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N-glycosylation on E protein and temperature regulate Zika virus infection in Aedes aegypti and Aedes albopictus mosquito cell

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N-glycosylation on E protein and temperature regulate Zika virus infection in Aedes aegypti and Aedes albopictus mosquito cell

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Content i
N-glycosylation on E Protein and Temperature
Regulate Zika Virus Infection in Aedes aegypti
and Aedes albopictus Mosquito Cell

by
Qianhui Du

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2018

Copyright 2018 Qianhui Du
ii

ACKNOWLEDGEMENT
I would first like to express my sincere gratitude to my mentor Dr. I-Chueh Huang and my
committee members, Dr. Peter Danenberg and Dr. Hyungjin Eoh, for their support of my
study and research.

I would also like to thank Dr. Huang and the lab members in Dr. Huang’s lab, for their
valuable guidance, patience and assistance.

Last but not the least, I would like to thank my family who always support me during my
study.

iii
Table of Contents
Table of Contents ......................................................................................................... iii
Abstract ......................................................................................................................... iv
Introduction .................................................................................................................... 1
1.Zika Virus Origin, Life Cycle and Structure ........................................................... 1
2.Role of Aedes mosquitoes in Zika Virus transmission ........................................... 3
3.N-glycosylation site on Zika Virus E protein ......................................................... 5
4.Temperature in regulation of ZIKV’s infectivity in mosquito cell ......................... 6
Results ............................................................................................................................ 8
1.Production of ZIKV sub-genomic replicon and single-cycle virus-like particle .... 8
2.Infection in C6/36 cell with the ZIKV virus-like particle South American
strain(Brazil WT,SpH2015) and African strain(MR WT,MR766) ............................. 9
3.Removal of N-154 glycosylation site on ZIKV E protein causes increase in
infectivity in C6/36 and Aag2 mosquito cell but does not affect the infectivity in
human glioblastoma cell SNB19 ............................................................................. 12
4.Domain III(DIII) of ZIKV E protein is receptor binding domain for both C6/36 cell
and SNB19 cells ....................................................................................................... 15
5.ZIKV infects C6/36 cell better in 37ºC than 28ºC ................................................ 16
6.Different infectivity causing by variances in temperature is related with post- entry
steps of ZIKV infection cycle .................................................................................. 18
Materials and Methods ................................................................................................. 21
Discussion .................................................................................................................... 25
References .................................................................................................................... 28

iv
Abstract
Zika virus (ZIKV) is a member of the virus family Flaviviridae and is transmitted by
Aedes mosquitoes, such as A. aegypti and A. albopictus. Being associated with serious
neurological and developmental pathologies, like congenital malformation during
pregnancy and Guillain-Barré syndrome, ZIKV has rapidly spread reaching a global
distribution pattern similar to that of dengue virus. Using the single-cycle ZIKVrep-based
VLPs which co-expressed the strain-specific structural (CprME) and identical non-
structural (NS1toNS5) proteins, it has been found that ZIKVs isolated in Africa could
infect mosquito C6/36 or Aag-2 cells more efficiently than that isolated in Southeast Asia
or in South America. We further confirm that the different infectivity among various strains
of ZIKV is attributed to N-linked glycosylation of ZIKV enveloped (E) protein. Sequence
analysis of different clinical and lab isolated strains has shown the existence of conserved
N-glycosylation motif on domain I of protein E, which is common to all flavivirus.
Infectivity of South American lineage ZIKVs in mosquito cells is strongly enhanced upon
removing N-linked glycosylation by either introducing mutations or exogenous enzyme
digestion. In contrast to mosquito cells, similar phenotypes could not be observed in human
glioblastoma SNB-19 cells. Our study also shows that domain III of E protein is the
receptor binding site for both human glioblastoma SNB-19 cells and Aedes mosquito
C6/36 cells. For both strains of ZIKV with or without E protein glycosylation site,
infectivity is significantly increased in 37°C compared with 28°C and this is post-entry
phenotype of ZIKV infection. Decrease of environmental temperature can play an
important role in suppression of ZIKV infection in mosquito cells, which may potentially
become a key in control of the viral transmission and spread.

