Abstract virus is now spread in about


Zika Virus (ZIKV) is a
flavivirus that rose to infamy in the past decade because of the outbreaks of
its infection, first in 2007 in Micronesia and then again in 2015 in Brazil.
The virus has been associated with the incidence of Guillain-Barre syndrome in
affected individuals and the occurrence of microcephaly in infants born of
infected mothers. Like other flaviviruses, ZIKV is adept at evading the innate
immune system of host by inhibition of interferon signalling. It does so by
utilising its protein NS5 which binds to and directs degradation of STAT2,
which is an important mediator in type I and III interferon signalling. It is
known that NS5 causes proteasomal degradation of STAT2; but the host proteins,
in particular, the E3 ubiquitin ligase involved in the process in not known.
Also, the region of NS5 necessary for binding STAT2 is also not known. I
propose to probe these questions by means of NS5 pull-down and analysis for
interacting partners, and binding site prediction and confirmation by creating
mutants of NS5. The study will help in understanding the STAT2 degradation
mechanism of ZIKV in depth, in identifying potential drug targets, and provide
ways for development of new vaccines.

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KEYWORDS: Zika Virus,
interferon signalling, NS5, STAT2 degradation



Zika Virus (ZIKV)
belongs to the family of Flaviviridae
along with many other disease causing viruses like Dengue Virus (DENV),
Spondweni Virus (SPOV), Japanese Encephalitis Virus (JEV), West Nile Virus
(WNV) etc. which have a single stranded RNA genome (Schmitt et al., 2017). It is most closely
related to SPOV (Hamel et al., 2015).
It is vector-borne and two species of mosquito are known to carry it, Aedes aegypti and Aedes albopictus (Schmitt et
al., 2017). The virus was first isolated from a sentinel Rhesus monkey from
the Zika forest in Uganda in April, 1947 and it was known to cause sporadic,
self-limiting outbreaks in regions of Asia and Africa (Haddow et al., 2012). However, in 2007, a major
outbreak occurred on the Yap Island in Micronesia and it spread to nearby areas
of South Pacific (Duffy et al.,
2009). A more severe outbreak occurred later in 2014-2015 in Brazil involving
thousands of people and it spread to large regions of the Americas (Heukelbach et al., 2016; Aliota et al., 2017). According to the WHO, the
virus is now spread in about 70 countries of South Americas, Southeast Asia and
Pacific islands (Zika Situation Report, 13th October, 2016).

The threat of ZIKV
faced by India has been talked about in the light of other mosquito-borne viral
diseases like Dengue being widespread in the country (Mourya et al., 2016; Doss et al., 2017). In May, 2017, three laboratory confirmed cases of
ZIKV were reported in India (WHO, 2017).

Most cases of ZIKV are
asymptomatic; when it does lead to clinical manifestations, the commonly
reported symptoms are fever, maculopapular rash, arthalgia, fatigue, headache,
myalgia and conjunctivitis (Plourde & Bloch, 2016). During the outbreak in
Brazil, cases of Guillain-Barre syndrome associated with ZIKV infection were
reported (Oehler et al., 2014). An
increase in the incidence of microcephaly in new born infants was also noted in
ZIKV infected areas during the time (Faccini et al., 2016). It was later confirmed that ZIKV can indeed cross
the placental barrier (Noronha et al.,

The virus, like other
flaviviruses can inhibit Type I interferon (IFN) signalling in the host (Grant et al., 2016). Interferon signalling
involves activation of interferon receptors which leads to the phosphorylation
and activation of associated Janus activated kinases (JAK), which further
causes activation of the JAK-STAT (Signal Transducer and Activator of
Transcription) signalling pathway. The pathway finally concludes in the
activation of IFN stimulated genes (ISGs) which confer an antiviral state to the
host cell (Platanias, 2005; Grant et al.,
2016). Flaviviruses are known to employ a number of different strategies to
inhibit IFN signalling (Versteeg &Garcia-Sastre, 2010). It allows these
viruses to evade the innate immune system of the host. ZIKV employs a scheme
similar to Dengue virus wherein the NS5 (Non-structural protein 5) of the virus
binds to and degrades STAT2 (Ashour et al.,
2009; Grant et al., 2016). NS5 is
formed as part of a polyprotein which is translated from the positive-sense RNA
genome of ZIKV which is processed co- and post-translationally to give rise to
three structural and seven non-structural proteins (Bollati et al., 2010). NS5 also functions as the
RNA dependent RNA polymerase of the virus.

