West Nile virus and dengue virus capsid protein negates the antiviral activity of human Sec3 protein through the proteasome pathway

Authors

  • Raghavan Bhuvanakantham,

    1. Flavivirology Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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  • Mah-Lee Ng

    Corresponding author
    • Flavivirology Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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For correspondence. E-mail micngml@nus.edu.sg; Tel. (+65) 65163283; Fax (+65) 67766872.

Summary

Flavivirus capsid (C) protein is a key structural component of virus particles. The non-structural role of C protein in the pathogenesis of arthropod-borne flaviviruses is not clearly deciphered. This study showed that West Nile virus (WNV) and dengue virus (DENV) utilized C protein to reduce human Sec3p (hSec3p) levels at post-transcriptional level through activation of chymotrypsin-like proteolytic function of 20S proteasome. Mutagenesis studies confirmed amino acids 14, 109–114 of WNV C protein and 13, 102–107 of DENV C protein played an important role in activating the proteolytic function of 20S proteasome. Amino acid residues at 14 (WNV) and 13 (DENV) of C protein were important for C protein-hSec3p binding and physical interaction between C protein and hSec3p was essential to execute hSec3p degradation. Degradation motif required to degrade hSec3p resided between amino acid residues 109–114 of WNV C protein and 102–107 of DENV C protein. Proteasomes, hSec3p binding motif and degradation motif on C protein must be intact for efficient flavivirus production. Clinical isolates of DENV showed more pronounced effect in manipulating the proteasomes and reducing hSec3p levels. This study portrayed the non-structural function of C protein that helped the flavivirus to nullify the antiviral activity of hSec3p by accelerating its degradation and facilitating efficient binding of elongation factor 1α with flaviviral RNA genome.

Introduction

The Flaviviridae family consists of several medically important pathogens such as West Nile virus (WNV) and dengue virus (DENV). While the majority of WNV infections are asymptomatic, it can cause debilitating disease in humans and animals, with symptoms ranging from febrile illness to fatal encephalitis (Petersen and Roehrig, 2001; Watson et al., 2004). DENV causes a wide range of diseases in humans, ranging from acute febrile illness dengue fever to life-threatening dengue haemorrhagic fever/dengue shock syndrome [(DHF/DSS) (Gubler, 1998; Halstead, 2007; Leong et al., 2007)].

A mature flavivirus particle is smooth and spherical with a diameter of approximately 50 nm. The mature virus is symmetrically icosahedral with no spiky surface extensions (Mukhopadhyay et al., 2003). Each virion is composed of a single positive-strand genomic RNA and encodes a polyprotein precursor, which are co-translationally and post-translationally processed by host cell signalases and viral proteases to form three structural and seven non-structural (NS) proteins. The structural proteins capsid (C), pre-membrane/membrane (prM/M) and envelope (E) constitute the virus particle while the NS proteins are involved in viral RNA replication, virus assembly and modulation of host cell responses (Chambers and Rice, 1987; Rice, 1990; Brinton, 2002; Lindenbach and Rice, 2003; Beasley, 2005).

The C proteins of arthropod-borne flaviviruses such as WNV and DENV were localized in the cytoplasm and nuclei (Makino et al., 1989; Tadano et al., 1989; Bulich and Aaskov, 1992; Westaway et al., 1997; Chang et al., 2001; Mori et al., 2005; Oh and Song, 2006). Protein kinase C-mediated phosphorylation of arthropod-borne flaviviruses C protein was shown to be essential to mediate efficient nuclear localization via its interaction with importins (Bhuvanakantham et al., 2010a). The nuclear phase has been shown to be important for efficient virus replication (Bhuvanakantham et al., 2009). However, the exact functions of flavivirus C protein in nucleus are unclear since positive-stranded RNA viruses are thought to utilize cellular components in the cytoplasm for replication. The C protein of WNV and DENV has been shown to interact with various host proteins that are related to apoptosis (Oh and Song, 2006; Yang et al., 2008; Netsawang et al., 2010).

The other non-structural functions of flavivirus C protein and its role in the pathogenesis of arthropod-borne flaviviruses are poorly understood. In a recent study (Bhuvanakantham et al., 2010a), potential host proteins interacting with arthropod-borne flavivirus C protein were identified using yeast two-hybrid (Y2H) library screening. Human Sec3 protein (hSec3p) was observed to interact with both WNV and DENV C protein. The Sec3 protein is one of the eight subunits of the exocyst complex (Lipschutz and Mostov, 2002). In yeast, the exocyst complex plays an important role in the secretory pathway and vesicular trafficking (TerBush et al., 1996; Hsu et al., 1999) by targeting vesicles to the plasma membrane as well as regulating the later phases of exocytosis process (Zhang et al., 2008). However, the functional role of human Sec3p remained to be defined. Based on the known functions of Sec3p in yeast, it was hypothesized that flavivirus C protein might exploit hSec3p for virus trafficking and release. Conversely, our study pinpointed that hSec3p functioned as a negative regulator of flavivirus infection by modulating viral RNA transcription and translation through sequestering elongation factor 1α. It was also observed that the amount of hSec3p and EF1α–hSec3p complex was significantly lowered in infected cells compared with uninfected cells (Bhuvanakantham et al., 2010a). This study was thus extended to investigate the mechanism used by flaviviruses to manipulate hSec3p levels in order to favour flavivirus infection.

Results

WNV and DENV infection increases hSec3p turn-over

HEK293 cells were infected with WNV/DENV to examine whether flavivirus infection regulated the levels of hSec3p. At the indicated timings, hSec3p expression was assessed at the protein level in WNV/DENV-infected HEK293 cells using Western blotting and cell-based fluorescence (CBF) assay. The results showed that hSec3p was downregulated in WNV/DENV-infected cells compared with that of uninfected cells [Fig. 1A(i), B(i), C and D]. The reduction in hSec3p levels showed a reasonable kinetics over time. It was noted that the amount of C protein in virus-infected cells increased gradually over time [Fig. 1E(i), F(i), G and H]. The levels of C protein inversely correlated with endogenous hSec3p levels in both WNV/DENV-infected cells. We investigated whether clinical isolates of DENV were also capable of reducing hSec3p levels and observed that hSec3p levels were significantly reduced following infections with DF and DHF clinical virus isolates compared with that with lab-passaged NGC strain (Fig. S1). Moreover, DHF virus isolate reduced the hSec3p levels much more prominently than that of DF virus isolate at the early time points (Fig. S1C and D).

Figure 1.

Effect of flavivirus infection on hSec3p expression. HEK293 cells were infected with (A and C) WNV or (B and D) DENV.