1
Introduction
1. Zika Virus Origin, Life Cycle and Structure
Zika virus (ZIKV), a member of the Genus Flavivirus, family Flaviviridae, is closely
related to the four serotypes of dengue (DENV) as well as other globally relevant viruses
including yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV)
viruses
1
. ZIKV was first isolated in 1947 from a rhesus monkey in Uganda(prototype
strain MR 766)
2
, and has become a world-wide health concern since recent re-emergent
outbreaks reported in Africa, Malaysia and Indonesia
2,3
. At the beginning of 2015, the
first local transmission of ZIKV was detected in the northeastern part of Brazil, and
extended to Central and South America in an unexpected rate
4
. It has gradually become a
threat to North America and even the whole world. The clinical symptoms associated
with the infection include serious neurological and developmental pathologies in humans,
like congenital malformation during pregnancy, fetal and newborn meningoencephalitis
and Guillain-Barré syndrome
5-7
.
Following the same way with other flavivirus, the life cycle of ZIKV begins with
attachment to the host extracellular surface, and the virus is then endocytosed and
encapsulated inside an endosomal vacuole
8,9
. Acidic environment of the endosomal
compartment results in the structural change of E protein, causing the fusion of the virus
with the endosome, and facilitates viral uncoating to release genome into the intracellular
compartment. The released viral genome undergoes replication or translation into a
polyprotein by either being converted into a negative-sense RNA to make positive-sense
RNA copies or transported to the endoplasmic reticulum(ER) where translation takes
place. The large polyprotein is post-translationally processed into structural and non-
2
structural components which are important for virus assembly and maturation. The
newly-made viral genome is packaged into a capsid and transported to the Golgi where it
will be coated by the E/M protein complex to form a mature virion before budding out
and the whole viral life cycle is complete.

Figure 1.1. Life cycle of flavivirus.
9
Taken from ‘Flaviviruses, an expanding threat in
public health: focus on dengue, West Nile, and Japanese encephalitis virus.’ J Neurovirol,
2014;20(6):539-560
Like all flavivirus, Zika virus has a single-stranded, positive-sense RNA genome
approximately 11 kilobases in length. The genome contains 5′ and 3′ untranslated regions
as well as a single open reading frame (ORF) that encodes a polyprotein which is cleaved
by host and viral proteases into three structural proteins: the capsid (C), pre-
membrane/membrane (prM), and envelope (E), and seven non-structural proteins (NS1,
NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5)
10,11
. The structural proteins along with
genomic RNA form viral particles, while the nonstructural proteins are involved in
replication, assembly, and evasion of the host immune system. Among the 3 structural
3
protein, C forms a nucleocapsid when bound to viral RNA; prM complexes with E
shortly after synthesis to facilitate folding and prevent premature fusion to host
membranes; E protein plays an important role in virus entry and it is the main target of
neutralizing antibodies induced immune response
12,13
. ZIKV E protein is divided into
three domains: a central β-barrel domain (domain I, DI), an extended dimerization
domain (DII), and an immunoglobulin-like segment (DIII)
14
. It has been shown that many
type-specific protective antibodies recognizing determinants in DIII
15
.

Figure 1.2. Structure of Zika virus (ZIKV) genomic ribonucleic acid (RNA)

