SARS-CoV 9b Protein Diffuses into Nucleus, Undergoes Active Crm1 Mediated Nucleocytoplasmic Export and Triggers Apoptosis When Retained in the Nucleus
Undergoes Active Crm1 Mediated
Nucleocytoplasmic Export and Triggers Apoptosis When Retained in the Nucleus. PLoS ONE 6(5): e19436. doi:10.1371/journal.pone.0019436
SARS-CoV 9b Protein Diffuses into Nucleus, Undergoes Active Crm1 Mediated Nucleocytoplasmic Export and Triggers Apoptosis When Retained in the Nucleus
Sara A kerstro m
Anuj Kumar Sharma
Vincent T. K. Chow
Stephanie A. Booth
Timothy F. Booth
Sunil K. Lal
Patricia V. Aguilar, University of Texas Medical Branch, United States of America
Background: 9b is an accessory protein of the SARS-CoV. It is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (NES), however the role of NES in 9b functioning is not well understood. Principal Findings/Methodology: In this report, we demonstrate that 9b in the absence of any nuclear localization signal (NLS) enters the nucleus by passive transport. Using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. Further, we found that 9b interacts with the cellular protein Crm1 and gets exported out of the nucleus using an active NES. We have also revealed that this NES activity influences the half-life of 9b and affects host cell death. We found that an export signal deficient SARS-CoV 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. Conclusion/Significance: Here, we showed that nuclear shuttling of 9b and its interaction with Crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. This phenomenon may be critical in providing a novel role to the 9b accessory protein of SARS-CoV.
Funding: This work was supported by internal funds from the International Centre for Genetic Engineering and Biotechnology, New Delhi, and a research grant
on SARS from the Department of Biotechnology to S.K.L. The FACS facility funded by Department of Biotechnology at ICGEB was very useful and is greatly
acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Severe acute respiratory syndrome (SARS) was a new respiratory
illness that emerged in China in 2003 and spread globally [1,2]. The
causative agent was identified as a new coronavirus and was named
SARS coronavirus (SARS-CoV) . The SARS-CoV genome
consists of approximately 29,700 nucleotides encoding 28 putative
proteins [6,7]. Just like other coronaviruses, the SARS-CoV genome
also contains several small open reading frames (ORFs) in addition
to those encoding for structural proteins . These small ORFs
are presumed to encode 8 group specific, accessory proteins viz.
ORF3a, 3b, 6, 7a, 7b, 8a, 8b and 9b .
One of these accessory proteins, the 9b protein is encoded by
ORF-9b of the SARS-CoV genome. Just like the internal (I) gene of
other group II coronaviruses, the ORF-9b of SARS-CoV overlaps
with its nucleocapsid ORF [8,1012]. However, there is no
homology between the SARS-CoV 9b and I protein of other
coronaviruses. The 9b protein has been shown to get expressed in
SARS-CoV-infected cells and antibodies against it have been found
in the sera of SARS infected patients, demonstrating that the
protein is produced during infection , but its actual function
is not yet determined. Studies on 9b-structure by Meier et al., (2006)
revealed a 2-fold symmetric dimer having a lipid binding cavity and
proposed its role in virus assembly . Cellular localization of 9b
has been previously reported to be predominantly cytoplasmic and
membranous. Also, a nuclear export signal (NES) present in its
46LRLGSQLSL-54 amino acid region has been suggested to be
responsible for its nucleocytoplasmic export .
Keeping this in mind, we studied the cellular localization
pattern of 9b and found that in addition to the cytoplasm, some of
the 9b protein was also present in the nucleus. This entry of 9b
into the nucleus was independent of cell cycle progression.
Further, we showed that 9b which lacks the nuclear localization
signal (NLS) continued to enter the nucleus passively and was able
to exit the nucleus due to its functional NES. Also, nuclear export
was found to be Crm-1 dependent and blocking NES based export
resulted in an increased half-life of 9b, which accumulated in the
nucleus. Finally, our studies revealed that when 9b remained
within the nucleus, it triggered caspase 3 mediated apoptosis in
transiently transfected mammalian cells. The requirement for
caspase 3 in apoptosis induction was further confirmed using the
cell permeant caspase inhibitors, Z-VAD-FMK (general caspase
inhibitor) and Z-DEVD-FMK (caspase 3 inhibitor). To the best of
our knowledge, this is the first report showing the nuclear
localization of 9b, its passive diffusion into and active Crm-1
dependent transport out of the nucleus. Also, this is the first report
associating 9b with nucleocytoplasmic export linked apoptosis.
Materials and Methods
The SARS-CoV (GenBank accession number NC_004718) 9b
gene was PCR amplified and cloned into pCDNA3.1/V5-His
TOPO vector (Invitrogen) using gene specific primers; F1 (59
GTAATGGACCCCAATCAAACCAAC 39) and R1 (59
TTTTGC-CGTCACCACCACGAA 39). In order to clone 9b into
pEYFPN1, it was PCR amplified using F2 (59
CGGGAATTCCTGATGGACCCCAATCAAACC 39) and R2 primers (59
GTATGG- ATCCCGTTTTGCCGTCACCACCAC 39). A mutant of
9b was made using commercially available, PCR based site directed
mutagenesis services (Banglore genei, Banglore) where all leucine
present in the NES region of 9b were replaced by alanine
(muNES-9bEGFPN1: 46-LRLGSQLSL-54 to
46-ARAGSQASA54) and the mutant was further cloned into EGFPN1 vector
(Clontech). Creatine phosphate, creatine phosphokinase, leptomycin
B (LMB), digitonin and cell-cycle inhibitors were purchased from
Sigma. Wheat germ agglutinin (WGA) was purchased from
Invitrogen. Caspase inhibitors were purchased from BD Pharmingen.