A and B (i). The hSec3p expression level was assessed at the indicated timings by immunoblotting with anti-hSec3p antibody. Reduced hSec3p level is detected in WNV/DENV-infected cells from as early as 4 h p.i.

A and B (ii). Actin loading controls are included to ensure equal loading. ‘−’ and ‘+’ represent uninfected and virus-infected samples respectively.

C and D. The hSec3p expression level was assessed at the indicated timings by CBF assay using anti-hSec3p and anti-actin antibody. The hSec3p levels are normalized against the actin control and the relative levels of hSec3p in virus-infected cells are shown. Reduced hSec3p level is detected in WNV/DENV-infected cells from as early as 4 h p.i.

E and F. Western blotting was performed to measure the levels of C proteins in virus-infected cells at 4, 8, 12 and 18 h p.i. The amount of C protein in virus-infected cells increases gradually over time.

G and H. The expression levels of C protein were assessed by CBF assay using anti-C and anti-actin antibody. The C protein levels are normalized against the actin control and the relative levels of C protein in virus-infected cells are shown.

I–L. HEK293 cells were treated with MG132 and infected with (I and J) WNV or (K and L) DENV. The hSec3p expression level was assessed at the indicated timings by Western blotting (I and K) and CBF assay (J and L) using anti-hSec3p and anti-actin antibody. Reduced hSec3p level is detected in WNV/DENV-infected cells from 4 h p.i. in the absence of MG132. In contrast, there are no significant differences in the levels of hSec3p in MG132-treated virus-infected cells. ‘−’ and ‘+’ represent uninfected and virus-infected samples respectively. In case of MG132, ‘−’ and ‘+’ represent absence and presence of MG132 respectively. *P < 0.05 compared with uninfected cells.

HEK293 cells were then infected with UV-inactivated WNV/DENV and hSec3p levels were measured to assess whether active virus replication played an essential role in reducing hSec3p levels. As expected, significantly higher level of virus proteins (both C and E) was observed in WNV/DENV-infected cells compared with that of UV-inactivated WNV/DENV-infected cells at 4 h post infection (p.i.) (Fig. S2). There was no significant difference (P > 0.05) in endogenous hSec3p levels between un-infected and UV-inactivated WNV/DENV-infected HEK293 cells in contrast to the significant reduction (P < 0.05) in WNV/DENV-infected cells. This indicated that active flavivirus replication was essential to reduce hSec3p level significantly and virus proteins were synthesized at as early as 4 h p.i.

After confirming that there were no significant changes (P > 0.05) in the mRNA levels of hSec3p in flavivirus-infected cells (Fig. S3A and B), host cell transcription was blocked by incubating HEK293 cells with actinomycin D. This was followed by infection with WNV/DENV. As shown in Fig. S3C and D, hSec3p level decreased in WNV/DENV-infected cells even in the presence of actinomycin D. The hSec6p levels were not affected in the presence of WNV/DENV infection (Fig. S3E and F). This indicated that the translation of hSec3 mRNA or the stability of hSec3p was specifically compromised following infection with WNV/DENV.

Proteasomal inhibitor, MG132 was then used to investigate if flavivirus infection interfered with hSec3p translation or its stability. The level of endogenous hSec3p was monitored after confirming that the levels of hSec3p and hSec6p were not altered significantly when MG132 was used (Fig. S3G). In the presence of MG132, hSec3p levels did not reduce significantly (P > 0.05) even after flavivirus infection (Fig. 1I, lanes 4, 8, 12 and 16 and Fig. 1L, lanes 4, 8, 12 and 16). Similar results were obtained from CBF assay (Fig. 1J and L). This supported the notion that intact proteasome activity was required to mediate the reduction of hSec3p levels and stability of hSec3p was compromised following flavivirus infection.

Flavivirus C protein mediated proteasome-dependent degradation of hSec3p

Since flavivirus C protein interacted with hSec3p (Bhuvanakantham et al., 2010a), the role of flavivirus C protein in reducing hSec3p level was evaluated by transfecting HEK293 cells with Myc-tagged plasmids encoding full-length WNV/DENV C gene (WMycC and DMycC) in the presence and absence of MG132. The plasmids used in this experiment were shown in Table S1. At 24 h post transfection, expression of hSec3p and full-length WNV/DENV C proteins were assessed using Western blotting. In the absence of MG132, the hSec3p level was markedly reduced following transfection with full-length WNV/DENV C protein [Fig. 2(i), lanes 6 and 8]. Densitometry analysis of the immunoblot showed that hSec3p levels were reduced by 35–40% following transfection with full-length C protein. In contrast, there were no significant differences (P > 0.05) in the levels of hSec3p in MG132-treated full-length C plasmids-transfected cells [Fig. 2(i), lanes 5 and 7]. The expression of full-length WNV/DENV C protein was confirmed using anti-Myc and actin antibodies by Western blotting [Fig. 2(ii)]. CBF assay also showed the similar results for the expression levels of hSec3p and C protein (data not shown). There were no significant changes in the expression levels of hSec6p in the absence or presence of MG132 (Fig. S4). This demonstrated that flavivirus utilized its C protein to specifically induce the proteasome-mediated degradation of hSec3p.

Figure 2.

Influence of flavivirus C protein on hSec3p expression. HEK293 cells were transfected with WMycC and DMycC in the absence and presence of MG132. (i) The hSec3p expression level was assessed by Western blotting using anti-hSec3p antibody. Reduced hSec3p level is detected in WNV/DENV C-transfected cells in the absence of MG132 (lanes 6 and 8). In contrast, there are no significant differences in the levels of hSec3p in MG132-treated WNV/DENV C-transfected cells (lanes 5 and 7). (ii) The expression of C protein is monitored by Western blotting using anti-Myc antibodies. ‘−’ and ‘+’ represent absence and presence of MG132 respectively. *P < 0.05 compared with mock-transfection.

