nucleocytoplasmic shuttling of the west nile virus rna

Wagstaff Kylie (Orcid ID: 0000-0003-4791-1410) Mackenzie Jason (Orcid ID: 0000-0001-6613-8350)

1Department of Microbiology and Immunology, University of Melbourne, at the Peter

Doherty Institute for Infection and Immunity, 2Department of Physiology, Anatomy &

Microbiology, La Trobe University, 3Faculty of Science, Biotechnology and Biomolecular

Sciences, University of New South Wales, Sydney, New South Wales, Australia and

4Department of Biochemistry, Monash University, Melbourne, Victoria, Australia.

Nucleocytoplasmic shuttling of the West Nile virus RNA-dependent RNA polymerase

NS5 is critical to infection

Adam J. Lopez-Denman1,2, Alice Russo3, Kylie M. Wagstaff4, Peter A. White3, David A.

Jans4 and Jason M. Mackenzie1,*

*Corresponding author: Ph. (+613) 9035 8376

email: [email protected]

Keywords: West Nile Virus, Nuclear import, nuclear export, nucleus, RNA-dependent RNA

polymerase, NS5

Running Title: Nucleocytoplasmic shuttling of WNVKUN NS5

This article is protected by copyright. All rights reserved.

This is the author manuscript accepted for publication and has undergone full peer review buthas not been through the copyediting, typesetting, pagination and proofreading process, whichmay lead to differences between this version and the Version of Record. Please cite this articleas doi: 10.1111/cmi.12848

http://dx.doi.org/10.1111/cmi.12848

http://dx.doi.org/10.1111/cmi.12848

http://crossmark.crossref.org/dialog/?doi=10.1111%2Fcmi.12848&domain=pdf&date_stamp=2018-04-24

2

Summary

West Nile Virus (WNV) is a single stranded, positive sense RNA virus of the family

Flaviviridae, and is a significant pathogen of global medical importance. Flavivirus

replication is known to be exclusively cytoplasmic, but we show here for the first time that

access to the nucleus of the WNV strain Kunjin (WNVKUN) RNA-dependent RNA

polymerase (protein NS5) is central to WNVKUN virus production. We show that treatment of

cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5

nuclear accumulation in WNVKUN-infected cells and NS5 transfected cells, indicative of

nucleocytoplasmic shuttling under normal conditions. We used site-directed mutagenesis to

identify the nuclear localisation sequence (NLS) responsible for WNVKUN NS5 nuclear

targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic

accumulation of NS5 even in the presence of LMB. Introduction of NS5 NLS mutations into

FLSDX, an infectious clone of WNVKUN, resulted in lethality, suggesting that the ability of

NS5 to traffic into the nucleus in integral to WNVKUN replication. This study thus shows for

the first time that NLS-dependent trafficking into the nucleus during infection of WNVKUN

NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import

reduce WNVKUN virus production, proving the principle that inhibition of WNVKUN NS5

nuclear import is a viable therapeutic avenue for antiviral drug development in the future.

Introduction


3

West Nile virus (WNV) is a single stranded positive sense RNA virus belonging to

the family Flaviviridae, and is closely related to other highly pathogenic viruses including;

dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and Zika

virus (ZIKV). WNV is transmitted to humans through a bird-mosquito-bird enzootic cycle.

Once infected most people are asymptomatic, however the infection can progress into febrile

illness with symptoms including; headaches, body/joint pains, vomiting, diarrhoea, or rash.

Complications are generally rare, however infection can further develop into significant

neurological illnesses including encephalitis, paralysis, and meningitis if the virus crosses the

blood brain barrier (Hayes et al., 2005).

WNV encodes for 10 genes that are co- and post translationally modified into 10

proteins required for viral replication, and immune system perturbation. Non-structural

protein 5 (NS5) is the largest viral protein, crucial for viral replication -containing both the

N-terminal Methyl transferase, and the C-terminal RNA-dependent RNA polymerase (RdRp)

domains (Grun & Brinton, 1987; Zhou et al., 2007). During infection, WNV replication is

associated with a cytoplasmic subcellular localisation, that is confined within intracellular

membranous structures, advantageous for efficient replication, and avoiding immune

detection (Gillespie, Hoenen, Morgan, & Mackenzie, 2010; Hoenen, Liu, Kochs, Khromykh,

& Mackenzie, 2007; Westaway, Mackenzie, Kenney, Jones, & Khromykh, 1997). Contrary

to this, during YFV, JEV, DENV, and more recently ZIKV infection, a significant proportion

of NS5 is observed within the nucleus, potentially contributing to replication and immune

perturbation (Buckley, Gaidamovich, Turchinskaya, & Gould, 1992; Grant et al., 2016; Pryor

et al., 2007; Uchil, Kumar, & Satchidanandam, 2006).


4

The nuclear localisation of DENV NS5 during infection has been demonstrated to be

a significant requisite for replication, as inhibition of nuclear import results in a severe

decrease in viral titres (Wagstaff, Sivakumaran, Heaton, Harrich, & Jans, 2012). This

suggests that while viral replication has been well documented to occur within the cytoplasm,

there is some functional need for NS5 to also gain access to the nucleus. Specific residues of

a putative NLS within DENV NS5 have also been mutated to interrogate the nuclear

trafficking functionality of NS5, identifying that import is mediated via the Importin

superfamily of proteins (Pryor et al., 2007). Importin proteins facilitate the highly regulated,

essential process of nucleo-cytoplasmic translocation for efficient transcription and

translation, and transport occurs via a large proteinaceous structure known as the nuclear pore

complex (NPC) (Davis & Blobel, 1986; Weis, 1998). However, a clear function of DENV

NS5 within the nucleus is not yet fully understood, but may have roles in inhibiting the host

antiviral response, in particular by modulating interleukin-8 levels (Rawlinson, Pryor,

Wright, & Jans, 2009), or interferon (De Maio et al., 2016) .

Like other flaviviruses, WNV replication occurs within the cytoplasm of infected

cells, (Gillespie et al., 2010; Westaway, Mackenzie, et al., 1997) and up to now the cellular

localisation of NS5 has previously observed to be at sites of cytoplasmic replication

(Mackenzie, Kenney, & Westaway, 2007). In this study, we have shown for the first time that

WNV strain Kunjin (WNVKUN) NS5 has the ability to access the nucleus. With the use of

small chemical inhibitors we have been able to visualise for the first time, an accumulation of

NS5 within the nucleus, and by blocking this localisation, have observed a decrease in viral

replication fitness and virus production. Through site-directed mutagenesis, we have


5

interrogated a putative bi-partite NLS found within NS5, identifying the crucial sequence

required for nuclear localisation. We have combined the inhibition and mutational

experiments with live cell microscopy to kinetically visualise these events in real time. Our

results indicate that WNVKUN NS5 has an integral role within the nucleus, and that inhibiting

its entry is severely detrimental to viral replication.