Cleaved caspase-3 antibody was purchased from Cell Signaling. The
SARS-CoV 9b specific antibody, Alexa FluorH488 goat-anti-rabbit
IgG and Crm 1 antibodies were purchased from Abgent, Invitrogen
and Abcam, respectively.
Cell culture and transfection
Vero cells were maintained in DMEM supplemented with
penicillin, streptomycin and 10% FBS. Approximately 0.6 million
cells were plated in a 60 mm dish. The total amount of transfected
DNA was 3 mg per 60 mm dish. The plasmid was mixed with
LipofectAMINE 2000 (Invitrogen) in serum-free DMEM media
(Invitrogen) and the cells were transfected as per manufacturers
protocol. For metabolic labeling, 36 hrs post-transfection, cells
were starved for 1 h in cysteine/methionine-deficient medium
(Invitrogen), and were then labeled with 100 mCi of [35S] cysteine/
[35S] methionine promix for a specific time period. After labeling,
cells were washed once in PBS and lysed in
radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP40, 0.5%
deoxycholate, 0.1% SDS and 50 mM Tris/HCl, pH 8.0 with
protease inhibitor cocktail). All transfection experiments were
performed in triplicates for at least three times. Standard deviation
was calculated wherever needed.
Vero E6 (VE6) cells (ATCC, Global Bioresource Center) were
maintained with Dulbeccos Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum, streptomycin, penicillin
and L-glutamine at 37uC and 5% CO2. Chamber-culture slides (BD
Falcon) were seeded with Vero E6 cells to form confluent
monolayers. Chambers were inoculated with SARS-CoV (Tor-3
strain) at an MOI of 5 TCID50 per cell. Infected cells were fixed in
2% paraformaldehyde in PBS, pH = 7, for 15 minutes at room
temperature, at 12, 18, 24, 36 and 48 hours post infection.
Immunofluorescence staining of SARS-CoV infected cells
Prior to use, culture slides were rinsed twice in PBS and
subjected to a 10 min permeabilization step with PBS containing
0.25% Triton X-100. After washing 365 min in PBS and blocking
with 1% (v/v) BSA in PBS containing 0.01% Tween-20 (PBST)
for 1 hour at RT, slides were incubated overnight at 4uC with
1:100 dilution of primary antibody in 1% BSA PBST (SARS virus
PUP6 polyclonal, N-terminal) (Abgent). Slides were rinsed
365 min in PBS, and then incubated with secondary antibody,
Alexa FluorH488 goat-anti-rabbit IgG (Invitrogen) for 1 h at RT
in 1% BSA PBST. After additional rinsing 365 min in PBS, slides
were air dried and mounted in 49,6-diamidino-2-phenylindole
(DAPI) antifade reagent (Jackson ImmunoResearch), mounted
with coverslips, and stored in darkness at 4uC until examination.
Slides were imaged using a Zeiss LSM700 Laser Scanning
Microscope with Zen2009 version 5.5 software.
Three-dimensional volume projections were rendered in Volume Viewer using
ImageJ version 1.43 g. Controls included mock-infected cells.
Confocal microscopy of 9b transfected cells
The 9b transfected cells were fixed 36 hrs post-transfection in
2% paraformaldehyde (in PBS, pH = 7) for 15 min at room
temperature. Paraformaldehyde was removed and cells were
washed once with 1xPBS and then rehydrated in 1xPBS for
20 min. Next, the cells were mounted using antifade reagent
having DAPI (Invitrogen). For localization studies, cells were
imaged using LSM 510 confocal microscope (Carl-Zeiss, AG,
Germany) fitted with Axiovert 200 M inverted microscope
(CarlZeiss). Images were captured with either 63x oil objective or 40x
objective using the 488 nm for GFP and 514 nm for YFP. Filter
used at the detector channel for the respective proteins were BP
505530 nm for GFP, and LP 530 nm for YFP. For (DAPI)
stained nuclei, 405 laser line was used for excitation and signals
were captured using LP 420 detector filter. The captured images
were processed using both Image J as well as LSM image
browser software. Approximately 200 cells were counted from
each sample and protein localization was scored. Data represents
analysis from 3 independent sets of experiments.
Vero cells were synchronized for 18 hrs by serum starvation.
Further, these cells were transfected in duplicates with the plasmid
of interest and were kept in complete media for 12 hrs. The cells
were treated for 16 hrs with either nocodazole or aphidicolin
(1 mg/ml and 1.5 mg/ml, respectively) and were placed in
complete media for the next 24 hrs. Subsequently, one set of
drug treated cells were processed for confocal microscopy and
another set were used for flow cytometry experiments. For
leptomycin B (LMB) treatment, cells were synchronized and were
treated with 25 nM drug for 16 hrs and further incubated in
complete media for another 24 hrs. For cyclohexamide treatment,
transfected cells were treated with 15 mg/ml of the drug 34 hrs
post-transfection. Two hours post-treatment, cells were processed
for microscopy. Z-FAfmk, Z-VAD-fmk and Z-DEVD-fmk were
dissolved in DMSO to give a final concentration of 50 mM and
stored at -80uC until use. Immediately prior to addition to cells,
the stock solutions were diluted into the culture medium.