Physical binding between capsid protein and human Sec3 protein is critical to reduce human Sec3 protein level

This study was extended to examine whether physical binding between flavivirus C protein and hSec3p is essential to mediate the reduction in hSec3p level. HEK293 cells were transfected with WMycC (WNV), DMycC (DENV) and hSec3p-binding defective mutants (first 15 amino acids deleted), WMycC5′Δ15 (WNV)/DMycC5′Δ15 (DENV) plasmids (Table S2). At 24 h post transfection, co-immunoprecipitation was performed using anti-hSec3p antibody followed by immunoblotting with anti-C antibody. Consistent to our earlier study (Bhuvanakantham et al., 2010a), immunoreactive bands were observed only in full-length WNV/DENV C-transfected cell lysates and there were no bands in WMycC5′Δ15/DMycC5′Δ15-transfected samples (Fig. S5). At 24 h post transfection, expression of hSec3p [Fig. 3(i)] and full-length/truncated WNV or DENV C proteins [Fig. 3(ii)] were also assessed. As mentioned previously, hSec3p levels were markedly reduced (P < 0.05) following transfection with full-length WNV/DENV C protein [Fig. 3(i), lanes 3 and 4]. In contrast, equivalent amounts of hSec3p were detected in mock-transfected, pCMV-Myc vector-transfected and WMycC5′Δ15/DMycC5′Δ15-transfected samples [Fig. 3(i), lanes 1, 2, 5 and 6]. This confirmed that physical binding between C protein and hSec3p was needed for C protein-mediated depletion of hSec3p.

Figure 3.

Measurement of hSec3p level in the presence of hSec3p-binding defective C mutants. HEK293 cells were transfected with WMycC, WMycC5′Δ15, DMycC and DMycC5′Δ15 plasmids. The expression levels of hSec3p and C protein were assessed using anti-hSec3p/anti-myc antibody. Reduced hSec3p level is detected in WMycC and DMycC-transfected cells (i, lanes 3 and 4). In contrast, there are no significant differences in the levels of hSec3p in WMycC5′Δ15 and DMycC5′Δ15-transfected cells (i, lanes 5 and 6). No difference is observed in the expression levels of C protein (ii). *P < 0.05 compared with mock-transfection.

Flavivirus C protein activated the chymotrypsin-like activity of 20S proteasome to degrade human Sec3 protein

HEK293 cells were transfected with full-length C protein or hSec3p-binding defective C mutants to demonstrate the specific proteolytic function of 20S proteasome. At 12 h post transfection, proteolytic activities of 20S proteasome was examined using Proteasome-Glo cell-based assay kits for chymotrypsin-like, trypsin-like and caspase-like activities. As shown in Fig. 4A and C, full-length C protein of WNV/DENV activated the chymotrypsin-like (A) and caspase-like (C) functions of 20S proteasome (P < 0.05). In contrast, hSec3p-binding defective C mutants failed to activate the proteolytic activities of 20S proteasome (P > 0.05). There was no significant difference in the trypsin-like activity (Fig. 4B) of 20S proteasome following transfection with full-length C protein or hSec3p-binding defective mutant C protein (P > 0.05). This inferred that full-length C protein of WNV and DENV triggered chymotrypsin-like and caspase-like activities of 20S proteasome to degrade hSec3p.

Figure 4.

A–C. Proteolytic activities of 20S proteasome following transfection with WNV/DENV C protein. HEK293 cells were transfected with full-length C protein (WMycC/DMycC) or hSec3p-binding defective C mutant (WMycC5′Δ15/DMycC5′Δ15) and the proteolytic activities of 20S proteasome was examined. (A) Chymotrypsin-like and (C) caspase-like functions of 20S proteasome are significantly increased following transfection with full-length C protein of WNV/DENV. (B) No significant difference in the trypsin-like activity of 20S proteasome is observed following transfection with WNV/DENV C proteins.

D and E. Influence of flavivirus C protein on hSec3p expression in the presence of lactacystin/YU-102. HEK293 cells were transfected with WMycC and DMycC in the absence and presence of lactacystin/YU-102. The hSec3p expression level was assessed using anti-hSec3p antibody. Reduced hSec3p level is detected in WNV/DENV C-transfected cells in the absence of lactacystin and there are no significant differences in the levels of hSec3p in lactacystin-treated WNV/DENV C-transfected cells (i). Reduced levels of hSec3p are observed in the presence or absence of YU-102 (i). The expression of C protein is monitored using anti-Myc antibodies (ii). Fig. 4E(ii) blot was obtained by merging 2 blots run separately.

To demonstrate the specific proteolytic function of 20S proteasome and to answer the question whether hSec3p is specifically required for the stimulation of the proteasome by C protein, hSec3pKD cells were transfected with full-length C protein or hSec3p-binding defective C mutants. As shown in Fig. S6A and C, full-length C protein of WNV/DENV activated the chymotrypsin-like (A) and caspase-like (C) functions of 20S proteasome in hSec3pKD cells at a much lower level. The magnitude of chymotrypsin-like and caspase-like activities were significantly lesser in hSec3pKD cells (P < 0.05) compared with that in HEK293 cells (Fig. 4A and C). We went onto perform co-immunoprecipitation in hSec3pKD cells transfected with full-length C protein. The hSec3pKD cells contained 20–25% of hSec3p compared with that of HEK293 cells. Cell lysates were immunoprecipitated with anti-hSec3p antibody followed by immunoblotting with anti-C antibody. Immunoreactive bands were observed in full-length C-transfected hSec3pKD cells only when SuperSignal Dura system (more sensitive substrate) was used in Western blotting (Fig. S6D, lanes 3 and 4). Overall, this indicated that full-length C protein must actively bind to hSec3p to trigger chymotrypsin-like and caspase-like activities of 20S proteasome.

To explore whether these two proteolytic activities are essential to execute the degradation of hSec3p, bioassays were performed using proteasome inhibitors (lactacystin, inhibitor of chymotrypsin-like activity and YU-102, inhibitor of caspase-like activity). HEK293 cells were incubated with 5 μM lactacystin/5 μM YU-102 and transfected with WMycC, DMycC, WMycC5′Δ15 or DMycC5′Δ15. At 24 h post transfection, expression of hSec3p and full-length WNV/DENV C proteins were assessed using Western blotting. In the absence of lactacystin/YU-102, the hSec3p level was markedly reduced (P < 0.05) following the transfection with full-length WNV/DENV C protein [Fig. 4D(i) and E(i), lanes 1 and 3] and not with hSec3p-binding defective C mutants [Fig. 4D(i) and E(i), lanes 5 and 7]. In contrast, there were no significant differences (P > 0.05) in the levels of hSec3p in lactacystin-treated full-length C plasmids-transfected cells compared with that of untreated cells [Fig. 4D(i), lanes 2 and 4]. YU-102 (inhibitor of caspase-like activity) could not restore the hSec3p level (P > 0.05) following transfection with full-length WNV/DENV C protein [Fig. 4E(i), lanes 2 and 4]. This indicated that caspase-like proteolytic function of 20S proteasome did not play a role in degrading hSec3p levels. The expression of full-length WNV or DENV C protein was confirmed using anti-Myc antibodies [Fig. 4D(ii) and E(ii)]. Overall, this confirmed that flavivirus C protein must physically bind to hSec3p to induce the chymotrypsin-like proteolytic function of 20S proteasome, which in turn is essential to mediate the degradation of hSec3p.