Experimental procedures

Cells & Virus stock

Vero C1008 cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium

(DMEM; Life Technologies) with the addition of 10% v/v Foetal Calf Serum (FCS; Thermo

Fisher) and 200 mM Glutamax (Glx; Gibco), termed DMEM complete. Cells were grown at

37ºC with 5% CO2. RepBHK cells were maintained in Dulbecco’s Modified Eagle’s Medium

(DMEM; Life Technologies) with the addition of 10% v/v Foetal Calf Serum (FCS; Thermo

Fisher) and 200 mM Glutamax (Glx; Gibco). 0.5 mg G418 (Geneticin) was also added as

selection to maintain persistent expression of WNVKUN replicon, as previously described

(Khromykh et al., 1999). RepBHK cells were then used as per other cell types.

WNVKUN MRM61C stocks were propagated and maintained in Vero cells

supplemented with 0.2% (w/v) BSA in DMEM at 37ºC 36 hours at which time the virus

containing supernatant was collected, centrifuged at 5,000 rpm for 5 mins to remove cell

debris, aliquoted and stored at -80ºC.


6

Plaque Assay

Vero cells were seeded in DMEM complete medium in six-well plates, followed by

incubation at 37°C overnight. The virus stock was diluted 10-fold in 0.2% BSA-DMEM,

used to infect previously seeded vero cells, in duplicate, 37°C, shaking for 60 min. Following

incubation, a semi solid overlay (DMEM, 2.5% v/v FCS, 0.9 M NaHCO3, Penicillin

G/streptomycin, Glx, 1 M HEPES (Thermo Fischer) and 0.35% w/v low-melting-point

agarose was added on top of the cells, and solidified at 4ºC for 30 min. Cells were incubated

at 37ºC for 72 hours, fixed with 10% formalin solution (Sigma-Aldrich) for 1 hr at RT, and

stained in 0.1% toluene blue (Sigma-Aldrich) at RT for 1 h. Plaques were manually counted,

and plaque forming units per mL (pfu/mL) were determined.

Transfection

Vero cells were seeded in DMEM complete onto methanol-sterilised 25 mm glass

coverslips (Menzel Glaser) and left to proliferate overnight. Cells were then transfected with

Lipofectamine 2000 (as per manufacturer’s instructions; Life Technologies) and plasmid

DNA. 4 hours post transfection (h.p.t), media was removed and replaced with 2% FCS in

DMEM. 24 h.p.t cells were fixed with 4% paraformaldehyde (PFA; Sigma Aldrich) in PBS.

Infection and drug treatment protocol

LMB treatment: Vero cells were seeded in 6-, 12-, and 24 well plates with 2%

FCS/DMEM. Cells were mock treated with the solvent vehicle Ethanol, or treated with 10

nM LMB (Cell signaling) in 2%FCS/DMEM for 2 h before infection. Media was replaced


7

with DMEM complete and cells infected with WNVKUN at MOI of 5 in the presence of the

compounds for a further 2 hours. Virus/drug inoculums were removed and fresh DMEM

containing 2% FCS was added. Culture medium was collected 24 hours post infection (h.p.i).

Gossypol, 4-HPR & 4-MPR Treatment: Vero cells were seeded in 6-, 12- or 24 well

plates with 2%FCS/DMEM. Cells were mock treated with the solvent vehicle Dimethyl

sulfoxide (DMSO), or treated with 10 mM of either chemical in 2% FCS/DMEM for 2 h

before infection. Media was replaced with DMEM complete and cells infected with WNVKUN

at MOI of 5 in the presence of the compounds for a further 2 hours. Virus/drug inoculums

were removed and fresh DMEM containing 2% FCS was added. Culture medium was

collected 24 h.p.i.

Antibodies

Mouse anti-flavivirus-NS1 (clone 4G4) and mouse anti-WNV-NS5 (clone 5H1.1)

monoclonal antibodies (Hall et al., 2009, Macdonald et al., 2005) were generously provided

by Roy Hall (University of Queensland). Rabbit anti-KPNA antibody was purchased from

BD antibodies. Mouse anti-GAPDH was purchased from Cell Signaling Technology. Anti-

rabbit and anti-mouse specific IgG-Alexa Fluor 488, 597 & 647 were purchased from

Molecular probes (Invitrogen).

Immunofluorescence analysis

Cells were fixed with 4% w/v paraformaldehyde (PFA) in PBS at RT for 10 min, then

permeabilised (in 4%PFA/PBS) with 0.1% w/v TritonX-100 at RT for 10 min. PFA was then


8

quenched with 0.2 M Glycine at RT for 10 min. Following washes with PBS, cells were

incubated with primary antibodies in 1% BSA/PBS for 1 hr at RT. Cells were then washed

twice in 0.2% BSA/PBS, then incubated with species specific secondary antibodies,

conjugated to either Alexa Fluor 647, 594, or 488 in 1% BSA/PBS at RT for 45 min. Cells

were then washed in 0.2% BSA/PBS, then incubated for 5 min with 4 ug/ml DAPI to stain

the nucleui. Coverslips were mounted with Ultramount no 3 (Fronine). Immunofluorescent

staining was visualized on Zeiss confocal laser scanning microscope (LSM 710) and Images

were collected and collated using Zen, Adobe Photoshop software, & Fiji Image analysis

software.

Time lapse Live cell confocal microscopy

Vero cell monolayers on µ-Dish 35mm high (Ibidi) were transfected with plasmid DNA

with Lipofectamine 2000 (as above), and samples were left for 24 hours before imaging on

Zeiss confocal laser scanning microscope (LSM700) at 37ºC/5%CO2. Cells were

subsequently left untreated, or treated with 10 nM LMB directly before being imaged. Images

were collected and collated by using Zeiss Zen, Adobe Photoshop software, and Fiji image

analysis software 2.0.0

Fluorescence recovery after photobleaching (FRAP) live cell confocal microscopy

Vero cell monolayers on 35mm Fluorodish (World precision instruments) were

transfected with plasmid DNA with Lipofectamine 2000 (as above). 12 h.p.t, media was

replaced with DMEM complete + 20 mM HEPES, and 10nM LMB. 12 h.p.t sample was


9

mounted onto a Nikon Andor WD spinning disk confocal microscope for imaging. The

nucleus was photo-bleached with 100% 488nm laser power for 5 pulses with 20 us dwell

time. Images were collected and collated using Metamorph (Molecular devises), Adobe

photoshop software (Adobe), and Fiji image analysis software 2.0.0]

Image Analysis

Images obtained by CLSM were analysed using Fiji 2.0.0 image analysis software.

TIFF images were imported and mean pixel values obtained. Laser power levels were kept

constant between sample acquisitions. Quantitation of the mean nuclear fluorescence (Fn) in

relation to that of the cytoplasm (Fc), referred to as the Fn/c was determined according to:

Fn/C = (Fn-Fb)/(Fc-Fb), where Fb is background fluorescence (Hu & Jans, 1999; Hübner,

Eam, Hübner, & Jans, 2006)

Western Blotting

WNVKUN infected Vero cells were lysed with radio immunoprecipitation assay buffer

(RIPA 150 mM sodium chloride, 1.0% NP 40, 0.5% sodium deoxycholate, 0.1% SDS, 50

mM Tris, pH 8.0) lysis buffer, containing protease inhibitor cocktail III (Astral Scientific).