Propidium iodide staining and cell-cycle analysis
For propidium iodide (PI) staining, cell pellets from Vero cells
were fixed in 70% ethanol at 4uC for 45 min. After being washed
twice with ice-cold PBS, the cell pellet was resuspended in 500 ml
PIsolution in PBS (40 mg/ml PI from 50x stock solution (2.5 mg/ml),
0.1 mg/ml RNase A and 0.05% Triton X-100) and incubated at
37uC for 30 min. For each sample, about 10,000 events were
acquired using a Cyan-ADP flow cytometer (Dako, Glostrup,
Denmark) at 488 nm excitation and the results were analyzed using
Summit (version 4.3) and FlowJo (version 6.4) software. All values
were expressed as mean 6 SD and a paired t-test was performed.
Fluorescent protein for in-vitro transport assay
Vero cells were transfected with the appropriate plasmid and were
processed 36 hrs post-transfection. The cells were washed at least two
times with cold phosphate buffer saline (PBS), pH 7.4, by
resuspension and centrifugation, 5000 rpm, 4uC. The cells were then washed
with 10 mM HEPES, pH 7.3, 110 mM potassium acetate, 2 mM
magnesium acetate, 2 mM DTT and then pelleted. The cell pellet
was lysed in 1.5 vol of lysis buffer (5 mM HEPES, pH 7.3, 10 mM
potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 1 mM
PMSF, and 1 mM Na3VO4 with a protease inhibitor cocktail)
(Amersham Biosciences, NJ, USA) and was processed for in-vitro
transport assay as described by Adam et al., 1990 .
Cell permeablization and in-vitro transport assay
In-vitro transport assay was performed as explained by Adam
et al., 1990 with minor modifications . Briefly, Vero cells were
grown on coverslips and were permeabilized by immersion in ice
cold transport buffer containing 40 mg/ml digitonin for 5 min.
After 5 min, cells were washed and kept in cold transport buffer.
The coverslips were then blotted to remove excess buffer and
inverted over a drop of complete transport mixture on a sheet of
parafilm in a humidified box. The complete transport mixture
contained 5075% fluorescent protein (freshly made as explained
above) diluted with transport buffer to give the following final
conditions: approx. 2535 mg/ml fluorescent-tagged fusion
protein + transport buffer (20 mM HEPES, pH 7.3, 110 mM
potassium acetate, 5 mM sodium acetate, 2 mM DTT, 1.0 mM
EGTA, 1 mM ATP, 5 mM creatine phosphate (Sigma), 20 U/ml
creatine phosphokinase (Sigma), and protease inhibitor cocktail
(Amersham Biosciences, NJ, USA)). The fluorescent protein was
prepared as explained above and the entire box was then floated in
a water bath at 30uC. As a positive control, we have used the NP
protein of H5N1 which is known to have two NLS . The 3a
protein of SARS-CoV has been used as a negative control as it has
been shown to localize in the extranuclear region . At the end
of the assay, each coverslip was rinsed and mounted in a small
amount of transport buffer (without DAPI) and was observed using
the confocal microscope.
Coverslips containing transfected Vero cells were incubated with
transport buffer containing 50 mg/ml wheat germ agglutinin (WGA)
for 15 min at room temperature. The coverslips were then blotted to
remove excess buffer, inverted on a drop of complete transport mix
and processed for in-vitro transport assay. At the end of the assay, each
coverslip was rinsed and mounted in a small amount of transport
buffer (without DAPI). Samples were observed by confocal
microscope (Carl-Zeiss) and processed using Image J software.
Nuclear extract preparation and Immunoprecipitation
Vero cells were resuspended in 400 ml buffer A (10 mM HEPES
pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
1 mM PMSF and protease inhibitor mix). Further, the mixture was
placed on ice for 15 min. After 15 min, 25 ml of 10% NP-40 was
added to the cells and vortexed for 10 seconds. Cells were centrifuged
at 1000 rpm for 10 min at 4uC. The supernatant was labeled as
cytoplasmic fraction. The pellet was resuspended in 200 ml of buffer
C (10 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 1 mM PMSF and protease inhibitor mix). The
samples were kept on ice for 20 min. Subsequently, the samples were
centrifuged, 14000 rpm, 10 min, at 4uC and the supernatant was
labeled as nuclear fraction. Immunoprecipitation was performed as
described previously . For SARS-CoV infected cells, nuclear and
cytoplasmic fractions were prepared using CelLyticTM
NuCLEARTM Extraction Kit (Sigma, MI, USA) according to
manufacturers protocol. Vero E6 cells infected by SARS-CoV (Frankfurt I
strain) at MOI = 1, were treated with or without 50 nM LMB for 24
or 48 hrs in 6 well plates. Western Blot was then performed on
nuclear and cytoplasmic fractions.