To demonstrate that the chymotrypsin-like proteolytic function of 20S proteasome observed in Fig. 4A was due to proteasome protease activity and not related to the activity of other proteases, HEK293 cells were treated with various inhibitors such as Bafilomycin A1 (lysosomal protease inhibitor), apoptosis inhibitor, lactacystin (chymotrypsin-like activity inhibitor) and YU-102 (caspase-like activity inhibitor) and transfected with full-length C protein or hSec3p-binding defective C mutants. As shown in Fig. S7, full-length C protein of WNV/DENV activated the chymotrypsin-like activity in the presence of all the inhibitors except lactacystin. The study was extended to examine the correlation between chymotrypsin-like proteolytic function of 20S proteasome and hSec3p levels. Chymotrypsin-like proteolytic function of 20S proteasome and hSec3p levels was inversely correlated with each other (data not shown). Overall, this demonstrated that chymotrypsin-like proteasome protease activity is specifically increased by flavivirus C protein.

Mapping the domains of flavivirus capsid protein responsible for activating chymotrypsin-like proteolytic function of 20S proteasome and degrading human Sec3 protein

Oh et al. (2006) reported that the last 15 amino acids of WNV C protein contained the cytotoxic and degradation inducing motif. Multiple sequence alignment of the amino acid sequences of WNV and DENV C protein was performed using CLUSTALW2 alignment software (Fig. S8). Alanine-scanning mutagenesis studies were performed on the hSec3p-binding domain of C protein (WC12, WC13, WC14 and WC15) and the conserved and semi-conserved regions in the last 15 amino acids (putative degradation motif) of WNV and DENV C protein [WC(a), WC(b), WC(c), DC(a), DC(b) and DC(c) mutants]. HEK293 cells were transfected with full-length C protein or various C mutants. At 12 h post transfection, activation of chymotrypsin-like function was significantly compromised following transfection with mutants such as WC14, WC(a), WC(b), DC13, DC(a) and DC(b) compared with that of full-length C protein and other mutants (Fig. 5A and B). The mutants WC14 and DC13 failed to activate any chymotrypsin-like function while the other mutants WC(a), WC(b), DC(a) and DC(b) activated the chymotrypsin-like activity at a much lower level (P < 0.05) compared with that of full-length C protein. As shown in Fig. 5C and D, the levels of hSec3p were markedly reduced following transfection with full-length WNV/DENV C protein (Fig. 5C and D, lane 3) and all the mutants (Fig. 5C and D, lane 6, 7 and 8) except WC14 (Fig. 5C, lane 5) and DC13 (Fig. 5D, lane 4) mutant C proteins. Equivalent amounts of hSec3p were detected in pCMV-Myc vector, WC14 and DC13-transfected samples. Although there was a significant reduction in hSec3p levels (P < 0.05) following transfection with WC(a), WC(b), DC(a) and DC(b) mutants, these reductions were much lower than that observed with full-length C proteins (P < 0.05). There were no significant differences in the level of C protein expression following various mutations (Fig. 5E and F). The results obtained from all the mutants were summarized in Table 1. Overall, these results defined that the amino acids 14, 109–114 of WNV C protein and 13, 102–107 of DENV C protein played an important role in activating the proteolytic function of 20S proteasome and mediating the degradation of hSec3p.

Figure 5.

A and B. Chymotrypsin-like activity of 20S proteasome following transfection with full-length or mutant C proteins. HEK293 cells were transfected with full-length C protein (WMycC/DMycC) or various C mutants and the proteolytic activities of 20S proteasome was examined. Chymotrypsin-like functions of 20S proteasome are significantly increased following transfection with full-length C protein and all the mutants of WNV/DENV C protein except WC14, WC(a), WC(b), DC13, DC(a) and DC(b) mutants. *P < 0.05 compared with mock-transfection. **P < 0.05 compared with full-length C protein.

C–F. HEK293 cells were transfected with WC, WC13, WC14, WC(a), WC(b), WC(c), DC, DC13, DC14, DC(a), DC(b) and DC(c) plasmids. The hSec3p protein expression levels and C protein expression levels were assessed by Western blotting. Similar level of reduction in hSec3p amount is detected in all the C-transfected experimental groups except with WC14, WC(a), WC(b), DC13, DC(a) and DC(b) mutants (C and D). There is no significant difference in the expression levels of C protein (E and F). *P < 0.05 compared with mock-transfection. **P < 0.05 compared with WC/DC transfection.

Table 1. Effect of representative mutation(s) on flavivirus C protein on chymotrypsin-like activity, hSec3p and C protein levels
PlasmidMutation at residueChymotrypsin-like activityhSec3p levelsC protein levels
  1. WC or DC represents WNV or DENV C protein. +++, strong chymotrypsin-like activity or high protein expression; ++, moderate chymotrypsin-like activity or moderate protein expression; +, mild chymotrypsin-like activity or low protein expression; −, no significant chymotrypsin-like activity.
Mock-transfection+++
pCMV-Myc vector+++
WNV
WC+++++++
WC1212+++++++
WC1313+++++++
WC1414++++++
WC1515+++++++
WC(a)109–111+++++++
WC(b)112–114++++++
WC(c)115–117++++++
DENV
DC+++++++
DC1212+++++++
DC1313++++++
DC1414+++++++
DC1515+++++++
DC(a)102–104+++++++
DC(b)105–107++++++
DC(c)108–110++++++

The mutations at the amino acid residues 14 of WNV C protein and 13 of DENV C protein fall within the hSec3p-binding motif on C protein (Bhuvanakantham et al., 2010a). We thus went onto examine the interaction status and the strength of interaction between hSec3p and C proteins carrying mutations in the first 15 amino acids by performing mammalian two-hybrid (M2H) assay. The results clearly showed that mutations introduced at amino acid residues 14 of WNV C protein and 13 of DENV C protein affected the interaction between flavivirus C protein and hSec3p (data not shown). Reduced chymotrypsin-like activity observed from WC14 and DC13 mutants (Fig. 5A and B) could have resulted as a consequence of no interaction between C protein and hSec3p. Taken together, this further supported the notion that physical binding between C protein and hSec3p was essential to activate chymotrypsin-like activity and to mediate the degradation of hSec3p.