2. Role of Aedes mosquitoes in Zika Virus transmission
As a member of mosquito-borne flaviviruses family, the process of transmission can
be completed through the bite of an infected Aedes species mosquito, or through vertical
transmission by passing from mother to fetus during pregnancy
6,16
. Spread of the virus
through blood transfusion and sexual contact also have been reported
16
. ZIKV is also
spread by Aedes mosquitoes, such as A. aegypti and A. albopictus
17,18
. While most of the
study focus on mechanisms of ZIKV’s infection in mammalian cell and tissue, the
regulation of viral infectivity in mosquito cell have received far less attention.
Mosquitoes are responsible for the transmission of numerous infectious diseases
including malaria, dengue fever, West Nile fever and Japanese encephalitis.
9
The
mosquito lifecycle comprises a series of life stages beginning with eggs laid on or near
4
water that hatch after a number of days into larvae
19,20
. The larvae obtain nutrition mostly
through filter-feeding but predation on other larvae and certain species of small
invertebrates. Mosquitoes develop through four major phases, from egg, larva to form
non-feeding pupae, which metamorphose into adults. While both male and female adult
mosquitoes can derive nutrition from nectar, in most species females require a blood meal
to promote egg development through the acquisition of protein and iron from blood,
which can provide the opportunity for pathogen transmission, particularly as female
mosquitoes take multiple feeds during their lifecycle. When a female mosquito bites an
infectious human or animal, the pathogen is ingested with the blood meal and
disseminates from the mosquito midgut, eventually reaching the mosquito’s salivary
glands for transmission to a new host. Cells can be obtained from each of these
developmental stages to generate cell lines appropriate for each experimental approach,
in which way various aspects of virus-mosquito host interaction can be analyzed in
vitro
19
.
The Asian tiger mosquito, Ae. albopictus, occupies urban areas with or without
vegetation, is a competent vector of many arboviruses including DENV, Chikungunya
virus and Eastern equine encephalitis virus (EEEV)
21
. The first cell lines developed from
Ae. albopictus, the C6/36 cell line, were generated from larvae in the mid 1960s
22
. C6/36
cells are shown to be susceptible to a wide-range of arboviruses, and this cell line is now
widely used to isolate or propagate arthropod-borne viruses
22,23
.Lineages of the C6/36
cell line have also been applied for studies in the interactions between arboviruses and
mosquito vectors, and the research of the infectious dengue-1 virus entry mechanism, for
instance, has been performed on this mosquito cell line
24
.
Aedes aegypti mosquitoes are also important vectors of viral diseases, such as
Yellow and Dengue fever, which have significant impact on human morbidity and
5
mortality. Recently, Aag-2, an Aedes aegypti cell lineage, has also been frequently used
as a model for studies of mosquito immunity
25
. As ZIKV use both Ae. Albopictus and
Ae. Aegypti mosquitoes as vectors, infection on both C6/36 and Aag2 cell lines could be
used to provide preliminary evidence for the vitro studies reflecting the potential of
infectivity of emerging ZIKV.
3. N-glycosylation site on Zika Virus E protein
N-linked glycosylation is one of the most common post-translational modifications of
secretory and membrane-associated proteins. In this process a high mannose core is
added to the side-chain group of an asparagine (N) residue present in the conserved
glycosylation motif N-X-S/T (where X is any amino acid with the exception of P)
26
.
Biosynthetic process of N-linked oligosaccharide occurs in the ER and Golgi. Research
on function of N-linked glycosylation in viral infection has received increasingly intense
attention in recent years due to its ability to affect viral survival and virulence by
interfering process of receptor binding or host immune response. It has been shown that
HIV and influenza rely on expression of specific oligosaccharides to evade detection by
the host immune system and mediate the interactions with host cell receptors
27-29
. In
addition, other viruses such as SARS-CoV, hepatitis virus and West Nile Virus rely on N-
linked glycosylation for crucial functions when entry into host cells
30,31
.
As the E proteins are responsible for receptor binding and membrane fusion, and they
are also the major targets of neutralizing antibodies, the functions of glycoprotein on viral
E protein are critical in the entry step of viral life cycle
14,32
. Usually, there are up to two
N-glycosylation sites on the E protein of vector-borne pathogenic flaviviruses, except for
YFV
33
. One highly conserved glycosylation site localizes at N153 or N154 (depending on
the particular virus) of E protein, while another one at N67 is only found on the dengue
6
virus
34
. ZIKV E protein is N-glycosylated at N154, which is located on a loop close to the
fusion peptide of a neighboring E protein
33,35
.
In the case ZIKV, it has been shown that one sequence motif, VNDT, containing an
N-linked glycosylation site in the envelope (E) protein central domain I(DI) is
polymorphic for being absent in many of the African isolated strains while present in all
South American strains in the recent outbreaks
36
. Studies indicate that N-linked
glycosylation of ZIKV E protein is relevant with expression and secretion of the soluble
ecto-domain from transfected cells, and also viral production and infectivity of
mammalian cells
37
. However, little is known about its role in regulation of infection in
mosquito cells.

Figure 2. Sequence alignment of various strains of ZIKV at E protein glycosylation site
4. Temperature in regulation of ZIKV’s infectivity in mosquito cell
The infectivity of Zika virus in mosquito cell could be affected not only by intrinsic
factors like genetic variation and innate immune response of the cell but also various
extrinsic factors like climate and temperature fluctuations as well
38

39
.Temperature is one
of the critical environmental factors affecting biological processes of mosquitoes,
including their interactions with viruses.
7
Temperature is closely related to the distribution pattern of mosquito cell in the
world. Variations among season and geographical location not only have influence on the
mosquito lifespan, population dynamics but also its susceptibility to arbovirus infection
40
.
Recent studies have confirmed that temperature-associated changes in morphology and
physiology may translate to altered permissibility of midgut and salivary gland barriers in
mosquito that must be overcome for virus transmission to subsequent hosts
38
. In addition,
the time that lapses from the ingestion of the infectious blood meal to transmission of the
virus is referred to as the Extrinsic Incubation Period (EIP)
19,41,42
. EIP is temperature-
dependent and often used as an index of vector competence. In order to successfully
transmit, the vector must survive the EIP of the virus. It has been shown that higher
temperatures have usually been associated with increased vector competence, thus even
enhance the viral replication and promote the transmission
43
. Nevertheless, the role of
temperature may differ among various vector-virus systems
38
. Research has shown that
elevated temperature during immature stages increase the susceptibility to infection and
dissemination of dengue viruses and other flavivirus in Ae. Aegypti
44
. The ZIKV
outbreak is more likely to take place in warm areas and seasons, which favors the
survival and activity of mosquito, however, whether the temperature variations play a
role in the ZIKV infection in the mosquito cell, and especially, the specific steps in the
viral infection that might be affected, remains unclear. It is of great importance to explore
how variances of temperature result in different ability of the ZIKV to infect the
mosquito cell.