Pulse chase assay
Asynchronously growing Vero cells were transfected with
appropriate constructs and incubated for 36 hrs. Subsequently,
transfected or untransfected cells were pulse-labeled for 30 minutes
and chased for 0, 30 and 60 minute time-points. Whole cell lysate
was then prepared by cell lysis as described above in fluorescent
protein preparation method. Immunoprecipitation was performed
using a polyclonal anti-9b antibody and band intensities were
quantified by Image J software.
TUNEL (terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labelling) assay
Cell death was detected using cell-death-detection TUNEL assay
kit (Roche Biochemicals), according to the manufacturers
instructions. Apoptotic cells were quantified by counting TUNEL-positive
cells in each set of experiments. At least 200 cells from three separate
images were inspected, and percentages were calculated.
Cell extracts from transfected cells were assayed for cleaved
caspase-3 by western blotting. Briefly, cell lysates were analyzed on
SDS-PAGE, transferred onto nitrocellulose membrane and a
western was performed using cleaved caspase-3, caspase-7 and
caspase-9 specific antibodies separately (Cell signaling, USA).
Band intensities were quantified using Image J software.
Cell viability assay (MTT assay)
Vero E6 cells were infected with SARS CoV (MOI = 1) for 1 hr,
then washed and treated with or without 50 nM LMB for 24 or
48 hrs. Cell viability was then determined using
(3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
colorometric detection assay from Cell BiolabsCytoSelect TM
Cell Viability and Cytotoxic Assay Kit (Cell Biolabs, Inc)
according to manufacturers protocol.
Experiments were performed in triplicates and were repeated at
least three times. Statistical analysis was done using the GRAPHPAD
software. Unpaired t-test was performed and two-tailed p-value was
calculated. Statistical significance of variations was considered.
SARS-9b protein localizes in the nucleus in addition to
Cells infected with SARS-CoV showed faint cytoplasmic staining
with antibodies directed at the 9b protein at 12 h post-infection
(Fig. 1a). By 18 h post-infection, many discrete cytoplasmic foci
were observed, and by 24 h post-infection, small foci of nuclear
staining appeared, while control uninfected cells showed no staining
(Fig. 1b). Typical infected cells showed numerous nuclear foci of 9b
protein by 36 h post-infection, as well as extensive, larger areas of
cytoplasmic inclusions (Fig. 1a and 1b). By 48 h post-infection,
many nuclei were seen to be changing their appearance and
undergoing apoptosis, and majority of the cells at this time point
contained fewer foci of the 9b protein (Fig. 1b, 48 h time point,
second panel). Stacks of confocal images were used to generate
three-dimensional projections, with merged data from the alexa
fluor and DAPI channels (Fig. 1b). When viewed from the side and
from the top, as well as sections through the volume projections, it
was observed that the antibody staining (in green) was within the
nucleus as well as the cytoplasm. These foci of fluorescence first
appeared in the nucleus at 24 h post infection to reach a maximum
at about 36 hr. p.i., and are somewhat reduced in numbers at 48 h
p.i. (Fig. 1b). After the observation that some proportion of the 9b
protein enters into the nucleus, we planned to check its localization
in 9b transfected cells. Vero cells were transfected with the plasmid
constructs pEYFPN1-9b (having 9b sequence fused to a yellow
fluorescent protein coding sequence at the C-terminus region).
Analysis of 9b-YFP localization showed that in addition to the
extranuclear region, some amount of 9b was also present within the
nucleus similar to the SARS-CoV infected cells (Fig. S1, panel (i), (ii)
and (iii)). In a parallel experiment, only pEYFPN1 vector was
transfected in order to rule out any effect due to the vector itself (Fig.
S1, panel (iv), (v) and (vi)). Only cells showing clear nuclear
localization of 9b were scored positive. Similar results were also
obtained with HEK-293T and COS-7 cells (data not shown).
In order to biochemically confirm the nuclear localization of the
9b protein, we transfected Vero cells with pCDNA3.1-V5 His-9b
construct. After 36 hrs, cells were processed for nuclear and
cytoplasmic extract preparation. As shown in Figure 2, 9b was
present both in cytoplasm as well as nucleus. The purity of the
extracts was checked using anti-actin and anti-polymerase II
antibodies for the cytoplasmic and nuclear extracts, respectively
(Fig. 2, upper and central panel).
SARS-9b localization pattern is independent of cell-cycle
The cellular localization of many proteins is cell-cycle specific
. In order to examine the effects of cell-cycle on 9b
localization, we blocked cells either in the G1 or G2 stage, treating
them with different cell-cycle inhibitors. For G1 phase, the cells
were treated with aphidicolin (G1-S phase inhibitor) at a
concentration of 1.5 mg/ml and processed for microscopy and
flow cytometry analysis. The microscopy results clearly showed
that the cellular localization pattern of 9b remained unchanged in
the presence of aphidicolin (Fig. 3a). Flow cytometer analysis was
done to confirm the efficacy of the drug. The percentage of cells
arrested at G1-S phase of the cell-cycle after aphidicolin treatment
were found to be 57.9% [60.794%] as compared to untreated
cells (44.8%60.361%) confirming a significant arrest at this phase
(p = 0.0015) (Fig. 3b). Blocking cell-cycle at the G2 phase (using
nocodazole) did not affect the cellular localization pattern of the 9b
protein, indicating that there was no effect of cell-cycle progression
on the cellular localization and nuclear transport of the 9b protein
(Fig. 3c). Flow cytometry analysis showed that the percentage of
cells arrested at the G2-M phase of the cell-cycle after nocodazole
treatment was 71.4% [62.689%] as compared to untreated cells
(35.8%61.453%) showing significant cell-cycle arrest at this phase
(p = 0.0027) (Fig. 3d).