Reverse genetics system to analyse the influence of capsid protein on the degradation of human Sec3 protein

The influence of C protein on the degradation of hSec3p was investigated using reverse genetics system. Mutations were introduced at the key residues of C protein in the full-length infectious clones of WNV and DENV [pWNSC13, pWNSC14, pWNSC(d), pDENVC13, pDENVC14 and pDENVC(d)]. HEK293 cells were infected with the viruses derived from these mutant infectious clones at multiplicity of infection (moi) of 1. The hSec3p level was markedly reduced following the infection with pWNS, pWNSC13, pDENV and pDENVC14 viruses [Fig. 6A(i) and B(i)], while the reduction in hSec3p level was significantly compromised (P < 0.05) in pWNSC(d) and pDENVC(d) viruses-infected cells compared with that obtained from pWNS and pDENV [Fig. 6A(i) and B(i)]. The amount of hSec3p in each experimental group inversely correlated well with the level of chymotrypsin-like activity in that particular experimental group [Fig. 6A(ii) and B(ii)]. In the absence of MG132, there was no significant differences (P > 0.05) in the expression levels of C protein [Fig. 6A(iii) and B(iii)] and hSec6p [Fig. 6A(iv) and B(iv)]. The presence of MG132 restored hSec3p levels in virus-infected cells comparable to endogenous hSec3p level in uninfected cells [Fig. 6A(i) and B(i)]. The C protein levels were increased following treatment with MG132 [Fig. 6A(iii) and B(iii)] as Jab1 could not degrade C protein in the presence of MG132 (Oh et al., 2006), while the chymotrypsin-like proteolytic activity decreased [Fig. 6A(ii) and B(ii)] significantly (P < 0.05) in virus-infected cells.

Figure 6.

Effect of mutations on degradation motif of C protein on hSec3p expression using reverse genetics system. HEK293 cells were infected with wild-type and mutant viruses in the presence or absence of MG132. (i) The hSec3p expression level was assessed by CBF assay using anti-hSec3p and anti-actin antibody. The hSec3p levels are normalized against the actin control and the relative levels of hSec3p in virus-infected cells are shown. Reduced hSec3p level is detected in [A(i)] pWNS, [A(i)] pWNS115-117, [B(i)] pDENV and [B(i)] pDENV108-110 compared with mock-transfection (*P < 0.05). The hSec3p levels are significantly higher in other mutants compared with that of pWNS or pDENV. (ii) Chymotrypsin-like activity of 20S proteasome following infection with wild-type or mutant viruses. Chymotrypsin-like functions of 20S proteasome are significantly increased following infection with [A(ii)] pWNS, [A(ii)] pWNS115-117, [B(ii)] pDENV and [B(ii)] pDENV108-110 viruses compared with mock-infection (*P < 0.05). The chymotrypsin-like activity is significantly lower in other mutants compared with that of pWNS or pDENV. (iii) The expression of virus C protein is monitored by CBF assay using anti-C and anti-actin antibodies. There is no change in C protein expression levels following infection with mutant viruses. (iv–vi) HEK293/hSec3pKD cells were infected with wild-type and mutant viruses and the levels of hSec3p, chymotrypsin-like activity and the levels of C protein are measured.

In virus-infected hSec3pKD cells, hSec3p levels and chymotrypsin-like activity were significantly lower [Fig. 6A and B (v–vi)] compared with that in HEK293 cell infection. The C protein levels were significantly higher in virus-infected hSec3pKD cells [Fig. 6A and B (vi)] as virus protein levels were increased in hSec3pKD cells (Bhuvanakantham et al., 2010a). Overall, this confirmed that the amino acid residues 14, 109–114 of WNV C protein and 13, 102–107 of DENV C protein played an essential role in activating the chymotrypsin-like activity of proteasomes and specifically mediating the proteasome-mediated degradation of hSec3p.

Influence of proteasomes on flavivirus production

HEK293 cells were infected with pWNS, pWNSC13, pWNSC14, pWNSC(d), pDENV, pDENVC13, pDENVC14 and pDENVC(d) viruses in the presence and absence of MG132 and lactacystin. Reduced virus titres were observed with MG132 and lactacystin treatment following infection with pWNS, pWNSC13, pDENV and pDENVC14 viruses (Fig. 7 A). The other mutant viruses pWNSC14, pWNSC(d), pDENVC13 and pDENVC(d) showed decreased virus titres even in the absence of MG132 and lactacystin (Fig. 7A). This confirmed that the proteasome activity, hSec3p-binding motif and hSec3p degradation motif on C protein must be intact for efficient flavivirus production.

Figure 7.

(A and B) Effect of mutations on degradation motif of C protein on flavivirus production using reverse genetics system. HEK293 cells were infected with viruses derived from infectious clones (A) or infected with WNV and DENV (NGC/DF/DHF) in the presence of MG132 and lactacystin (B). Presence of MG132 and lactacystin reduces virus titres compared with that of DMSO-treated cells following infection with pWNS, pWNSC13, pDENV and pDENVV14 viruses. The mutant viruses pWNSC14, pWNSC(d), pDENVC13 and pDENVC(d) show reduced virus titres in the presence or absence of MG132 and lactacystin. Virus titres are reduced in the presence of MG132 and lactacystin following infection with wild-type WNV and DENV (both clinical virus isolates and NGC strain). *P < 0.05 compared with DMSO-treated wild-type WNV/DENV-infected cells. **P < 0.05 compared with DMSO-treated pWNS and pDENV viruses.

This study was extended to investigate if proteasomes played a similar role during wild type infection. HEK293 cells were infected with WNV and DENV (NGC/DF clinical virus isolate/DHF clinical virus isolate) in the presence and absence of MG132 and lactacystin. MG132 and lactacystin reduced virus titres compared with that of DMSO-treated cells following infection with both WNV and DENV (Fig. 7B). Decreased virus titres [WNV: 2.2 to 2.3 log units reduction (28–30% reduction); DENV: 1.7 to 3 log units reduction (26–42% reduction)] were observed with MG132 and lactacystin treatment (Fig. 7B). Moreover, the degree of reduction in virus titre varied among the lab strain (DENV NGC) and the clinical virus isolates (DENV DF and DENV DHF). The percentage reduction in virus titre was significantly higher for the clinical virus isolates (35–42%) compared with the lab strain DENV NGC (26–28%). The hSec3p levels and C protein levels were shown in Fig. S9. Overall, this reaffirmed that clinical DENV isolates exploited proteasome system effectively for efficient flavivirus production.