8
Results
1. Production of ZIKV sub-genomic replicon and single-cycle virus-like particle
In order to investigate whether the E protein glycosylation site influence the infection
in human cell and mosquito cell, we generated ZIKV sub-genomic replicon and virus-like
particle (VLP) system, which can be used to determine viral factors in infection
efficiency of ZIKV in target cells. This ZIKV-GFP based replicon(ZIKVrep-GFP) was
generated by replacing the coding region of capsid (C), pre-membrane (prM), and E
proteins with the GFP reporter. Another sub-genomic replicon from WNV (WNVrep-
GFP) created in a similar way, was also applied in the study
45
. To generate single-cycle
ZIKV VLPs which are unable to replicate, we co-transfected Human embryonic kidney
cells 293 cells with the sub-genomic replicon (either ZIKVrep-GFP or WNVrep-GFP)
and a protein-expressing plasmid encoding ZIKV C, prM, and E proteins from various
ZIKV strains. VLPs carrying various C, prM, and E protein variants from MR766 ZIKV
and SPH2015 ZIKV, isolated in Brazil, were generated.

Figure 3. Schematic of the three plasmids used to produce virus-like particle(VLP):
replicon plasmid encoding the ZIKV or WNV nonstructural proteins (NS1 to NS5), with
9
GFP in place of the three structural proteins (C, prM, and E), and plasmid expressing C,
prM, and E from various ZIKV strains. The cDNA fragments were synthesized and
assembled in the PCAGGS vector under the CMV promoter. An HDV ribozyme was
inserted downstream from the 3′ end of the ZIKV genome.

2. Infection in C6/36 cell with the ZIKV virus-like particle South American
strain(Brazil WT,SpH2015) and African strain(MR WT,MR766)
South American strain(Brazil WT,SpH2015) and African strain(MR WT,MR766)
ZIKV virus-like particle were harvested from the 293T supernatant after transfection and
applied for infection on C6/36 for 2 days. To evaluate the infectivity of both strains,
infected target cells C6/36 were imaged by confocal microscope and analyzed by flow
cytometry. 20,000 cells were collected for analysis and percentage of green cells which
expressed the GFP reporter were counted. As shown below, the infectivity of SpH2015
was significantly lower than MR766, either by using ZIKVrep-GFP or WNVrep-GFP
virus-like particle.

10

Figure 4.1. Two days after infection by ZIKV VLP(ZIKVrep-GFP), C6/36 cells were fixed
and labeled with 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged by confocal
microscopy(A) and green cells were quantified by flow cytometry(B). Error bar represents
standard deviation. Statistics were performed using unpaired Student’s t test; **very
significant (p < 0.01).

11

Figure 4.2. Two days after infection by ZIKV VLP(WNVrep-GFP), C6/36 cells were fixed
and labeled with 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged by confocal
microscopy(A) and green cells were quantified by flow cytometry(B). Error bar represents
standard deviation. Statistics were performed using unpaired Student’s t test; **very
significant (p < 0.01).

0
10
20
30
40
50
60
70
80
Brazil WT(WNVrep) MR WT(WNVrep)
Green cell percentage%
C6/36 Infectiviry
12

3. Removal of N-154 glycosylation site on ZIKV E protein causes increase in
infectivity in C6/36 and Aag2 mosquito cell but does not affect the infectivity in
human glioblastoma cell SNB19
The N154 glycosylation site has a conserved amino acid motif: N-X-T/S, while X
can be any one of the amino acid except proline. Once the T(Threonine) has been
replaced by I(Isoleucine), the glycan cannot be added on the N(Asparagine). Thus the
glycosylation is absent in those amino acid sequence of NDI. In order to investigate
whether the difference of infectivity between African strain and South American strain
ZIKV is attributed by E protein N-154 glycosylation site, an E-T156I mutation was
introduced to delete the E protein N-154 glycosylation motif in South American strain
(Brazil WT, SpH2015), while an E-I156T mutation was made to create the glycosylation
motif on E N154-glycosylation site in African strain(MR WT,MR766).In this way, the
mutated South American strain(Brazil mut) is without N154-glycosylation site while the
mutated African strain(MR mut) is added with glycan on E protein. The existence of the
glycosylation on E protein was confirmed by western blotting. As anticipated, infectivity
of Brazil WT in both C6/36 and Aag2 mosquito cells was strongly enhanced upon
removing N-linked glycosylation introducing mutations or exogenous enzyme digestion.
In contrast, similar phenotypes could not be observed in human glioblastoma SNB-19
cells.