SARS-9b protein lacks an active NLS and enters the
nucleus by passive mode of transport
Cellular localization of some well known proteins like p27Kip1,
transcription factor lymphoid enhancer factor 1 (LEF-1), T-cell
factor 1 (TCF-1), cystinosin and some viral proteins have been
confirmed by in-vitro transport assays using
digitonin-permeabilized cells . To investigate the mode of nuclear transport of
9b, an in-vitro transport assay was performed. Initially, the 9b-YFP
fusion protein was expressed in Vero cells separately and then
their lysate was added to digitonin permeabilized cells. The cells
were supplied with all necessary components needed for in-vitro
transport as detailed in materials and methods. Results showed
that both H5N1-NP (a positive control in the assay) and 9b-YFP
were able to enter the nucleus in digitonin permeabilized cells
(Fig. 4, panel (i) and (ii), respectively). On the contrary, the 3a-GFP
protein (negative control) was not able to get into the nucleus
(Fig. 4, panel (iii)). A protein sorting signal analysis of the 9b
sequence (using PSORT program, http://psort.org/) predicted
absence of NLS in it and suggested its nuclear import to be passive
. Further, in order to rule out the possibility that the 9b protein
uses an active transport mode to enter the nucleus, we used wheat
germ agglutinin (WGA), a lectin known to inhibit NLS dependent
active nuclear transport . When WGA was added to the cells,
H5N1-NP (known to undergo NLS dependent nuclear import)
was found to get restricted to cytoplasm indicating that WGA was
able to inhibit the active transport of H5N1-NP protein through
the nuclear membrane (Fig. 4, panel (iv)). However, SARS-CoV
9b showed similar nuclear localization even in the presence of
WGA (Fig. 4, panel (v)). Thus, our data clearly shows that
although the SARS-CoV 9b protein lacks a NLS, it enters the
nucleus by passive transport. As expected, the negative control,
SARS-3a remained cytoplasmic (Fig. 4, panel (vi)).
SARS-9b protein gets exported from the nucleus using
the Crm-1 dependent NES
The nuclear export signal (NES) has been reported to be present
in the C-terminal region of the 9b protein . To confirm the
involvement of the NES in 9b export, we mutated the essential
amino-acids of the NES region (46-LRLGSQLSL-54 to
46ARAGSQASA-54) and cloned it into the pEGFPN1 vector (now
onwards called as mu-NES-9b). Cells were transfected with this
mutant and were analyzed 36 hrs after transfection. Microscopic
analysis clearly revealed that most of the fluorescence accumulated
in the nucleus (Fig. 5a, panel i-iii). Further, in order to check the
role of NES in nuclear export of 9b, we treated the 9b-EYFPN1
transfected cells with Leptomycin-B (LMB); a drug known to
inhibit active NES based export [32,33]. We found that most of
the 9b protein accumulated within the nucleus upon LMB
treatment, confirming the role of NES in its nuclear export
(Fig. 5b, panel ivvi). In order to reduce the cytoplasmic staining
resulting from the newly synthesized 9b protein, transfected cells
were treated with cyclohexamide and processed for microscopy.
The reduction in cytoplasmic fluorescence in lower panels of
Figure 5A and 5B confirms the effect of cyclohexamide treatment
(Fig. 5a, panel ivvi and Fig. 5b, panel viiix). The data obtained
was confirmed by performing biochemical analysis of the
fractionated lysates of transfected cells. Nuclear and cytoplasmic
extracts were prepared 36 hrs post-transfection and were analyzed
on 15% SDS-PAGE gel followed by a western blot using anti-9b
polyclonal antibody. Purity of the extracts was checked as
described before. Results confirmed our previous observations
(Fig. 5c). Taken together, all these results proved that 9b follows a
NES dependent, active nuclear export. In order to confirm the
localization pattern of 9b in the presence or absence of LMB,
SARS-CoV infected cells were processed for western blotting at
two different time points, 24 hrs and 48 hrs. Calnexin was used as
purity control for nuclear and cytoplasmic extract preparation.