Influence of mutations on flavivirus production

Human Sec3p was shown to function as a negative regulator of flavivirus infection by modulating flaviviral RNA transcription and translation through the sequestration of elongation factor-α (EF1α) (Bhuvanakantham et al., 2010a). HEK293, hSec3p293OE and hSec3p293KD cells were infected with WNV/DENV, pWNS/pDENV and various mutant viruses. Reduced virus titres resulted from the mutations at 14 (pWNSC14), 109–114 of WNV C protein [pWNSC(d)] and 13 (pDENVC13), 102–107 of DENV C protein [pDENVC(d)] in HEK293 cells (Fig. 8). In all the other experimental groups, reduced virus titres were observed with hSec3p293OE cells and higher virus titres were observed with hSec3p293KD cells (Fig. 8).

Figure 8.

Effect of mutations on flavivirus production. HEK293, hSec3p293OE and hSec3p293KD cells were infected with WNV/DENV or mutant viruses. Reduced virus titres are obtained with pWNSC14, pWNSC(d), pDENV13 and pDENV(d)-infected HEK293 cells. In addition, reduced virus titres are observed with hSec3p293OE cells and higher virus titres are observed with hSec3p293KD cells in all the experimental groups. *P < 0.05 compared with DMSO-treated wild-type WNV/DENV-infected cells.

Discussion

Viruses containing their nucleic acid enclosed in a protein shell are parasites of their host organisms. Although viruses possess simple structures, they employ sophisticated mechanisms to replicate within their hosts. Viruses, in general, rely on host cellular components for replication and transcription of their genomic RNAs. They utilize their structural and non-structural proteins to hijack and manipulate the host cell environment to support their life cycle. These virus-host interactions are complex and dynamic. Understanding this multifaceted bio-molecular process has the potential to yield new and exciting strategies for therapeutic intervention.

The capsid (C) protein of flavivirus is the first viral protein synthesized in an infected cell. The capsid protein is a key structural component of virus particles. They are nucleic acid-binding proteins whose known function is packaging viral genomes into nucleocapsids. However, it is not required as a structural component until the later phases of assembly pathway. Growing evidences showed that capsid protein of RNA viruses have non-structural functions in addition to their structural roles (Yang et al., 2002; 2008; Oh et al., 2006; Hunt et al., 2007; Ko et al., 2010; Bhuvanakantham et al., 2010a). These non-structural functions were achieved by modulating host cell signalling pathways. This is possible since the C protein is the first viral protein to contact host cell proteins in the cytoplasm.

Although flavivirus replication is confined to the cytoplasm of host cells and virus assembly occurs on the endoplasmic reticulum (Mukhopadhyay et al., 2005), the C proteins are observed to have a nuclear phase (Makino et al., 1989; Tadano et al., 1989; Bulich and Aaskov, 1992; Westaway et al., 1997; Chang et al., 2001; Mori et al., 2005; Oh and Song, 2006; Bhuvanakantham et al., 2009; 2010b; Cheong and Ng, 2010). As there is no obvious role for nuclear localization of C protein in virus assembly, this nuclear phase may presumably modify the host cell environment in such a way that virus replication and dissemination is enhanced (Mori et al., 2005; Bhuvanakantham et al., 2009). This suggested that the dual cellular distributions (nucleus and cytoplasm) of C proteins place them in a strategic position to act as a modulator of the host cell environment.

The reported principle non-structural function of C protein in arthropod-borne flaviviruses is mainly associated with apoptosis (Lee et al., 2006; Oh and Song, 2006; Oh et al., 2006; Limjindaporn et al., 2007; Yang et al., 2008; Liao et al., 2010; Netsawang et al., 2010; Bhuvanakantham et al., 2010b). The cellular functions of C protein in arthropod-borne flaviviruses other than its role as a major structural component and its involvement in apoptosis are poorly defined. This study aimed to investigate if C protein of WNV and DENV possess any other novel non-structural functions apart from its role in initiating apoptosis. We have reported earlier (Bhuvanakantham et al., 2010a) that hSec3p functioned as a novel antiflaviviral factor. The hSec3p battled against flavivirus infection by forming more hSec3p–EF1α complex and decreasing the amount of free EF1α available to bind with viral replicative complex. As a result, decreased viral RNA and proteins were synthesized, which then led to decreased virus production (Bhuvanakantham et al., 2010a).

If hSec3p truly posed a hindrance to flavivirus replication, flaviviruses should have evolved some strategies to circumvent the anti-viral effects of hSec3p in a natural infection. This study has unveiled how flavivirus C protein overcome the anti-viral effects of hSec3p. Investigation into the molecular mechanism revealed that flavivirus utilized its capsid protein to degrade hSec3p through proteasome degradation pathway.

The proteasome degradation pathway plays a crucial role in various biological processes in cells such as antigen processing, apoptosis, signal transduction, transcriptional regulation and help to eliminate non-functional, misfolded or unwanted proteins (Coux et al., 1996; Pickart, 1997; Glickman, 2000; Niedermann, 2002). Several viruses exploited the proteasomal pathway to support efficient virus production such as to escape from the immune system, during release from cells or to suppress apoptosis (Kikkert et al., 2001; Sakurai et al., 2004; Hassink et al., 2006; Urata et al., 2007; Mack et al., 2008; Lagunas-Martinez et al., 2010; Raaben et al., 2010).

Analysis of hSec3p stability in the presence of MG132, a proteasomal inhibitor, indicated that intact proteasome activity was required to mediate the reduction of hSec3p levels. The stability of hSec3p was compromised following flavivirus infection. This study clearly demonstrated that flavivirus C protein was capable of reducing hSec3p levels in the absence of other viral components and flaviviruses subverted the proteasome degradation pathway for their own advantage. Studies performed with mutants of C protein revealed that this protein must physically bind to hSec3p to execute the degradation of hSec3p via proteasome-mediated pathway. The degradation motifs important to mediate the degradation of hSec3p resided between the amino acid residues 109–114 of WNV C protein and 102–107 of DENV C protein.

After confirming the involvement of proteasomes in decreasing hSec3p levels using MG132, this study was extended to dissect which of the three proteolytic functions of 20S proteasome was activated by flavivirus C protein. Analysis of the specific proteolytic activities of 20S proteasome delineated that chymotrypsin-like and caspase-like activities of 20S proteasome were activated by WNV/DENV C protein. Further detailed analyses using inhibitors discovered that the chymotrypsin-like proteolytic function of 20S proteasome was activated by C protein to execute the proteasome-mediated degradation of hSec3p and caspase-like function had no effect on modulating endogenous hSec3p levels. Based on our previous publication (Bhuvanakantham et al., 2010a) and the data generated from this study, we proposed a model as depicted in Fig. 9. Flavivirus C protein induced the degradation of hSec3p by activating the chymotrypsin-like proteolytic function of 20S proteasome. As a result, hSec3p levels were reduced in flavivirus-infected cells, which subsequently resulted in the decreased formation of EF1α–hSec3p complex and rendered free EF1α readily available to interact with flaviviral RNA to aid viral RNA transcription and translation.

