1 in binding, internalization, and infection of HPV16, a high-risk variant subtype of HPV. WAVE1 knock out (KO) and overexpression cell lines were created from wildtype (WT) HeLa cells. WAVE1 KO significantly reduced HPV16 infection while overexpression significantly increased the rate of HPV16 infection. The absence of WAVE1 did not affect HPV binding or early internalization, but it did play a role in HPV intracellular trafficking. Utilizing immunofluorescence microscopy, we demonstrated that WAVE1 is crucial for HPV trafficking to the Golgi apparatus, and not for HPV endocytosis as we initially hypothesized.

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Asset Metadata

Creator Cheng, Stephanie LokYee (author)

Core Title WAVE1 is a novel mediator of HPV trafficking to the Golgi apparatus

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2021-08

Publication Date 07/18/2021

Defense Date 06/07/2021

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

Tag endocytosis,HPV,macropinocytosis,OAI-PMH Harvest,WASH,WASP,WAVE1,Wiskott-Aldrich syndrome protein

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Advisor Kast, Martin (committee chair), Machida, Keigo (committee member), Yuan, Weiming (committee member)

Creator Email chengsl@usc.edu,stephee0804@gmail.com

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