Lysates were loaded onto a 10% Tris-glycine polyacrylamide gel and separated at 100 V for

2 hours. Separated proteins were then transferred to Nitrocellulose membrane (GE

Healthcare) at 100 V for 75 min and blocked at RT with 5% skim milk/TBS (Diploma) with

0.05% Tween (TBS-T). After blocking, membranes were washed for 15 min in TBS-T, then

incubated overnight at 4ºC with primary antibodies diluted in 5%BSA/TBS-T. Membranes


10

were then washed three times in TBS-T then incubated with secondary antibodies conjugated

to either Alexa Fluor 488 or 647 at RT for 2 h protected from light. After two washes in TBS-

T, and one in TBS, membranes were viewed on the PharosFX fluorescent scanner (Biorad).

Site-directed Mutagenesis of WNVKUN NS5 protein putative Nuclear localisation sequence

Alanine mutations were generated in the WNVKUN cDNA infectious clone FLSDX,

and a pcDNA3.1 NS5-GFP fusion protein via site directed mutagenesis (primers available on

request) using Q5 hotstart high fidelity polymerase (NEB). Clones were then isolated and

transformed into DH5-alpha chemically competent E. coli, and DNA isolated using

PureYield midiprep kit (Promega) as per manufactures descriptions. FLSDX-NS5-GFP

plasmid creation:

GFP was amplified from pre-existing plasmid (pcDNA3.1-GFP; Primers available on

request). FLSDX-NS5-GFP was created by insertion of GFP in-frame into the Sma1

restriction endonuclease site at the C- end of NS5 within the FLSDX WNVKUN infectious

clone. Ligation reactions were constructed with T4 DNA ligase (Promega) and transformed

into Chemically competent JM109 E. coli cells. Plasmid DNA was extracted as previously

described, and construct validated through sanger sequencing.

In-vitro RNA transcription & RNA electroporation

FLSDX clones (including FLSDX, FLSDX-NS5(KYmut), FLSDX-NS5(REKmut) and

FLSDX-NS5-GFP) were linearised by XhoI treatment, and DNA purified using

Phenol:Chloroform extraction. RNA was transcribed from 0.5 ug of each linearised clone


11

using an SP6 RNA polymerase kit (Ambion) and electroporated into Vero or repBHK cells

using the Neon electroporation system (Life tech) (2x 1150 V pulses, 20 ms pulse with

infinite resistance) with 100 ul tips. Cells were recovered in growth medium (2% FCS, 200

mM Glutamax) at room temperature for 5 mins then plated out into 6 or 24 well plates. Cell

lysates and tissue culture fluid were collected 24, 48, & 72 h post electroporation (h.p.e).

RNA-dependent RNA polymerase activity assay

NS5 and NS5(REKmut) constructs were created in pET24b to incorporate a

hexahistidine affinity tag at the 5’ end with NEBuilder HiFi DNA assembly cloning kit

(NEB). Protein expression was performed in T7 Express competent E. coli (NEB), after

induction with 0.5 mM IPTG at 37°C. RdRp purification was performed by lysing (50 mM

Tris-HCl pH 7.5, 300 mM NaCl, 0.1% Triton™-X 100, 2 mM β-mercaptoethanol) E. coli,

and clearing on HiTRAP TALON crude column (GE Healthcare). Eluted fractions were

concentrated with Amicon Ultra-Centrifugal filter (Merck). Protein concentration was

determined using a Pierce® BCA Protein Assay Kit (Pierce Biotechnology).

WNVKUN NS5 and REKmut RdRp activity was determined through fluorescence based

assay as described (Eltahla, Lackovic, Marquis, Eden, & White, 2013).

Results

WNVKUN NS5 localises to the nucleus and cytoplasm during infection

NS5 from DENV1-4 has been reported to show nucleocytoplasmic trafficking in

infected cells (Tay et al., 2013), with virus infection being reduced by inhibitors of nuclear


12

import such as 4-HPR (Fraser et al., 2014). Our previous observations indicated that 4-HPR

also inhibited WNV replication, therefore we examined the ability of the WNVKUN NS5

protein to traffic to the nucleus. Initially, we examined the effect of the small molecular

inhibitor of nuclear export Leptomycin B (LMB) on localisation of the WNVKUN proteins

NS3 and NS5 during infection (Fig.1). In untreated, infected cells WNVKUN NS3 and NS5

were predominantly cytoplasmic as previously observed (Mackenzie et al., 2007; Westaway,

Mackenzie, et al., 1997) (Mackenzie et al., 2007, Westaway et al., 1997). NS3 displayed a

primarily perinuclear staining pattern with some smaller foci also observed (Fig.1 i). NS5

was diffusely cytoplasmic, although some faint nuclear localisation was also observed (Fig. 1

ii). Upon 10nM LMB treatment, nuclear accumulation of NS5, but not NS3, was clearly

evident (Figs 1 iv-vi). These results indicate that WNVKUN NS5, in contrast to NS3, cycles

continually between nucleus and cytoplasm under normal conditions. These results also

clearly demonstrate that NS3 does not translocate to the nucleus as no accumulation was

observed even in the presence of the nuclear export inhibitor LMB.

To determine the mechanism of nuclear trafficking, we probed WNVKUN localisation

with the additional inhibitors of nuclear import 4-HPR, shown to block DENV NS5:Importin

interactions (Fraser et al., 2014), and gossypol (GSP). GSP is a small molecule nuclear

import inhibitor we identified to be specific for the nuclear import protein importin

(Wagstaff, Rawlinson, Hearps, & Jans, 2011) (manuscript in preparation). Thus, Vero cells

were mock treated or pre-treated 2 h prior to infection with nuclear transport inhibitors then

subsequently infected with WNVKUN, at 24 h.p.i. cells were fixed for IFA, and dual labelled

with anti-NS5 and anti-Karyopherin alpha (KPNA) antibodies (Fig. 2A). In untreated and


13

infected cells (Fig. 2A i-iv), NS5 labelling was observed primarily within the cytoplasm, with

a faint signal within the nucleus. Upon treatment with LMB (Figs. 2A v-vii), NS5 staining

was also observed within the nucleus, reaffirming that WNVKUN NS5 can access to the

nucleus. Pre-treatment of infected cells with the nuclear import inhibitors 4-HPR (Fig. 2A ix-

xii) or Gossypol (Fig. 2A xvii-xx), revealed NS5 labelling of the cytoplasm alone, indicating

that nuclear import was impeded. Cells were additionally treated separately with 4-MPR (Fig.

2A xiii-xvi), a closely related analogue of 4-HPR, which has been shown not disrupt nuclear

localisation (Fraser et al., 2014) and the NS5 staining was similar to that of the untreated

samples.

Quantification of nuclear fluorescence intensity was performed to determine the

proportion of nuclear NS5 throughout treatments (Fig. 2B). Mean fluorescence intensity of

the nucleus (Fn), and the cytoplasm (Fc) was performed and ratio of nuclear to cytoplasmic

intensity (Fn/c) determined. NS5 nuclear fluorescence is observed to be low during infection

of untreated samples, and levels remains similar or decrease with treatment of nuclear import

inhibitors. Upon treatment with LMB, an increased level of nuclear NS5 was detected. These

results would indicate that nuclear import of WNVKUN NS5 is mediated via the cellular

importin protein; a process that can be prevented with available import inhibitors.