Figure 9.

A model depicting the biological consequences of flavivirus C protein-hSec3p interaction. Flavivirions enter the cell by receptor-mediated endocytosis and release the nucleocapsid in the cytoplasm followed by its dissociation into RNA and C proteins. The RNA is translated into a polyprotein which subsequently forms structural and non-structural proteins. The non-structural proteins together with some cellular proteins bind flaviviral RNA and form the viral replicative complex. Free EF1α then interacts with viral replicative complex and results in efficient viral RNA and viral protein synthesis. This results in enhanced virus assembly and virus secretion. Flavivirus C protein sequesters hSec3p and degrades it. This results in the decreased formation of hSec3p–EF1α complex. Free EF1α readily interacts with flaviviral RNA genome and potentiates flavivirus production. The effects mediated by hSec3p are shown in red and the effects mediated by C protein are shown in blue. The broken arrow represents the interference caused by hSec3p (red) at particular step(s).

Drug inhibition studies showed that virus titres were significantly reduced in the presence of MG132 and lactacystin following infection with wild-type or mutant WNV/DENV. In addition, the reduction in virus titres and the levels of hSec3p were significantly higher with DENV clinical isolates (DENV DF/DENV DHF) compared with that of lab strain (DENV NGC). This demonstrated that clinical virus isolates from DF and DHF patients had more pronounced effect in manipulating the proteasome function and thereby reducing hSec3p levels significantly. Perhaps the expression levels of hSec3p could be used as an indicator for diseases pathogenesis. Much lowered hSec3p levels could create a better micro-environment for clinical virus isolates to establish infection. This warrants further investigation with more clinical isolates of varied disease severity outcome.

Consistent with this study, the involvement of proteasomes to favour flavivirus infection have been reported for efficient WNV, DENV, HCV and JEV replication (Lin et al., 2005; Fink et al., 2007; Krishnan et al., 2008; Dutta et al., 2009; Gilfoy et al., 2009; Kanlaya et al., 2010). Flaviviruses have also utilized proteasome-dependent pathway to invade the host immune system by degrading STATs (Ashour et al., 2009). It is thus clear that proteasomes' involvement plays an essential role in favouring flavivirus infection. Similar scenario was observed during human immunodeficiency virus (HIV) infection. The deoxycytidine deaminase, APOBEC3G protein functioned as the anti-retroviral agent. One of the viral proteins encoded by HIV, Vif induced the proteasome-dependent pathway to degrade APOBEC3G and nullified the anti-viral function of APOBEC3G (Mehle et al., 2004; Albin and Harris, 2010; Fourati et al., 2010; Ooms et al., 2010; Wissing et al., 2010; Zangari et al., 2011). This showed that viruses are capable of utilizing proteasomal functions to degrade the anti-viral cellular proteins to favour the infection.

Studies have also shown that host proteins such as Jab1, subunit of COP9 signalosome and Makorin ring finger protein 1 induced the degradation of flavivirus C protein in a proteasome-dependent manner (Oh et al., 2006; Ko et al., 2010). Consistent with these findings, we also observed the increase in the levels of C protein in the presence of proteasomal inhibitors. Overall, this suggested that proteasomal degradation pathway played a dual role during flavivirus infection. The host cell uses it as a tool to defend against the virus and the virus exploits it to support successful infection. This ambivalent behaviour of proteasomal system suggested that this could have resulted from the co-evolution of the host and flavivirus.

By inhibiting proteasomal activity, it is possible to impair several processes such as cell proliferation, inflammatory responses and apoptosis. The proteasome inhibitor, bortezomib, has been approved by FDA to use in cancer therapy and organ transplantation (Everly et al., 2010; Kennedy et al., 2010; Zangari et al., 2011; Flechner et al., 2010). It will therefore be of great interest to investigate further on targeting the chymotrypsin-like proteolytic function of 20S proteasome in an effort to strengthen the host anti-viral state against flavivirus.

Experimental procedures

Cells and viruses

HEK293 (human embryonic kidney), hSec3pOE (hSec3p overexpressing 293 cells) and hSec3pKD (293 cells knocked down for hSec3p) cells were maintained at 37°C in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal calf serum (PAA Laboratories) and Geneticin (Sigma). Both hSec3pOE and hSec3pKD cells were generated in our previous study (Bhuvanakantham et al., 2010a). West Nile virus (Sarafend) and Dengue 2 virus (NGC) were gifts from late Emeritus Professor E. G. Westaway (Sir Albert Sakzewski Virus Research Centre, Australia). DENV (serotype 2) DF and DENV (serotype 2) DHF clinical isolates (isolated during 2005 epidemics in Singapore) were kindly provided by Dr Lee Ching, Head of Environmental Health Institute, Singapore. Virus infection was carried out at moi of 1 or 0.1 using wild-type or mutant WNV/DENV viruses.

Cloning

The full-length and truncated C constructs generated earlier (Bhuvanakantham and Ng, 2005; Bhuvanakantham et al., 2010a) were used in this study. In addition, QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used to mutate the required residues on WNV/DENV Myc-tagged or HA-tagged C protein. To introduce mutations in the full-length infectious clones of WNV (Li et al., 2005) and DENV (gift from Dr Andrew Davidson, Bristol University, UK), mutations were first introduced on a 1.3 kb carrier plasmid of WNV and DENV as described earlier (Li et al., 2005). The 1.3 kb carrier plasmid contains 5′ UTR, C, prM and partial envelope sequences of flavivirus in pBR322 vector. After confirming the presence of introduced mutations by sequencing, the remaining purified plasmids were subjected to RE digestion with 1 μl of BsiWI and MluI (New England Biolabs, USA) each at 37°C for 2 h. The reaction was subjected to agarose gel electrophoresis and the 1.3 kb fragment carrying the mutations was excised from the gel and purified. The full-length infectious clones of WNV/DENV were also RE-digested and subjected to electrophoresis in the same manner and the 11 kb fragment was purified from the gel. Approximately 7.5 μl of the 1.3 kb fragment and 0.5 μl of 11 kb DNA fragments were ligated using 1 μl T4 DNA ligase and transformed into DH5α cells. Positive clones were then sequenced and propagated. The mutants generated in this study were shown in Table S3.