13

Figure 5.1. ZIKV VLP(WNVrep-GFP) were harvested on Day2 after transfection and
analyzed by Western blot. Supernatant of HEK293T cell were collected, concentrated,
pelleted down and treated with PNGase F for 4 h at 37°C. Western blot analysis of
enzyme digested E protein was assessed by 10% SDS-PAGE using mouse anti-E IgG as
primary antibody.

14

15
Figure 5.2. ZIKV VLP(WNVrep-GFP) were harvested on Day2 after transfection.
Supernatant of HEK293T cell were collected, concentrated, pelleted down and treated with
PNGase F for 4 h at 37°C.Pellets were re-suspended in RPMI 1640 medium or Minimum
Essential Medium (MEM) before being applied for the following infection. After infection
(48 hours for mosquito cells; 24 hours for SNB-19 cells), cells were harvested and fixed,
and green cells were quantified by flow cytometry(B).

4. Domain III(DIII) of ZIKV E protein is receptor binding domain for both C6/36 cell
and SNB19 cells

It has been confirmed that the receptor binding site of ZIKV in mammalian cell is on
DIII of ZIKV E protein
15
, while the N-154 glycosylation site on Domain I(DI)
33
, however,
can cause significant differences of infectivity in mosquito cells. In order to further
examine whether identical receptor binding sites are involved in viral entry in mosquito
cells, anti-ZIKV antibody ZV-13 and ZV-54, which target domain I and domain III of E
protein, respectively, are applied for neutralizing essay
15
. Our results show that ZV-54 can
not only block the ZIKV infection in human glioblastoma SNB-19 cells but also in Aedes
mosquito C6/36 cells, which means domain III of E protein is the receptor binding site for
both 2 cell lines.

16

Figure 6. Neutralizing essay for test of receptor binding domain. Virus-like particle(MR
766) were mixed with antibody(10ug/mL) and incubated 1h in 37 ℃ before infection in
SNB human cells and C6/36 mosquito cell. After infection (48 hours for mosquito cells;
24 hours for SNB-19 cells), cells were harvested and fixed, and green cells were quantified
by flow cytometry. Error bar represents standard deviation. Statistics were performed using
unpaired Student’s t test; **very significant (p < 0.01).

5. ZIKV infects C6/36 cell better in 37ºC than 28ºC
As the ZIKV outbreaks always take place in hot summer of tropical and sub-tropical
17
region, we compare the viral infectivity under different temperatures. C6/36 cells were
infected with ZIKV MR 766 or ZIKV FLR Colombia at an MOI of 0.05 in 37ºC or 28ºC,
respectively. The results demonstrated that both of the African ZIKV strain MR 766 and
the South American Strain ZIKV FLR Colombia exhibited similar temperature sensitivity.
The infectivity was higher in 37 ºC but significantly lower in 28ºC for both strains. The
existence of glycosylation on E protein does not play a role leading to variances of
infectivity under the 2 different temperature conditions.

Figure 7. Comparison of infectivity of C6/36 cells under different temperatures. C6/36 cells
were infected with ZIKV MR 766 or ZIKV FLR Colombia at an MOI of 0.05 in 37ºC or
28ºC, respectively. After 48h-infection, viral titers were measured using plaque assays on
Vero cells. Error bar represents standard deviation. Statistics were performed using
unpaired Student’s t test; **very significant (p < 0.01).

18

6. Different infectivity causing by variances in temperature is related with post- entry
steps of ZIKV infection cycle

To further confirm whether the different infectivity causing by temperature sensitivity
is entry phenotype or post-entry phenotype, four strains of ZIKV VLP(ZIKVrep-GFP)
including Brazil WT, Brazil E mut, MR WT, MR E mut were applied in the experiment.
Entry steps of flavivirus including virus-host cell receptor binding and endocytosis usually
take up to 4-6 hours
46
. In this experiment, C6/36 cells were infected by the ZIKV VLP for
6 hours in 37ºC or 28ºC, respectively, before switching temperature conditions to complete
the rest 2-day infection. Therefore, there were 4 combinations in total:28-28,28-37,37-37
and 37-28. The results suggested that the temperature of the first 6-hour infection cannot
cause significant differences in infectivity in C6/36 cells, instead, it was the temperature
for the rest 2-day infection that played a decisive role in causing the infectivity variances.
In other words, temperature for the post-entry steps of ZIKV life cycle is critical for the
viral infectivity in C6/36 cells.