WNVKUN NS5 protein production and viral secretion decreases when nuclear import is

inhibited

To elucidate the contribution of nuclear-cytoplasmic shuttling during WNVKUN

replication, we treated cells with the respective nuclear import and export inhibitors (4-HPR,


14

4-MPR, GSP and LMB; respectively) and cell lysates and supernatants were collected for

analyses (Fig. 3). Vero cells were treated with the inhibitors for 2 hrs and subsequently

infected with WNVKUN for an additional 24 hrs. Our western blot analyses revealed that pre-

treatment of the WNVKUN-infected cells with the nuclear import inhibitors (GSP and 4-HPR)

resulted in a significant decrease in detectable NS5 when compared to the vehicle controls

(Ethanol and DMSO) and cells treated with 4-MPR and LMB (Figs 3A). This reduction in

viral protein production was also reflected in a significant reduction in the production of

infectious WNVKUN particles upon pre-treatment with GSP and 4-HPR (Fig. 3B).

Surprisingly no significant changes in WNVKUN replication were observed during inhibition

of nuclear export, indicating that NS5 must enter the nucleus for efficient replication,

however does not necessarily need to leave it.

Overall these observations further support the critical role of nuclear import of the

WNVKUN NS5 for efficient virus replication. This also supports previous published reports,

highlighting the role for nuclear involvement of NS5 in facilitating efficient flavivirus

replication (Pryor et al., 2007).

Mutation of a putative nuclear localisation sequence within the WNVKUN NS5 linker

region reduces nuclear accumulation of an NS5-GFP fusion protein in transfected cells.

Previous reports have identified a bipartite NLS in the DENV NS5 protein and our

gene comparison analyses revealed the presence of a similar motif within the WNVKUN NS5

protein (Fig. 4A)(Pryor et al., 2007). Two segments of the putative WNVKUN NLS were

separately mutated via site-directed mutagenesis and incorporated into the recombinant NS5-


15

GFP cDNA expression plasmids. Amino acids Lys-Tyr (aa 373-374 - termed KYmut) were

substituted to Ala-Ala and Arg-Glu-Lys (aa 389-391 - termed REKmut) to Ala-Ala-Ala (Fig.

4A). IF analysis of the expressed mutant NS5(KYmut)-GFP protein showed NS5 both

cytoplasmic and nuclear staining (Fig 4B g-i), similar to that of the wild-type NS5-GFP (Fig

4B i-iii). Conversely, the expressed mutant NS5(REKmut)-GFP protein displayed a total

abolition of nuclear fluorescence (Fig. 4B xiii-xv). Transfected cells were also treated with

LMB to promote accumulation of NS5 within the nucleus, and to discount any rapid shuttling

of the mutant NS5 protein out of the nucleus. We observed that the NS5(KYmut)-GFP protein

was distributed similarly to wild-type NS5-GFP protein (compare panels 4B iv-vi and x-xii),

with an increase in nuclear fluorescence after treatment. Conversely, the NS5(REKmut)-GFP

protein was confined to the cytoplasm and displayed no nuclear staining of NS5 even in the

presence of this nuclear export inhibitor (Fig 4B xvi-xviii). Quantitation of the extent of

nuclear localisation revealed that NS5(REKmut)-GFP was abolished in its ability to

translocate to the nucleus, even in the presence of LMB (Fig. 4C).

These observations further demonstrate compelling evidence that WNVKUN NS5 is

actively transported into the nucleus via the use of an active NLS located at Arg-Glu-Lys (aa

389-391).

WNVKUN NS5 protein displays a kinetic nuclear translocation

To further analyse NS5 protein kinetics after transfection, cDNA recombinant

plasmids encoding NS5-GFP and NS5(REKmut)-GFP fusion proteins were transfected into

Vero cells, treated with 10 nM LMB 12 h.p.t, then live cells were imaged for 600 minutes at


16

10 minute time intervals (Fig 5A, Supplementary Movies 1 to 4). We observed that the

untreated NS5-GFP cellular distribution in live cells was analogous to that of the fixed

samples, i.e. primarily cytoplasmic with minimal nuclear signal. However, after treatment

with LMB, nuclear accumulation of NS5 can be observed as early as 380 minutes, with a

significant increase by 600. Our nuclear fluorescence quantification clearly shows

accumulation of NS5-GFP in the presence of LMB but minimal accumulation for

NS5(REKmut)-GFP either in the presence and absence of LMB (Fig 5B).

WNVKUN NS5 repopulates the nucleus after photo-bleaching when treated with LMB

To gain further insight into NS5 nuclear trafficking kinetics, FRAP live cell

microscopy was utilised (Fig. 6, Supplementary movie 5). NS5-GFP transfected cells were

treated with LMB to accumulate NS5 within the nucleus. The nucleus was then photo-

bleached and images taken at 5 min intervals. Within 40 minutes, fluorescence repopulation

of the nucleus was observed, and nuclear fluorescence returned to pre-bleach state by 120

minutes (Fig. 6A). Image analysis was also performed to quantify the rate of nuclear

fluorescence recovery (Fig. 6B). This recovery suggests that total NS5 protein pool may

access the nucleus and cycles between the nucleus and cytoplasm. However, we do

acknowledge that these experiments utilise overproduced NS5-GFP protein examined in

isolation (i.e. outside of the context of infection).

NS5 NLS mutations in the WNVKUN full length infectious clone FLSDX inhibit viral

replication


17

To interrogate the role of the NS5 NLS during virus replication, NLS mutations were

introduced into the WNVKUN cDNA infectious clone, FLSDX (A. A. Khromykh, Kenney, &

Westaway, 1998). Vero cells were electroporated with in-vitro transcribed RNA, and samples

collected 24, 48 & 72 h.p.e. IF analysis for the WNVKUN viral protein NS1 revealed that

FLSDX and FLSDX-KYmut were observed to replicate throughout all time points (72 h.p.e

shown; Figs. 7A i, ii, iv, and v). This is in stark contrast to FLSDX-REKmut, where

replication was not observed at any time point over the course of the experiments (Fig. 7A iii

and vi). Western blot analyses revealed that by 48 h.p.e, a clear signal for both WNVKUN NS1

and NS5 was visible in the FLSDX and FLSDX-KYmut electroporated samples (Figs. 7B),

whilst no protein production was evident for FLSDX-REKmut (Figs. 7B). These observations

correlated with a steady increase in viral particle secretion in the FLSDX and FLSDX-KYmut

electroporated samples, whilst no detectable virus could be observed from the FLSDX-

REKmut electroporated cells (Fig 7C). Intriguingly, we were unable to efficiently rescue virus

replication via complementation of WT NS5 in a BHK replicon cell line by 72 hours (Egloff,

Benarroch, Selisko, Romette, & Canard, 2002; Khromykh et al., 1998; Khromykh, Sedlak, &

Westaway, 1999) (Supplementary figure 1).

Interestingly, this difficulty in restoring NS5 function is a feature also noted for NLS

mutations in DENV (Kumar et al., 2013). It came to consideration that the inclusion of this

mutation may be affecting NS5 RNA polymerase functionality. To this end we produced two

NS5 constructs (WT NS5 and NS5-REKMUT) to analyse RNA polymerase activity (Fig 7D).