In vitro synthesis of infectious RNA

About 5 μg of full-length/mutant infectious clones of WNV and DENV were linearized with XbaI (Promega, USA). The linearized DNA was purified by phenol-chloroform extraction and ethanol precipitation and reconstituted in 15 ml of RNase-free water. The linearized DNA was used for in vitro synthesis of infectious RNA using T7 RibomaxTM large-scale RNA production system and cap analogue according to the manufacturer's protocol (Promega, USA). Briefly, the linearlized DNA was mixed with T7 reaction components and incubated at 37°C for 4 h. The resulting RNA was purified using phenol : chloroform : isoamyl alcohol (25:24:1, v/v, Invitrogen) and precipitated with isopropanol on ice. The precipitated RNA was then pelleted at 16 000 g for 30 min (Sigma, USA) and washed with 70% ethanol in RNase-free water. The RNA was resuspended in RNase-free water and quantitated with Nanodrop (Thermo Scientific, USA).

Western blotting

For Western blotting, samples were electrophoresed in 10–15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred on to PVDF. The membrane was then incubated with anti-C (1:200) or anti-V5 (1:1000) or anti-HA (1:1000) or anti-Myc (1:1000) or anti-hSec3p (1:300) or anti-hSec6p (1:300) or anti-actin (1:2000, Millipore) antibodies followed by goat anti-rabbit or anti-mouse IgG conjugated with horseradish-peroxidase (Pierce Biotechnology) and the immunoreactive bands were developed using SuperSignal Pico system (Pierce Biotechnology).

Cell-based fluorescence assay

HEK293/hSec3pKD cells were seeded onto poly-D-lysine (Sigma, USA) coated 96-well plates and transfected with various WNV/DENV full-length/truncated/mutated plasmids or infected with WNV/DENV or pWNS/pDENV mutants viruses at moi of 1. At various timings post transfection or post infection, culture supernatant was removed and the cells were washed with PBS and fixed with 4% paraformeldyhyde for 15 min and permeabilized with 0.2% Triton-X for 5 min at RT. Cells were then blocked with 1% BSA in PBS. This was followed by the addition of mouse monoclonal anti-hSec3p (Abnova, USA) and rabbit polyclonal anti-actin (Sigma, USA) antibodies and incubated at 37°C for 2 h. Following three washes with PBS, the samples were treated with anti-mouse or anti-rabbit Alexa488 or Alexa594 secondary antibodies (Invitrogen, USA) and washed thrice in PBS before analysing samples in a fluorescence spectrophotometer. Similar assays were developed to detect hSec6p, viral C/E proteins and Myc-tagged C proteins using anti-hSec6p (Abnova, USA), anti-flaviviral E (4G2, Chemicon, USA), anti-C (kind gift from Late Emeritus Professor E.G. Westaway), anti-HA and anti-Myc (Clontech, USA) antibodies.

In vitro translation assay to study hSec3p degradation

The full-length C plasmids such as WMycC (WNV) and DMycC (DENV) or the hSec3p-binding defective C mutants (first 15 amino acids of C protein deleted), WMycC5′Δ15 (WNV) and DMycC5′Δ15 (DENV) were added to the rabbit reticulocyte mix in the TNT quick-coupled transcription/translation system (Promega, USA) together with hSec3p RNA. The in vitro translation assay was performed at 30°C for 1.5 h following manufacturer's instructions (Promega, USA). The amount of hSec3p was then analysed by Western blotting using anti-hSec3p antibody (Abnova, USA).

Drug inhibition studies

Host cell transcription was blocked by incubating HEK293 cells with 2 μg ml−1 of actinomycin D (Sigma, USA) for 1 h. This was followed by infection with WNV/DENV. Actinomycin D was included throughout the infection period. MG132 is the reversible cell-permeable proteasome inhibitor. HEK293/hSec3pKD cells were treated with 10 μM MG132 (Calbiochem, USA) for 1 h. This was followed by infection with WNV/DENV or transfection with recombinant WNV/DENV C proteins. MG132 was included throughout the infection or transfection period. HEK293/hSec3pKD cells were incubated with various concentrations of lactacystin (inhibitor of chymotrypsin-like activity) and YU-102 (inhibitor of caspase-like activity) at 37°C for 2 h and observed for cytotoxic effects using the fluorescent MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega, USA).

Generation of mutant capsid viruses

HEK293 cells were electroporated with 25 mg of RNAs synthesized from pWNS/mutant pWNS clones using RiboMAX RNA synthesis kit (Promega) as described earlier (Li et al., 2005). The culture supernatants collected from the electroporated cells were passaged twice before used in downstream experiments.

Plaque assay

HEK293, hSec3pOE and hSec3pKD cells were treated with DMSO, MG132 or lactacystin and infected with wild-type WNV/DENV (NGC, DF, DHF) or mutant pWNV/pDENV viruses at moi of 1. At the indicated timings (18 h p.i. for WNV and 48 h p.i. for DENV if one timing is used), cell culture supernatants were collected for plaque assay.

Measurement of proteolytic activities of 20S proteasome

HEK293/hSec3pKD cells were seeded on to 96-well plate and transfected with various full-length/truncated/mutated WNV/DENV C plasmids or RNAs in vitro transcribed from infectious clones of WNV/DENV. At 12 or 24 h post transfection, Proteasome-Glo cell-based assay kits for chymotrypsin-like, trypsin-like and caspase-like activities (Promega, USA) were used to measure the chymotrypsin-like, trypsin-like and caspase-like activities of 20S proteasome following manufacturer's instructions.

Detection of intracellular viral RNA by real-time RT-PCR

HEK293 cells were infected with wild-type WNV/DENV or mutant viruses. At indicated timings, cells were washed with PBS, incubated with an alkaline/high-salt solution (1 M NaCl, 50 mM Na bicarbonate, pH 9.5) and washed three times in PBS to remove surface-bound viruses. Total RNA was extracted and analysed by real-time RT-PCR with GAPDH mRNA as the endogenous control. Plus- and minus-strand viral RNA levels from various mutant viruses-infected cells were determined relative to that from wild-type WNV/DENV-infected HEK293 cells as described by Davis et al. (2007).

Acknowledgements

This work is supported by the grants from Biomedical Research Council, Singapore (BMRC/06/1/21/19/451), National University of Singapore (R-182-000-115-112) and NMRC/NRF (R-182-000-220–275). The authors thank the EDEN team especially Yee Sin Leo, Lee Ching Ng and Eng Eong Ooi for providing the clinical virus isolates. The authors do not have any conflict of interest.

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