19

20

Figure 8. C6/36 cells were infected by 4 different strains of ZIKV VLP(ZIKVrep-GFP)
including Brazil WT, Brazil E mut, MR WT, MR E mut, under 4 different temperature
conditions: 28-28,28-37,37-37 and 37-28 ºC. Temperature switch were carried on 6-h post-
infection. Two days after infection, cells were harvested and fixed, and green cells were
quantified by flow cytometry. Error bar represents standard deviation. Statistics were
performed using unpaired Student’s t test; **very significant (p < 0.01).

21
Materials and Methods
Cells and Antibodies

HEK293T cells were grown in high-glucose Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin,1%
sodium pyruvate and 1% Non-Essential Amino Acids (NEAA). Human glioblastoma cells
SNB-19 were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL
penicillin, and 100 µg/mL streptomycin (ThermoFisher Scientific). Vero cells are
maintained in a DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, 1%
NEAA at 37 °C with 5% CO2, C6/36 and Aag-2 cells were cultured in ATCC-formulated
Eagle's Minimum Essential Medium(MEM) containing 10% FBS and 1%
penicillin/streptomycin at 28 °C with 5% CO2. Antibodies used in this study: first antibody
murine anti-ZIKV E protein IgG2b (GeneTex, GTX634155, 1:200), secondary antibody
Alex 647-conjugated goat anti-mouse IgG2b. Mouse monoclonal anti-Zika Virus, clone
ZV-54, Cat. No. MABF2046, and anti-Zika virus, clone ZV-13, Cat. No. MABF2043 were
applied in antibody neutralizing assay. The ZIKV strain MR766 and FLR was obtained
from ATCC and propagated in Vero cells for 4 days. Viral supernatant was filtered via
0.45 µM syringe filters (VWR). Virus titers were determined by a standard plaque assay.

Plasmid construction

The E I156T or E T156I mutations were introduced to the ZIKV sub-genomic cDNA
clone as described previously in the Results part. The DNA fragments between unique
restriction sites NotI and XhoI could be obtained using the corresponding enzymes for
digestion. Insert genes were ligated with modified PCAGGS vectors before being
22
amplified by E.coli. All restriction enzymes were purchased from New England BioLabs
(Ipswitch, MA).
Production of ZIKV VLP, Flow cytometry and Confocal Microscope

Standard calcium phosphate transfections were performed on HEK-293T cells as
previously described
47
. The 293T supernatant containing the ZIKV VLP(WNVrep-GFP)
or ZIKV VLP(ZIKVrep-GFP) were harvested on Day2 or Day3, respectively after
transfection and applied for infection. After infection (48 hours for mosquito cells; 24 hours
for SNB-19 cells), cells were harvested, washed 3 times with DPBS before being fixed
with 2% formaldehyde (Polysciences), and analyzed by flow cytometry. The cells were
mounted with DAPI (4’, 6-diamidino-2-phenylindole; Vector Laboratories, Inc.) before
confocal imaging and analysis.

Infection by replicating ZIKV and Plaque assay

C6/36 cells (7 × 10^6 cells/well) were seeded in 6-well plates 24 h prior infection.
Cells were inoculated with ZIKV strain MR766 and FLR at an MOI of 0.05; infections
were executed in triplicates. Twenty-four hours prior infection, Vero cells were seeded into
a 6-well plate (7 × 10^5cells/well). Viral samples were 10-fold serially diluted six times in
DMEM before being added to the Vero cells seeded in a 6-well plate; infections were
executed in duplicates. After 6-hour incubation at 37°C, and the plates were incubated at
37°C for 5 days. Following incubation, the cells were fixed with 2% formaldehyde for 30
min at room temperature. After removing the fixative, the plate was stained with 1% crystal
violet for 10 min. Visible plaques were counted, and viral titers (PFU/ml) were calculated.