On comparing both the WT and mutant NS5, no significant difference was observed between


18

the samples, indicating that the introduced NLS mutation did not yield a possible RNA

polymerase dysfunction responsible for the lethal phenotype.

Overall, these results indicate that the import of WNVKUN NS5 protein into the

nucleus is a critical requirement for virus replication. Our observations have additionally

shown that NS5 protein nuclear import is facilitated by a conserved functional NLS at amino

acids 389Arg-Glu-Lys391 within the WNVKUN NS5 protein.

Discussion

Flavivirus replication has been extensively studied, and is associated with significant re-

assortment of the intracellular membrane architecture within the cytoplasm (Gillespie et al.,

2010; Leary & Blair, 1980; Mackenzie, 2005; Westaway, Mackenzie, & Khromykh, 2002).

However, recently it has also been documented that select flavivirus proteins can also cross the

nuclear pore during infection (Bulich & Aaskov, 1992; Lopez-Denman & Mackenzie, 2017;

Mori et al., 2005; Westaway, Khromykh, Kenney, Mackenzie, & Jones, 1997). Whilst DENV,

JEV and YFV NS5 protein nuclear localisation has been documented during infection, the

same observations have not previously been corroborated with WNVKUN (Buckley et al., 1992;

Edward & Takegami, 1993; Forwood et al., 1999; Mackenzie et al., 2007). In this study, we

utilised a variety of techniques, including chemical inhibitors, mutagenesis studies and real

time via live cell microscopy (Figs 1-7), to enable the first time visualisation of WNVKUN NS5

nuclear localisation. Additionally, we have identified a conserved NLS within NS5 (Fig 4), and

mutation of this motif within a recombinant NS5 plasmid and the WNVKUN infectious clone

impaired nuclear localisation and was lethal to virus replication, respectively (Fig 7). Overall,


19

our results support and extend the crucial role the nucleus plays during flavivirus replication

and indicates that nuclear import of NS5 (and perhaps other factors) is critical for efficient

virus replication, although intriguingly their export from the nucleus is not.

The most important observation from these studies is that inhibition of nuclear import,

via either chemical inhibition or via mutagenesis of the NS5 NLS, severely impacted on

WNVKUN replication, and that mutation of the identified conserved NLS within NS5 is in fact

lethal to the virus. This suggests that the, yet unidentified, role of NS5 within the nucleus is

an absolute requirement for WNVKUN and presumably other flaviviruses. Of course we

cannot discount the potential role of WNVKUN capsid protein within the nucleus (Westaway,

Khromykh, et al., 1997), as we did not investigate capsid in this study. However, our

mutagenesis studies would indicate a direct effect by NS5 alone. We believe the nuclear

import of NS5 is not directly affecting WNVKUN RNA replication, as (i) dsRNA is not

observed within the nucleus of infected cells and (ii) NS5 within the nucleus does not need to

be exported back out to enable virus replication (Fig. 3). This would intimate that the pool of

NS5 that enters the nucleus is not required for viral replication and that NS5 associated with

the RC most likely remains resident and immobile within this viral compartment. We have

previously shown that many of the viral proteins are recycled during genome amplification

(Westaway, Khromykh, & Mackenzie, 1999), and earlier seminal studies have indicated that

only a very small faction of the NS5 pool is utilised during viral RNA replication (Chu &

Westaway, 1987). Thus, we would postulate that NS5 is affecting some cellular function to

promote replication, potentially effects on cellular transcription.


20

However, we do note that we have only confirmed that the NLS mutation does not

affect NS5 RdRp activity and cannot conclusively discount other additional functions of NS5

that could be induced via mutation in this area. Additionally, we acknowledge that the

transport inhibitors may have off-target effects and their influence on virus replication could

be due to alteration in the localization/function of host cell proteins in virus replication.

Previous studies have indicated that NS5 can influence the regulation of immune-

related genes (De Maio et al., 2016; Medin, Fitzgerald, Alan, & Rothman, 2005; Rawlinson

et al., 2009), and certainly for flavivirus NS5 is known to contribute significantly to evasion

of the host innate immune system (31–37). Although, most of these functions are attributable

to mechanisms performed in the cytoplasm of infected cells. Our use of the nuclear export

inhibitor LMB has suggested that NS5 cycles quickly in and out of the nucleus, potentially

attributing to the fact that nuclear localised WNVKUN NS5 has not been previously observed.

Intriguingly, we observed that treatment of WNVKUN-infected cells with LMB had no

significant impact on virus replication or the production of infectious particles. This also

suggests that the pool of NS5 within the nucleus does not need to be exported, although this

does occur during replication. These observations also suggest that other nuclear proteins or

components that are exported via CRM1/exportin1 are not required for efficient replication,

however, this most likely discounts the export of mRNA and micro-RNAs that may

contribute to the replication cycle. Transcriptomic studies may reveal if in fact nuclear-

located NS5 is regulating gene transcription and/or the production of different RNA species

influencing cellular function and immune capacity.

Our live cell imaging and FRAP analyses revealed that repopulation of NS5-GFP within


21

the nucleus occurred by 45 min post bleach, and returned to pre-bleach levels by 120 minutes.

In contrast, the nuclear accumulation of NS5-GFP occurred at around 380 minutes after

treatment with LMB. However, an explanation for this lag time of accumulation is the time

required for LMB to saturate the exportin molecules within the cell. Again, this may also shed

light as to why WNVKUN NS5 has not been observed within the nucleus before, as it would

appear that WNVKUN NS5 cycles in and out of the nucleus, and does not accumulate within the

nucleus like DENV NS5 protein. However, we also note that we cannot assume NS5

localization reflects the LMB bound state of CRM1. Particularly as we have not investigated

that NS5 binds directly to CRM1. Thus, it is possible that the response of NS5 to LMB is

indirect and caused by the redistribution of host proteins affected by LMB.

One thing that is evident is that WNVKUN NS5 nuclear import is facilitated via a

conserved and functional NLS located at amino acids 389Arg-Glu-Lys391 within the WNVKUN

NS5 protein. It is apparent from our drug treatments that this NLS promotes an interaction

between NS5 and the Importin proteins. Perhaps more important is our observations that

treatment of WNVKUN-infected cells with these compounds significantly reduces replication

efficiency and dramatically reduces the production of infectious virus. This then offers the

possibility of targeting and developing small molecule inhibitors of nuclear import. This also

offers the advantage that they target a key step in the viral life-cycle and not a viral protein

reducing the potential of the development of antiviral resistance. Recently there has been

evidence of another NLS in the C- terminal of DENV NS5. However sequence comparison

between DENV2 and WNVKUN has shown that conservation is not fully shared between the

two, and further study on the importance of this segment in WNVKUN is needed (Tay et al.,


22

2016).

Overall, we have identified a critical role for the nuclear import of viral, and most

likely host factors, into the nucleus of WNVKUN-infected cells. Surprisingly though protein

export from the nucleus is not a strict requirement. As the flavivirus RC and replicative

events are solely confined to the cytoplasm we speculate that the involvement of the nucleus

must centre on modulation of host transcriptional events that drive the antiviral response in

infected cells. In addition, our observations suggest that targeting nuclear import would be a

suitable antiviral therapy.