The 9b protein was seen in both nucleus as well as cytoplasm at
both 24 hrs and 48 hrs (lane 2 and 5, Fig. 5d, upper panel). This
shows that 9b localizes both in the nucleus as well as in the
cytoplasm in both transfection and infection conditions. Further,
the effect of LMB was checked and the proportion of 9b in nuclear
and cytoplasmic extracts was compared in reference to total
protein using densitometric analysis (Fig. 5d, lower panel). As
shown in Figure 5d, total 9b in LBM treated cells seem slightly
lower compared to untreated cells. This is most probably due to
more cells in apoptotic phase. However, densitometry graphs
clearly show that the percentage of nuclear 9b gets increased on
LMB treatment which further supports our transfection data. The
effect seems more pronounced at the 48 hrs time point. The only
difference we observed between transfection and infection
conditions was that even without LMB, a lot of 9b was seen in
the nucleus 48 hrs post infection. This may be attributed to the
impact of other SARS-CoV proteins (expressed during infection)
on 9b localization.
anti-9b polyclonal antibody. Left panel shows purity of extracts. Results in right panel show that both mu-NES-9b as well as LMB treated 9b have
increased nuclear localization of 9b. C and N represent cytoplasmic and nuclear extracts respectively. D. Biochemical analysis of the localization
pattern of 9b protein in SARS-CoV infected cells. Synchronized cells were infected with the virus. Nuclear and cytoplasmic extracts were prepared 24
and 48 hrs post infection and were analyzed on 15% SDS-PAGE gel followed by western blotting using appropriate antibody. The upper panel shows
that 9b is present in both nuclear as well as cytoplasmic extracts. Calnexin was used to check the purity of extracts. Lower panel shows the
densitometric comparison of nuclear and cytoplasmic 9b using Image J program. Results show that LMB treatment (50 nM) leads to an increased
nuclear localization of 9b as compared to untreated cells. E. The SARS-CoV 9b protein interacts with Crm-1. Synchronized cells were transfected with
the appropriate constructs and the lysate was immunoprecipitated using the mentioned antibodies. Next, western blotting was performed using
either anti-9b or anti-Crm1 antibodies as per the case. Lane 2 in the lowermost panel clearly shows that 9b is able to pull out the endogenous Crm-1
protein. However, mutated NES clearly shows inhibition of binding of 9b with Crm-1 further confirming the presence of NES in 4654 amino acid
region. Lane 1 represents pEYFP-N1 vector which has been used as a negative control. Uppermost and central panel shows the expression level of
Crm-1 and 9b respectively in the transfected cells. IP represents immunoprecipitation; W represents western blotting.
Active nuclear export takes place by interaction of the nuclear
export signal (NES) with a protein, known as Crm-1 and is critical
for RNA and protein export from the nucleus [32,33].
Synchronized cells were transfected with the appropriate
constructs and the lysate was immunoprecipitated using the
mentioned antibodies. The complex was subjected to SDS-PAGE
followed by western blotting using either anti-9b or anti-Crm1
antibodies. As a control, the levels of Crm-1 were measured by
performing immunoprecipitation using anti-Crm-1 antibody
followed by western blotting with the same antibody. Results
show an interaction of 9b with the Crm-1 protein, clearly
supporting our previous observations (Fig. 5e, lane 2). Further,
mu-NES-9b was unable to bind Crm-1, confirming the presence of
NES activity in the 46 to 54 amino-acid region (Fig. 5e, lane 3).
NES dependent nuclear export facilitates intracellular
degradation of the 9b protein
Many cellular proteins are known to follow the ubiquitin/
proteasome pathway for their degradation . For shuttle
proteins, nuclear export is known to promote degradation .
Our data has already indicated that 9b behaves like a shuttle
protein, going into the nucleus passively and coming out using the
NES. Keeping these facts in mind, we designed experiments to
investigate whether nuclear export influences the intracellular
stability of 9b protein. Vero cells transiently expressing
9bEYFPN1 or mu-NES-9b were metabolically pulse-labeled
followed by a chase period for 0, 30 and 60 min. Compared to 9b,
we observed a significantly prolonged half-life for mu-NES-9b,
indicating that nuclear export is continuously supplying substrate
for the degradation machinery. Similar results were observed for
9b-EYFPN1 transfected cells treated with a nuclear export
inhibitor, LMB (Fig. 6). The experiment was performed in
triplicates and the significance of the data was calculated using
Graphpad software. The differences between 9b, mu-NES-9b
and 9b+LMB for time point of 30 min and 60 min were
significant as the p value ranged from p = 0.0004 to p = 0.0013.
Blocking active nuclear export of 9b protein induces
apoptosis in transfected cells
During our pulse-chase analysis explained above, it was
discovered that a number of 9b-EYFPN1 transfected cells died
on treatment with LMB. For quantification of the apoptotic cells,
TUNEL assay was performed (Fig. 7a, left panel). The number
was significantly higher for LMB treated 9b transfected cells as
compared to untreated cells. Similar results were observed for
muNES-9b (Fig. 7a, right panel). These results indicated that blocking
9b nuclear export may induce cell death by apoptosis. Similar
results were obtained when we checked the cell viability in
SARSCoV infected cells by MTT assay. Maximum viability of
SARSCoV infected cells was lost on LMB treatment suggesting that 9b
nuclear export may be important for cell survival (Fig. 7b).
However, the role of other viral proteins in this process can not be
ignored. Saponin was used as control. To check the pathway thus
involved in induction of apoptosis, we determined the levels of
various cleaved caspases (caspase 3, 7 and 9) in transiently
transfected cells. Western blot results show that 9b-EYFP induce
apoptosis (only if its nuclear export is blocked) by increasing the
cellular levels of cleaved caspase-3 (Fig. 7c, lanes14). As expected,
mu-NES-9b induced apoptosis even in the absence of LMB. To
confirm that the apoptosis is caspase dependant, Z-FA-fmk,
ZVAD-fmk and Z-DEVD-fmk (200 mM each) were added to cells
16 hrs post-transfection, and then every 4 hrs thereafter, for a total
of four doses. The lysates were checked for the levels of cleaved
caspase-3. Results confirmed that apoptosis induced by 9b
(blocked in the nucleus) was caspase dependent (Fig. 7c, lanes 5
8). All these results collectively showed that the export deficient 9b
protein lead to caspase-3 dependent apoptosis of the host cells.