As mentioned previously,
ZIKV uses a mechanism similar to DENV to target STAT2 for degradation. DENV NS5
recruits UBR4 which is an E3 ubiquitin ligase to target STAT2 for proteasomal
degradation (Morrison et al., 2013).
Although ZIKV NS5 also mediates proteasomal degradation of STAT2, it does not
utilise UBR4 to achieve it (Grant et al.,
2016). The host proteins that could be involved in STAT2 degradation by ZIKV
are not known. Identifying these proteins would lead to a better understanding
of the mechanism. I propose to do this using pull down assays. The binding site
for STAT2 in NS5 is also not known. The site can be identified by using
truncations of NS5 and by 3D structure enumerations and prediction.

Hypothesis and Specific Aims

The degradation of
STAT2 by ZIKV NS5 requires the ubiquitin-proteasome system (Grant et al., 2016). Hence, it follows that
there should be an E3 ubiquitin ligase that is involved in the process. Evasion
of the host’s innate immune response by inhibiting interferon signalling is
necessary for extensive viral propagation (Munoz-Jordan & Fredericksen,
2010). This means that the E3 ubiquitin ligase involved in STAT2 degradation is
utilised by the virus and is necessary for its propagation. If identified, this
protein can serve as a potential drug target for ZIKV infections.

The NS5 protein of ZIKV
shares structural and functional similarity with that of DENV and other
flaviviruses (Sun et al., 2017; Best,
2016). Studies on the DENV NS5 have shown that the first 10 amino acids of NS5
are necessary for STAT2 binding (Ashour et
al., 2009). It will be interesting to investigate if the the N-terminal
region of the NS5 of ZIKV is involved in STAT2 binding. The specific amino acids
involved in binding can be utilised for creating ZIKV mutants which will be
deficient in inhibition of interferon signalling. These mutants could serve as
potential vaccines (Grant et al.,
2016). Also, different strains of ZIKV can be compared for their NS5 sequence
which may show differences in critical sites involved in binding. I intend to
address these questions through the following specific aims/objectives:

To identify host
protein/s involved in STAT2 degradation by NS5 of ZIKV.

To validate the function
of the identified host proteins and its importance for viral replication and

To identify the
region of NS5 involved in STAT2 binding by preliminary mapping.

Prediction of
the binding site and identification of specific amino acids in NS5 that are needed
to bind STAT2.

To determine if
the binding site is conserved across all strains of ZIKV.

Work plan

Methodology for the first objective

For the identification
of host protein/s involved in STAT2 degradation by ZIKV NS5, plasmid constructs
of ZIKV NS5 along with a Strep-tag will be generated. Also, plasmids for the
overexpression of STAT2 will be constructed. These plasmids will be transfected
into cultured 293T cells and the NS5 will be purified using strep-protein
interaction experiment i.e. SPINE (Herzberg et
al., 2007). Briefly, this method involves tagging of a bait protein with
Strep-tag; in this case, the bait is NS5. This is followed by formaldehyde
treatment to cross-link the interacting proteins and then the bait along with
its interacting partners is isolated using a sepharose column with Streptactin
as the ligand. Further, the column is washed a few times with a buffer followed
by elution of the proteins. The eluted proteins are run on SDS-PAGE after
heating with Laemmli buffer to break the cross-links. The protein band/s
obtained, other than those of NS5 and STAT2 will be identified by mass

Generation of plasmid

The NS5 sequence of the
French Polynesian ZIKV isolate with GenBank genome sequence KJ776791.1 will be
cloned into the plasmid pSF-Puro-COOH-TEV-Strep (Sigma-Aldrich) in order to
obtain its expression along with a C-terminal Strep-tag. Human STAT2 sequence
will be cloned into pSF-Puro-COOH-FLAG (Sigma-Aldrich) to express it along with
a C-terminal FLAG tag.

     TEV site: Tobacco Etch Virus
protease site


Transfection of
plasmids into cells and protein purification

The plasmid constructs
will be transfected into cultured 293T cells. The cells shall be lysed for
protein purification 48 hours after transfection. Prior to lysis, formaldehyde
treatment will be given to allow cross-linking. The NS5 protein purification
using SPINE will be done both in the presence and absence of overexpressed
STAT2-FLAG (Morrison et al., 2013).

Identification of
interacting partners of NS5

After trapping of the
Strep-tagged NS5 on the streptactin-sepharose column, the column will be washed
with buffer W (Herzberg et al., 2007)
and the protein will be released by treatment with Tobacco Etch Virus (TEV)
protease. This will separate the NS5 protein and its interacting partners from
the Strep-tag. The released proteins will be further processed for SDS-PAGE.
Any bands other than those of NS5 and STAT2 (assessed according to the
molecular weight) will be analysed by mass spectroscopy (Shevchenko et al., 1996).

Methodology for the second objective

Confirmation of the
function of the identified protein/s

Once a host protein/s
is identified, its function will be confirmed by ascertaining its requirement
for STAT2 degradation. This will be done by mutating the protein or knocking
down its expression using a shRNA, and then checking for the degradation of
STAT2 in 293T cells. Degradation will be checked for by using immune-blot with
anti-STAT2 antibody.