Western blots and de-glycosylation treatment
23

The 293T supernatant containing the ZIKV VLP was harvested on Day2 after
transfection and concentrated by centricon (MilliporeSigma UFC910008 Amicon® Ultra-
15 Centifugal Filter Concentrator with Ultracel® 100 Regenerated Cellulose Membrane,
NMWL: 100,000) before being pelleted down with 4°C centrifuge for 2h. The VLP pellet
was washed by DPBS followed by 2µl of GlycoBuffer 2 (10X) and water added to make a
20 µl total reaction volume, then was treated with Peptide N-Glycosidase F (PNGase F) in
accordance with the manufacturer’s instructions (New England BioLabs, Inc.). Proteins we
analyzed under denaturing conditions in 10% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. Blots
were then blocked in TBST buffer (10 nM Tris-HCl, pH 7.5, 150 nM NaCl, and 0.1%
Tween 20) supplemented with 1% skim milk for 1 h, followed by probing with primary
antibodies (1:200 dilution) for 3 h at room temperature. After three washes with TBST
buffer, the blots were incubated with goat anti-mouse secondary antibody (1:5000 dilution)
in TBST buffer with 1% milk for 1 h, followed by three washed with TBST buffer before
images were obtained.

Antibody neutralization assay

Pellets of ZIKV VLP MR766 were obtained as described previously, and re-
suspended in the medium for SNB 19 cells or C6/36 cells. The virus-like particle were
incubated with anti-Zika virus, clone ZV-13, or clone ZV-54, respectively, at 37°C for 2 h
at a concentration of 10ug/mL. Antibody-virus complexes were added to C6/36 cells in 24-
well plates. Forty-eight-hour post-infection, cells were harvested and fixed, and green cells
were quantified by flow cytometry.

24
Data analysis

Data was obtained from at least three independent experiments done in duplicate or
triplicate. SPSS 18.0 software was used to analyze all of the data. Results were expressed
as the mean ± standard deviation (SD). An unpaired two-tailed Student’s t test was applied
for statistical analyses and a P value of <0.05(marked with *) and a P value of
<0.01(marked with **) respectively indicates statistically significant or very significant.

25
Discussion
Glycosylation is the most common protein modification in viral cycle. N-
glycosylation of envelope or surface proteins can either promote proper folding and
subsequent trafficking using host cellular chaperones and folding factors, or affect the
interaction with the host cellular receptor, host innate immune system and antibody
recognition, thus have an impact on viral infectivity and replication. For the flavivirus, the
E protein is a major surface glycoprotein that is critical in receptor binding and membrane
fusion for the viral entry. Previous studies have already shown that the WNV E
glycosylated protein leads to in neuro-invasiveness in mice but has no effect in infectivity
in mosquito cells
48
.For the DENV, depletion of the N67 glycosylation site of E protein
could reduce its capability to make the infectious virus particles in mammalian cells but
not mosquito cells, while the other N153/154 glycosylation site positively regulate the viral
replication in mosquito C6/36 cells but does not show significant effect in vivo mosquito
sudies
49
. Likewise, the ZIKV glycosylation is also common, especially in the strains
isolated in South America. In our study, we compare the infectivity between the South
American strain SpH 2015 isolated in Brazil, which contains the N154 glycosylation site
and an original African strain MR 766 which is lack of the E protein glycosylation, and
switch the mutation sites before infection in the mosquito cell line C6/36, Aag-2, and
human glioblastoma cell SNB19 as well. In this way we narrowed down our focus on the
role of the glycosylation site on ZIKV E protein. Interestingly, the infectivity of ZIKV in
the 2 mosquito cell lines could be significantly increased upon the removal of glycosylation
on ZIKV E protein, which is confirmed either by mutagenesis study or exogenous enzyme
digestion. By contrast, similar results could not be observed in the human SNB19 cell. This
phenomenon arouses the possibility that the glycosylation on ZIKV E protein could have
an effect in interfering the binding of the virus with the host cell receptor, and the
26
mechanisms might differ from human cells to mosquito cells. It should be pointed out that
our conclusion is drawn mainly based on the phenotype observed upon the single-cycle
ZIKV virus-like particle, study on infectious ZIKV with identical full-length replicon will
be necessary for further confirmation.

It has been reported that the carbohydrates on viral E protein act as the initial binding
component on the host cell surface via C-type lectin, a family of host protein with
carbohydrate-binding activity
50
. However, lectin molecules from human or mosquito host
cells associate with the glycosylated E protein through different mechanisms. In contrast
to mammalian lectin DC-SIGNs that are presented on the cellular surface through their
transmembrane domains, mosquito lectins are not directly associated with the cell surface
but are soluble, cell-free that could interact with the glycan of flaviviral E proteins to form
a lectin-virus complex, which could then bind to specific lectin-binding proteins located
on the mosquito cell surface, leading to viral attachment and entry
51,52
.The different
patterns of the lectin molecules presenting on the host mosquito and human cell involved
in viral binding could be one possible explanation for the discrepancies in the infectivity
of the ZIKV in the two different cell lines.