References

Ashour, J., Laurent-Rolle, M., Shi, P.-Y., & Garcia-Sastre, A. (2009). NS5 of Dengue Virus

Mediates STAT2 Binding and Degradation. Journal of Virology, 83(11), 5408–5418.

https://doi.org/10.1128/JVI.02188-08

Best, S. M., Morris, K. L., Shannon, J. G., Robertson, S. J., Mitzel, D. N., Park, G. S., …

Bloom, M. E. (2005). Inhibition of interferon-stimulated JAK-STAT signaling by a tick-

borne flavivirus and identification of NS5 as an interferon antagonist. Journal of

Virology, 79(20), 12828–12839. https://doi.org/10.1128/JVI.79.20.12828-12839.2005

Buckley, A., Gaidamovich, S., Turchinskaya, A., & Gould, E. A. (1992). Monoclonal

antibodies identify the NS5 yellow fever virus non-structural protein in the nuclei of

infected cells. The Journal of General Virology, 73 ( Pt 5), 1125–30. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/1534119

Bulich, R., & Aaskov, J. G. (1992). Nuclear localization of dengue 2 virus core protein


23

detected with monoclonal antibodies. J Gen Virol, 73 ( Pt 11(1992), 2999–3003.

https://doi.org/10.1099/0022-1317-73-11-2999

Chu, P. W. G., & Westaway, E. G. (1987). Characterization of Kunjin virus RNA-dependent

RNA polymerase: Reinitiation of synthesis in Vitro. Virology, 157(2), 330–337.

https://doi.org/10.1016/0042-6822(87)90275-3

Davis, L. I., & Blobel, G. (1986). Identification and characterization of a nuclear pore

complex protein. Cell, 45(699–709), 699–709. https://doi.org/10.1016/0092-

8674(86)90784-1

De Maio, F. A., Risso, G., Iglesias, N. G., Shah, P., Pozzi, B., Gebhard, L. G., … Gamarnik,

A. V. (2016). The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and

Modulates Splicing. PLoS Pathogens, 12(8).

https://doi.org/10.1371/journal.ppat.1005841

Edward, Z., & Takegami, T. (1993). Localization and functions of Japanese encephalitis

virus nonstructural proteins NS3 and NS5 for viral RNA synthesis in the infected cells.

Microbiol Immunol, 37(3), 239–243. Retrieved from


Egloff, M. P., Benarroch, D., Selisko, B., Romette, J. L., & Canard, B. (2002). An RNA cap

(nucleoside-2′-O-)-methyltransferase in the flavivirus RNA polymerase NS5: Crystal

structure and functional characterization. EMBO Journal, 21(11), 2757–2768.

https://doi.org/10.1093/emboj/21.11.2757

Eltahla, A. A., Lackovic, K., Marquis, C., Eden, J.-S., & White, P. A. (2013). A

Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of


24

the Genotype 3a Hepatitis C Virus RNA Polymerase. Journal of Biomolecular

Screening, 18(9), 1027–1034. https://doi.org/10.1177/1087057113489883

Eltahla, A. A., Lim, K. L., Eden, J. S., Kelly, A. G., Mackenzie, J. M., & White, P. A. (2014).

Nonnucleoside inhibitors of norovirus rna polymerase: Scaffolds for rational drug

design. Antimicrobial Agents and Chemotherapy, 58(6), 3115–3123.

https://doi.org/10.1128/AAC.02799-13

Forwood, J. K., Brooks, a, Briggs, L. J., Xiao, C. Y., Jans, D. a, & Vasudevan, S. G. (1999).

The 37-amino-acid interdomain of dengue virus NS5 protein contains a functional NLS

and inhibitory CK2 site. Biochemical and Biophysical Research Communications, 257,

731–737. https://doi.org/10.1006/bbrc.1999.0370

Fraser, J. E., Watanabe, S., Wang, C., Chan, W. K. K., Maher, B., Lopez-Denman, A., …

Jans, D. A. (2014). A nuclear transport inhibitor that modulates the unfolded protein

response and provides in vivo protection against lethal dengue virus infection. Journal of

Infectious Diseases, 210(11), 1780–1791. https://doi.org/10.1093/infdis/jiu319

Gillespie, L. K., Hoenen, A., Morgan, G., & Mackenzie, J. M. (2010). The endoplasmic

reticulum provides the membrane platform for biogenesis of the flavivirus replication

complex. Journal of Virology, 84(20), 10438–10447. https://doi.org/10.1128/JVI.00986-

10

Grant, A., Ponia, S. S., Tripathi, S., Balasubramaniam, V., Miorin, L., Sourisseau, M., …

Garc??a-Sastre, A. (2016). Zika Virus Targets Human STAT2 to Inhibit Type i

Interferon Signaling. Cell Host and Microbe, 19(6), 882–890.

https://doi.org/10.1016/j.chom.2016.05.009


25

Grun, J. B., & Brinton, M. A. (1987). Dissociation of NS5 from cell fractions containing

West Nile virus-specific polymerase activity. Journal of Virology, 61(11), 3641–3644.

Hall, R. A., Tan, S. E., Selisko, B., Slade, R., Hobson-Peters , J., Canard, B., Hughes, M.,

Leung, J. Y., Balmori-Melian, E., Hall-Mendelin, S., Pham, K. B., Clark, D. C., Prow, N.

A. & Khromykh, A. A. (2009). Monoclonal antibodies to the West Nile virus NS5

protein map to linear and conformational epitopes in the methyltransferase and

polymerase domains. Journal of General Virology, 90(12), 2912-2922.https://doi:

10.1099/vir.0.013805-0

Hayes, E. B., Sejvar, J. J., Zaki, S. R., Lanciotti, R. S., Bode, A. V., & Campbell, G. L.

(2005). Virology, pathology, and clinical manifestations of West Nile virus disease.

Emerging Infectious Diseases. https://doi.org/10.3201/eid1108.050289b

Hoenen, A., Liu, W., Kochs, G., Khromykh, A. a., & Mackenzie, J. M. (2007). West Nile

virus-induced cytoplasmic membrane structures provide partial protection against the

interferon-induced antiviral MxA protein. Journal of General Virology, 88(11), 3013–

3017. https://doi.org/10.1099/vir.0.83125-0

Hu, W., & Jans, D. a. (1999). Efficiency of importin alpha/beta-mediated nuclear localization

sequence recognition and nuclear import. Differential role of NTF2. The Journal of

Biological Chemistry, 274(22), 15820–15827. Retrieved from


Hübner, S., Eam, J. E., Hübner, a, & Jans, D. a. (2006). Laminopathy-inducing lamin A

mutants can induce redistribution of lamin binding proteins into nuclear aggregates.

Experimental Cell Research, 312(2), 171–83.


26

https://doi.org/10.1016/j.yexcr.2005.10.011

Khromykh, A. A., Kenney, M. T., & Westaway, E. G. (1998). trans -Complementation of

Flavivirus RNA Polymerase Gene NS5 by Using Kunjin Virus Replicon-Expressing

BHK Cells. Journal of Virology, 72(9), 7270–7279.

Khromykh, A. A., Sedlak, P. L., & Westaway, E. G. (1999). trans-Complementation analysis

of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal

half in RNA replication. Journal of Virology, 73(11), 9247–55.