Role of the protein in
viral replication

To determine if the
protein is important for viral replication, Vero cell line (derived from African
green monkey kidney cells) will be used in which the protein will be mutated or
knocked down using a shRNA. The cells will be then be assessed for their
susceptibility to ZIKV infection. The viral replication within the cells will
be monitored by calculating the titre using qRT-PCR for the viral genome.

Methodology for the third objective

Preliminary mapping of
the region of NS5 involved in STAT2 binding will be done by using N-terminal
truncations of NS5. To express truncated protein, the appropriate truncated
gene sequence of NS5 will be cloned into pSF-Puro-COOH-FLAG and the plasmid
will be transfected into 293T cells. The NS5 truncations that will be generated
are: 1-10, 1-40, 1-100, 1-250, and 1-350; the numbers indicate the amino acid
residues. The truncated protein will be expressed with a C-terminal FLAG tag
and will be immunoprecipitated using anti-FLAG antibody. The precipitated
fraction will be immuno blotted and checked for STAT2 using anti-STAT2
antibodies. Anti-STAT1 antibody will be used as negative control. Whole cell
extracts will also be immune blotted using the two antibodies. GAPDH shall be
used as control in all Western blots.

Methodology for the fourth objective

Prediction of binding

The binding site in NS5
for STAT2 will be predicted using a method developed by Guo et al. (Guo et al., 2012). Briefly, this method computes all the possible 3D
configurations between two proteins and each configuration is scored using
Atomic Contact Energy function. The configurations with the lowest scores are
more likely and the corresponding interacting residues are the predicted
binding sites.

Identification of
specific amino acids involved in binding

Based on the predicted
binding sites, the residues involved will be specifically mutated. The NS5
sequence cloned into pSF-Puro-COOH-FLAG will be mutated using the protocol of
QuikChange site-directed mutagenesis by Stratagene (Khasawneh et al., 2006). The mutant proteins will
then be analysed for their ability to bind and degrade STAT2 by previously
described methods.

Methodology for the fifth objective

Bioinformatic analysis

To check for the
conservation of the binding site residues across all ZIKV strains, multiple
sequence alignment (MSA) of the NS5 sequences from different strains will be
done. The various ZIKV strains that will be used for this analysis are:

Lineage of the strain

GenBank sequence ID

Place and year of isolation



Polynesia, 2013



Malaysia, 2012



Canada, 2013






Cambodia, 2010






Uganda, 1947



Nigeria, 1968



Senegal, 1968



Senegal, 2001



Senegal, 2001



Senegal, 1984


Potential problems/limitations

If NS5 purification
using SPINE yields too many bands due to non-specific protein binding,
alternative method for purification like tandem affinity purification (TAP) tag
will be used. TAP identifies more specific binding than SPINE (Berggard et al., 2007). If the problem persists,
then a database of abundant proteins that are usually found as contaminants
will be used and the presence of these proteins will be checked for in the TAP
purified extract (Schirle et al.,
2003). In case of NS5 truncations, if expected results are not obtained, then
C-terminal truncations will be generated and their function shall be assessed.
The binding site prediction should provide cues for this.

Expected outcome

I anticipate that one
or more host proteins associated with NS5-STAT2 will be found. Their function
will need to be validated by mutation or knock down of expression. Once
confirmed, the mechanism of STAT2 degradation will become clearer and also
potential new drug targets will be identified. Elucidation of the binding site
for STAT2 on NS5 will provide an insight into the functioning of NS5 as it also
serves as the RNA dependent RNA polymerase, needed for viral replication. I
expect that the N-terminal region of NS5 will be involved in the binding like
the NS5 of DENV. Determining exactly which amino acids are involved in the
binding will provide a way of developing attenuated ZIKV by mutating these
residues. Such attenuated viruses will be deficient only in interferon
signalling and the function of NS5 in replication will not be compromised, so
such viruses can serve as potential vaccines. By comparing the NS5 sequences
for conservation of the STAT2 binding site across all ZIKV strains will give
information about the importance of the mechanism for viral propagation. If differences
in the NS5 sequence especially in the binding site are found, further
investigation into their different binding potential to STAT2 can be done.

Projection of progress

Work to be

Year 1

Year 2

Year 3

and transfection of plasmid constructs


purification by SPINE and identification by mass spectroscopy

of the function of the protein/s and importance for viral replication

of truncations of NS5 and validation of their function


Prediction of
binding site, creation of mutants of binding site residues and validation of
their function



Milestones for each

mapping of the region of NS5 involved in STAT2 binding.

of host proteins bound to NS5-STAT2.

Confirmation of
the function of the bound proteins; elucidation of the binding site for STAT2
in NS5. 


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