On the other hand, studies have revealed that among the three domains of the Zika
virus envelope protein, domain III is actually the decisive component to induce specific
and neutralizing immune responses against ZIKV and contains the cellular receptor-
binding motifs
14,53
.More importantly, domain III has been found to be targeted by several
different ZIKV-specific antibodies with distinct yet potent neutralizing activities
15
.The
N154 glycosylation site which elicits such huge differences of viral infectivity, however,
is on the domain I of the ZIKV E protein
33
.Based on the apparent different infectivity
observed from human cells and mosquito cells, we hypothesized that different receptor
27
binding sites might be involved in mosquito cell and mammalian cell during ZIKV
infection. In our experiment, the two antibodies, ZV-13 and ZV-54
10,15
, are applied for
testing the neutralizing activity in SNB19 and C6/36 cells, respectively. Contrary to our
anticipation, only ZV-54, which targeted the domain III of the ZIKV, possesses the ability
to inhibit the ZIKV infection.

Temperature is one of the critical environmental factors that heavily influence the
emergence and transmission of the vector-borne diseases. Detection of infectivity of ZIKV
in mosquito cells could provide the preliminary evidence to unveil the underlying
relationship of temperature variation and viral infection rate. As ZIKV outbreaks
frequently take place in summer around tropical and sub-tropical regions, in this study, we
infect the C6/36 mosquito cells under 28°C and 37°C with ZIKV South American strain
FLR and African strain MR 766, respectively. As expected, it is found that the infectivity
is significantly higher in 37°C for both of the ZIKV strains with or without glycosylation
site on E protein. We further identify that the trend is contributed by post-entry steps of the
viral life cycle, as described previously. Nevertheless, our study is constrained within the
one mosquito cell line and might not be able to reflect the phenotype in viral infection for
the real mosquitos. Previous studies on West Nile Virus(WNV), Chikungunya virus,
Dengue Virus(DENV), and Yellow Fever Virus(YWV) indicated that increased vector
competence, often characterized by Extrinsic Incubation Period (EIP), as well as the viral
binding to specific receptors in different organs were positively correlated with higher
temperature, while under which condition the mosquito lifespan was usually
unfavorable
38,54-57
. Optimal temperature for the ZIKV dissemination requires further
research on real mosquito and even the mosquito-mammalian animal model for
transmission study, to provide additionally potential clues for the role that temperature
plays in ZIKV spread.
28
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Abstract (if available)

Abstract Zika virus (ZIKV) is a member of the virus family Flaviviridae and is transmitted by Aedes mosquitoes, such as A. aegypti and A. albopictus. Being associated with serious neurological and developmental pathologies, like congenital malformation during pregnancy and Guillain-Barré syndrome, ZIKV has rapidly spread reaching a global distribution pattern similar to that of dengue virus. Using the single-cycle ZIKVrep-based VLPs which co-expressed the strain-specific structural (CprME) and identical non-structural (NS1toNS5) proteins, it has been found that ZIKVs isolated in Africa could infect mosquito C6/36 or Aag-2 cells more efficiently than that isolated in Southeast Asia or in South America. We further confirm that the different infectivity among various strains of ZIKV is attributed to N-linked glycosylation of ZIKV enveloped (E) protein. Sequence analysis of different clinical and lab isolated strains has shown the existence of conserved N-glycosylation motif on domain I of protein E, which is common to all flavivirus. Infectivity of South American lineage ZIKVs in mosquito cells is strongly enhanced upon removing N-linked glycosylation by either introducing mutations or exogenous enzyme digestion. In contrast to mosquito cells, similar phenotypes could not be observed in human glioblastoma SNB-19 cells. Our study also shows that domain III of E protein is the receptor binding site for both human glioblastoma SNB-19 cells and Aedes mosquito C6/36 cells. For both strains of ZIKV with or without E protein glycosylation site, infectivity is significantly increased in 37°C compared with 28°C and this is post-entry phenotype of ZIKV infection. Decrease of environmental temperature can play an important role in suppression of ZIKV infection in mosquito cells, which may potentially become a key in control of the viral transmission and spread.

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Asset Metadata

Creator Du, Qianhui (author)

Core Title N-glycosylation on E protein and temperature regulate Zika virus infection in Aedes aegypti and Aedes albopictus mosquito cell

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/26/2018

Defense Date 05/03/2018

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

Tag E protein,mosquito cell,N-glycosylation,OAI-PMH Harvest,temperature,Zika virus

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Huang, I-Chueh (committee chair), Danenberg, Peter (committee member), Eoh, Hyungjin (committee member)

Creator Email qdu@tulane.com,qianhui.du@foxmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-26782

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