Kumar, A., Bühler, S., Selisko, B., Davidson, A., Mulder, K., Canard, B., … Bartenschlager,

R. (2013). Nuclear localization of dengue virus nonstructural protein 5 does not strictly

correlate with efficient viral RNA replication and inhibition of type I interferon

signaling. Journal of Virology, 87(8), 4545–57. https://doi.org/10.1128/JVI.03083-12

Laurent-Rolle, M., Boer, E. F., Lubick, K. J., Wolfinbarger, J. B., Carmody, A. B., Rockx,

B., … Best, S. M. (2010). The NS5 protein of the virulent West Nile virus NY99 strain is

a potent antagonist of type I interferon-mediated JAK-STAT signaling. Journal of

Virology, 84(7), 3503–3515. https://doi.org/10.1128/JVI.01161-09

Leary, K., & Blair, C. D. (1980). Sequential events in the morphogenesis of Japanese

encephalitis virus. Journal of Ultrastructure Research, 72(2), 123–129.

https://doi.org/10.1016/S0022-5320(80)90050-7

Lin, R.-J. J., Chang, B.-L. L., Yu, H.-P. P., Liao, C.-L. L., & Lin, Y.-L. L. (2006). Blocking

of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5 through a

protein tyrosine phosphatase-mediated mechanism. Journal of Virology, 80(12), 5908–

18. https://doi.org/10.1128/JVI.02714-05


27

Lopez-Denman, A. J., & Mackenzie, J. M. (2017). The IMPORTance of the Nucleus during

Flavivirus Replication. Viruses, 9(1), 14. https://doi.org/10.3390/v9010014

MacDonald, J., Tonry, J., Hall, R. A., Williams, B., Palacios, G., Ashok, M. S., Jabado, O.,

Clark, D., Tesh, R. B., Briese, T. & Lipkin, W. I. (2005). NS1 Protein Secretion during

the Acute Phase of West Nile Virus Infection. Journal of Virology, 79(22), 13924-13933.

https:// doi.org/10.1128/JVI.79.22.13924-13933.

Mackenzie, J. (2005). Wrapping Things up about Virus RNA Replication. Traffic, 6(11),

967–977. https://doi.org/10.1111/j.1600-0854.2005.00339.x

Mackenzie, J. M., Kenney, M. T., & Westaway, E. G. (2007). West Nile virus strain Kunjin

NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA

synthesis. Journal of General Virology, 88(4), 1163–1168.

https://doi.org/10.1099/vir.0.82552-0

Mazzon, M., Jones, M., Davidson, A., Chain, B., & Jacobs, M. (2009). Dengue virus NS5

inhibits interferon-alpha signaling by blocking signal transducer and activator of

transcription 2 phosphorylation. The Journal of Infectious Diseases, 200(8), 1261–1270.

https://doi.org/10.1086/605847

Medin, C. L., Fitzgerald, K. A., Alan, L., & Rothman, A. L. (2005). Dengue Virus

Nonstructural Protein NS5 Induces Interleukin-8 Transcription and Secretion. The

Journal of Virology, 79(17), 11053–11061. https://doi.org/10.1128/JVI.79.17.11053

Mori, Y., Okabayashi, T., Yamashita, T., Zhao, Z., Wakita, T., Yasui, K., … Matsuura, Y.

(2005). Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances

Viral Replication. Society, 79(6), 3448–3458. https://doi.org/10.1128/JVI.79.6.3448


28

Pryor, M. J., Rawlinson, S. M., Butcher, R. E., Barton, C. L., Waterhouse, T. A., Vasudevan,

S. G., … Davidson, A. D. (2007). Nuclear Localization of Dengue Virus Nonstructural

Protein 5 Through Its Importin α/β-Recognized Nuclear Localization Sequences is

Integral to Viral Infection. Traffic, 8(7), 795–807. https://doi.org/10.1111/j.1600-

0854.2007.00579.x

Rawlinson, S. M., Pryor, M. J., Wright, P. J., & Jans, D. A. (2009). CRM1-mediated nuclear

export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and

virus production. Journal of Biological Chemistry, 284(23), 15589–15597.

https://doi.org/10.1074/jbc.M808271200

Tay, M. Y. F., Fraser, J. E., Chan, W. K. K., Moreland, N. J., Rathore, A. P., Wang, C., …

Jans, D. A. (2013). Nuclear localization of dengue virus (DENV) 1-4 non-structural

protein 5; protection against all 4 DENV serotypes by the inhibitor Ivermectin. Antiviral

Research, 99(3), 301–306. https://doi.org/10.1016/j.antiviral.2013.06.002

Tay, M. Y. F., Smith, K., Ng, I. H. W., Chan, K. W. K., Zhao, Y., Ooi, E. E., … Vasudevan,

S. G. (2016). The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its

Subcellular Localization and Contains a Conserved Arginine Residue Essential for

Infectious Virus Production. PLOS Pathogens, 12(9), e1005886.

https://doi.org/10.1371/journal.ppat.1005886

Uchil, P. D., Kumar, A. V. A., & Satchidanandam, V. (2006). Nuclear Localization of

Flavivirus RNA Synthesis in Infected Cells. Journal of Virology, 80(11), 5451–5464.

https://doi.org/10.1128/JVI.01982-05

Wagstaff, K. M., Rawlinson, S. M., Hearps, A. C., & Jans, D. a. (2011). An AlphaScreen®-


29

based assay for high-throughput screening for specific inhibitors of nuclear import.

Journal of Biomolecular Screening  : The Offi

Biomolecular Screening, 16(2), 192–200. https://doi.org/10.1177/1087057110390360

Wagstaff, K. M., Sivakumaran, H., Heaton, S. M., Harrich, D., & Jans, D. A. (2012).

Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit

replication of HIV-1 and dengue virus. Biochemical Journal, 443(3), 851–856.

https://doi.org/10.1042/BJ20120150

Weis, K. (1998). Importins and exportins: How to get in and out of the nucleus. Trends in

Biochemical Sciences, 23(May), 185–189. https://doi.org/10.1016/S0968-

0004(98)01204-3

Westaway, E. G., Khromykh, a a, Kenney, M. T., Mackenzie, J. M., & Jones, M. K. (1997).

Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus.

Virology, 234(234), 31–41. https://doi.org/10.1006/viro.1997.8629

Westaway, E. G., Khromykh, a a, & Mackenzie, J. M. (1999). Nascent flavivirus RNA

colocalized in situ with double-stranded RNA in stable replication complexes. Virology,

258(1), 108–117. https://doi.org/10.1006/viro.1999.9683

Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K., & Khromykh, a a. (1997).

Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with

double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.

Journal of Virology, 71(9), 6650–6661.

Westaway, E. G., Mackenzie, J. M., & Khromykh, a a. (2002). Replication and gene

function in Kunjin virus. Current Topics in Microbiology and Immunology, 267, 323–51.


30

https://doi.org/10.1007/978-3-642-59403-8

Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., … Li, H. (2007). Structure and

Function of Flavivirus NS5 Methyltransferase. Journal of Virology, 81(8), 3891–3903.

https://doi.org/10.1128/JVI.02704-06


31

Acknowledgements

We thank Roy Hall for generously providing the WNV antibodies and Alexander Khromykh for

providing the WNVKUN infectious clone, FLSDX. The authors acknowledge the facilities, and

the scientific and technical assistance, of the University of Melbourne's Biological Optical

Microscopy Platform (BOMP). This research was supported by a Project Grant (No. 1081756) to

J.M.M. from the National Health and Medical Research Council of Australia.