Therefore, an NES based nuclear export of 9b appears to be very
significant for host survival and may play a vital role in supporting
the SARS-CoV life cycle.
The SARS-CoV 9b protein passively diffuses into the
nucleus and undergoes active Crm-1 mediated
The present study uncovers an important step in understanding
the roles of accessory proteins in the SARS-CoV life-cycle. In this
study, we characterized the nucleocytoplasmic shuttling of the 9b
protein and found that 9b enters the nucleus by a passive mode of
transport whereas its export is active and NES-Crm1 interaction
dependent. Further, in transfection experiments, we showed that
this localization pattern of 9b was independent of cell-cycle stages.
A time course study of 9b revealed that the pattern of its cellular
localization remained unchanged irrespective of post-transfection
time (data not shown). Disruption of 9b-Crm1 binding after
mutating the critical residues within the NES, and nuclear
accumulation of 9b on treatment with LMB, a Crm1 antagonist,
reconfirmed our hypothesis.
Earlier studies on 9b show that it localizes in the cytoplasm
[17,18]. We also found the cytoplasmic localization of 9b, however
our biochemical as well as confocal analysis clearly showed that
9b, in addition to cytoplasm, also localizes in the nucleus. This
import is not active and is NLS independent as 9b is able to enter
into the nucleus even in the presence of WGA. WGA is known to
bind to the nuclear pore complex and inhibit the nuclear
localization signal (NLS)-dependent intracellular transport [8,36].
Proteins below 50 kDa have been reported to get into the nucleus
passively [37,38]. SARS-CoV 9b is a small protein of about
13 kDa, thus there is a fair possibility of passive diffusion of 9b
even as a dimer. However, the possibilities of exposure of a hidden
NLS after conformational changes cannot be ignored. Our
preliminary data suggests that 9b interacts with many nuclear
proteins (Fig. S2AC). These interactions may also provide a
possible reason for the presence of 9b in the nucleus.
Cell-cycle inhibitors are known to affect the cellular localization
of some proteins [24,25,39]. To confirm the effect for 9b, we used
various cell-cycle inhibitors. Aphidicolin, arrests G1-S phase
progression of the cell-cycle by specially inhibiting DNA
polymerase a, responsible for DNA replication [25,40,41].
Nocodazole is an anticancer drug that interferes with the structure
and function of microtubules during interphase and blocks
cellcycle progression at the G2-M phase . Our results show that
the localization of 9b is independent of cell-cycle stages and the
pattern of distribution of 9b remains unaltered in the presence of
the drugs used. A low level of apoptosis observed for nocodazole
treated cells is a well established effect of this drug .
LMB inhibits NES dependent protein export [32,33]. The
suggested mechanism involves direct binding of LMB to Crm1
(exportin 1), which blocks the binding of Crm1 to proteins
containing the NES. Deletion of the NES present in 9b as well as
LMB treatment of cells has been reported to retain 9b in the
nucleus . We observed similar results when we repeated these
experiments. Further when we mutated the essential amino-acids
within the NES by site-directed mutagenesis or used cells treated
with LMB; we found accumulation of 9b in the nucleus. We
performed a BLAST analysis of the NES region of SARS-CoV 9b
protein which revealed that this region was well conserved (except
the replacement of Q by N at amino-acid position 51 in a few
cases) irrespective of the host of the virus.
NES dependent nuclear export facilitates intracellular
degradation of the 9b protein
Interestingly; this phenomenon of nuclear transport exhibited
by 9b is similar, but not identical to some other known proteins.
Proteins like MAPK and survivin are reported to localize in both
cytoplasm as well as the nucleus [43,44]. Similar to 9b protein,
survivin protein does not have NLS but contains NES. Survivin
also enters passively into the nucleus. However, blocking nuclear
export of survivin enhances its half-life . Our results show that
the 9b behaves similar to the survivin protein. We found that the
half-life of 9b protein in cells is about 20 minutes and an active
nuclear export is mandatory for proper degradation of the 9b
protein. Our pulse-chase analysis clearly shows an approximate
two-fold increase in the half-life of 9b protein, when its export
from nucleus is blocked. The dependence of 9b degradation on
NES is also supported by increased half-life of both; mutant
(lacking essential amino acids of its NES region) as well as 9b
treated with LMB. The NES based nuclear export has been shown
to promote degradation of shuttle proteins like nuclear factor
kappa B or inhibitory kappa B by the proteasome or ubiquitin
pathway . Similar to 9b, the degradation of many shuttle
proteins has been reported to get delayed if their export is
The SARS-CoV 9b protein triggers caspase 3 mediated
apoptosis when retained in the nucleus of mammalian
While performing pulse-chase assays, we found that a significant
number of Vero E6 cells, in which nuclear export of 9b has been
inhibited (either by treating with LMB or using NES deficient 9b),
were showing caspase 3 dependent apoptosis. The absence of
significant cell death in case of EYFP-N1 transfected, LMB treated
cells, ruled out the possibility of drug induced cell death. All these
results indicate that 9b if blocked in the nucleus, activates
apoptotic signaling pathway/s. However, the possibilities for this
observation to be cell-type specific can not be ignored.