32

Figure Legends

Figure 1. WNVKUN NS5 protein accumulates in the nucleus upon nuclear export

inhibition. Vero cells were infected with WNVKUN at MOI 5 for 24 hours and were left

untreated (i-iii), or treated with 10nM LMB at 2 h.p.i (iv-vi). Cells were subsequently fixed

for IF analysis and labelled with either anti-NS3 (red; panels i and iv) or anti-NS5 (green;

panels ii and v) antibodies. The nucleus is counterstained with DAPI (blue; panels iii and vi)

and the merged images are provided (panels iii and vi). The white arrows highlight nuclear

accumulation of NS5, and not NS3, in the LMB-treated cells.

Figure 2. Nuclear import inhibition restricts WNVKUN NS5 to the cytoplasm in infected

cells. (A) Vero cells were left untreated, or treated with nuclear transport inhibitors (LMB, 4-

HPR, 4-MPR or GSP), and infected with WNVKUN at MOI 5 for 24 hours. Cells were

subsequently fixed and stained for IF analysis with anti-NS5 (red; panels i, v, ix, xii, and

xvii) and anti-KPNA (green; panels ii, vi, x, xiv, and xviii) antibodies. The nucleus was

counterstained with DAPI (blue; panels iii, vii, xi, xv, and xix) and merged images are

provided (panels iv, viii, xii, xvi, and xx). (B) Mean nuclear fluorescence was quantified

using the software FIJI 2.0.0 and Fn/c determined. 90 cells per sample were analysed over

triplicate experiments. Error bars represent ±SD.

Figure 3. Nuclear import is critical for efficient WNVKUN replication. Vero cells were

treated with nuclear transport inhibitors (LMB, 4-HPR, 4-MPR and GSP) and infected with

WNVKUN at MOI 5 for 24 h.p.i. (A) Cell lysates collected at 24 h.p.i were analysed by


33

Western blot for changes in viral NS5 protein production. The membrane was probed with

anti-NS5 and anti-GAPDH antibodies. (B) Tissue culture fluid was also collected at 24 h.p.i

and analysed for the production of infectious virus via plaque assay (n=3). Titre was

determined as pfu/ml. Significance was determined through Student’s t-test. Results represent

the mean ±SD.

Figure 4. Nuclear localisation of WNVKUN NS5 is dependent on a functional nuclear

localisation sequence. (A) Schematic diagram of the WNVKUN NS5 protein highlighting the

known enzymatic domains. The putative bi-partite NLS is indicated in bold and underlined.

The introduced mutations are indicated in bold and red and labelled NS5(KYmut)-GFP or

NS5(REKmut)-GFP. (B) Vero cells were transfected with the recombinant cDNA expression

plasmids encoding WNVKUN NS5-GFP (panels i and iv), NS5(KYmut)-GFP (panels vii and x)

or NS5(REKmut)-GFP (panels xiii and xvi), untreated (panels i-iii, vii-ix, and xiii-xv) or

treated with LMB (panels iv-vi, x-xii, and xvi-xviii) at 12 h.p.t and subsequently fixed for IF

analysis at 24 h.p.t.. The nucleus was counterstained with DAPI (panels ii, v, viii, xi, xiv, and

xvii) and merged images are provided (panels iii, vi, ix, xii, xv, and xviii). (C) Mean nuclear

fluorescence was quantified using the software FIJI 2.0.0 and Fn/c determined. Results

represent mean ±SD of 90 cells per sample over triplicate experiments.

Figure 5. Live cell microscopy of NS5-GFP nuclear translocation. Vero cells were

transfected with the recombinant cDNA expression plasmids encoding WNVKUN NS5-GFP

(A and C) or NS5(REKmut)-GFP (B and D) and were imaged live via confocal microscopy


34

every 10 minutes for 12 hours total in the absence (A and B) or presence (C and D) of LMB.

(E) Mean nuclear fluorescence was quantified using the software FIJI 2.0.0 and Fn/c

determined. Results reflect mean±SD over triplicate experiments. Significance was

determined via Student’s t-test.

Figure 6. WNVKUN NS5-GFP repopulates the nucleus during FRAP indicating nucleo-

cytoplasmic shuttling. (A) Vero cells were transfected with the recombinant cDNA

expression plasmid encoding WNVKUN NS5-GFP, and at 12 h.p.t the cells were treated with

10nM LMB to accumulate NS5 within the nucleus. At 24 h.p.t the nucleus was photo-

bleached with 488 laser line and the cells were subsequently imaged on a spinning disk

confocal microscope with image acquisition every 5 min thereafter. Images collected at 2m0

min intervals are shown. (B) Mean nuclear fluorescence was quantified using the software

FIJI 2.0.0 and Fn/c determined. Results reflect mean ±SD over triplicate experiments

Figure 7. WNVKUN NS5 NLS motif is critical for virus replication. In vitro transcribed

RNA was generated from cDNA plasmids encoding FLSDX, FLSDX-KYmut or FLSDX-

REKmut and electroporated into Vero cells. (A) At 48 h.p.e. the electroporated cells were

fixed for IF analysis and visualised with anti-NS1 antibodies and anti-IgG 488 (Green; panels

iv-vi) The nucleus was counterstained with DAPI (Blue; panels i-iii). (B) Western blot

analysis of cell lysates collected from electroporated cells at 24, 48 & 72 h.p.e was

performed. The membrane was probed with anti-NS1, anti-NS5 & anti-PDI antibodies. (C)

Tissue culture fluid was collected from the electroporated cells at 24, 48 & 72 h.p.e and


35

analysed for the virus production via plaque assay. Results reflect mean ±SD over triplicate

experiments. (D) To determine the RNA-dependent RNA polymerase activity of the

WNVKUN WT and REKmut proteins, a fluorescence-based assay was used as previously

described (Eltahla et al., 2014). Reactions contained 400 ng of the target protein. Enzymatic

activity was determined by subtracting a blank containing heat-inactivated enzyme.


CMI_12848_F1.tif


CMI_12848_F2.tif


CMI_12848_F3.tif


CMI_12848_F4.tif


CMI_12848_F5.tif


CMI_12848_F6.tif


CMI_12848_F7.tif


Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

Lopez-Denman, AJ; Russo, A; Wagstaff, KM; White, PA; Jans, DA; Mackenzie, JM

Title:

Nucleocytoplasmic shuttling of the West Nile virus RNA-dependent RNA polymerase NS5 is

critical to infection

Date:

2018-08-01

Citation:

Lopez-Denman, A. J., Russo, A., Wagstaff, K. M., White, P. A., Jans, D. A. & Mackenzie, J.

M. (2018). Nucleocytoplasmic shuttling of the West Nile virus RNA-dependent RNA

polymerase NS5 is critical to infection. CELLULAR MICROBIOLOGY, 20 (8),

https://doi.org/10.1111/cmi.12848.

Persistent Link:

http://hdl.handle.net/11343/283766

File Description:

Accepted version

nucleocytoplasmic shuttling of the west nile virus rna

Documents