Summing up, we propose a hypothesis describing the possible
role of the SARS-CoV 9b protein in maintaining the cell survival
Figure 8. Hypothesis showing probable role of 9b localization. There are certain proteins which function as transcription factor and on
activation (by stress or infection) move into the nucleus. Nuclear localization of such proteins further regulates the genes that play a role in apoptosis
and cell cycle progression. We propose that NES of 9b may remain unexposed until it binds to the nuclear proteins (represented as nuclear
protein = X). Our preliminary data shows that 9b binds with the nuclear proteins (Fig. S2). This interaction may be exposing NES of 9b leading to the
export of X proteins along with 9b using Crm1 dependent pathway. This event may stop host cell response against stress caused by the viral
after viral infection (Fig. 8). There are certain proteins which
function as transcription factor and on activation (by stress or
infection) move into the nucleus [45,46]. Nuclear localization of
such proteins further regulates the genes that play a role in
apoptosis and cell-cycle progression. We propose that the NES of
9b may remain unexposed until it binds to the nuclear proteins.
Our preliminary results show that 9b binds with the nuclear
proteins (Fig. S2). These interactions may be exposing the NES of
9b leading to the export of X proteins along with 9b using the
Crm1 dependent pathway. This event may stop host cell response
against stress caused by the viral infection.
Recent studies on accessory proteins of coronaviruses including
SARS-CoV reveal that these proteins are not essential for virus
replication and pathogenecity , but studies are there to
support the fact that accessory proteins do affect virus release,
stability, pathogenesis, and finally contribute towards virulence
. In MHV, I protein (an accessory viral structural protein) can
contribute to plaque morphology . Also, SARS-CoV (9b
protein) has been shown to be a structural component of the
virions which again indicates the importance of 9b protein for the
virus . However, more studies need to be performed on the
functional analysis of the 9b protein.
Thus, besides other mechanisms like dimerization, lipid binding
or interaction with other proteins, export of 9b seems to be a very
important phenomenon for both functional activities of 9b as well
as the SARS-CoV life-cycle. In-vivo studies as well as identification
of the nuclear protein/s specifically binding to 9b will help in
further understanding the role of 9b in nucleocytoplasmic
shuttling. While it is not sure whether SARS-CoV will again
emerge in the human population, it has spurred on the awareness
against it. Hopefully, this study will add a step towards the better
understanding of the behavior and function of the 9b accessory
protein in the life-cycle of the SARS-CoV.
Figure S1 Microscopy results show that the SARS-9b
protein localizes in both cytoplasm as well as nucleus
when expressed in transfected mammalian cells. Vero
cells were transfected with either pEYFPN1-9b or pEYFPN1
alone. After 36 hrs, cells were fixed and mounted. Arrow in panel
(i) indicates that some protein also enters into the nucleus. Panel
(iv) shows the localization pattern of pEYFPN1 vector as a control.
Panel (ii) and (v) corresponds to the DAPI staining of panel (i) and
(iv) respectively. Panel (iii) and (vi) show a merge image. Arrows in
various panels show nucleus of the cell.
Figure S2 SARS-CoV 9b pulls down some specific
proteins from nuclear extract of Vero cells. A. The
pCDNA3.1/V5-His TOPO-9b was used for in-vitro transcription
and translation. When ran on a 15% SDS-acrylamide gel, dried
and processed by autoradiography, a band of approx. 17 kDa was
seen on the autoradiogram (lane 2). Lane 1 shows the mock lysate.
B. The TNT expressed 9b protein was immunoprecipitated using
anti-9b specific antibody (Abgent). The antibody was able to
recognize the 9b protein (lane 2). M represents mock lysate. C.
Vero cells were processed and the nuclear proteins were extracted
as explained in material and methods. The TNT expressed 9b
protein was added to the nuclear extract and a pull down assay
was performed using 9b specific antibody (Abgent). In parallel, one
control reaction having nuclear extract incubated with the TNT
product of an empty pCDNA 3.1 vector (labeled as mock lysate)
was also assembled. The pulled-out proteins were run on a 15%
SDS PAGE followed by Coomassie blue staining. Lane 1 shows
the proteins pulled out with 9b protein. Lane 2 shows the proteins
pulled out with mock lysate. Lane 3 shows a pull-down using a
non-specific antibody. The protein ladder is shown in lane 4. N.E.
We thank Ravinder Kumar for lab support; Md. Atif Bin Towheed,
Manjusha and Shailja for technical assistance and Swapnil Sharma for
assistance in manuscript preparation. K.S. is a Research Associate of the
Department of Biotechnology (DBT), India.
Conceived and designed the experiments: KS TFB AM SKL. Performed
the experiments: KS Ss AKS ST BA. Analyzed the data: KS Ss BA SAB
TFB AM SKL. Contributed reagents/materials/analysis tools: VTKC
TFB AM SKL. Wrote the paper: KS TFB AM SKL.
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