Rift Valley Fever Virus NSs Protein Promotes Post-Transcriptional Downregulation of Protein Kinase PKR and Inhibits eIF2α Phosphorylation

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-β mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or α-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)–mediated eukaryotic initiation factor (eIF)2α phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2α accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2α phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2α phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.


Introduction
Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen, which is distributed in sub-Saharan Africa [1] and has also caused large outbreaks in Madagascar [2], Egypt [3,4,5], Saudi Arabia [6], and Yemen [6]. In endemic areas, RVFV naturally circulates among mosquitoes and ruminants, such as sheep, goat and cattle [7]. RVFV infection in adult ruminants causes febrile illness and a high rate of abortions, while some newborn animals less than 1-2 weeks of age develop an acute infection which results in higher mortality rates than those in adults [8]. Humans infected with RVFV usually develop an acute febrile myalgic syndrome; however, a small percentage of patients have a lethal illness that results in hepatic damage, hemorrhagic fever-like illness, encephalitis and/or retinal vasculitis [8].
Due to the exotic origin of the virus, the potential for the aerosol transmission [9,10,11,12] and serious consequences for humans and livestocks, wild-type (wt) RVFV is classified as a Risk Group 3 pathogen, that needs to be handled in a high-containment facility, e.g., a biosafety level (BSL)-4 laboratory, whereas a highly attenuated MP-12 strain of RVFV, produced after 12 serial passages of wt RVFV ZH548 in MRC-5 cells in the presence of 5fluorouracil [13], is a Risk Group 2 pathogen. RVFV, which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome composed of S, M and L segments [14]. The S segment encodes N and NSs genes and uses an ambi-sense strategy to express the N and NSs proteins in infected cells; N mRNA encoding N protein is transcribed from the viral-sense (negative-sense) S segment, while NSs mRNA encoding NSs protein is transcribed from the antiviral-sense (positive-sense) S segment. Monocistronic M mRNA and L mRNA are transcribed from the viral-sense M and L segments, respectively. M mRNA has one large open-reading frame (ORF) which encodes the nonstructural NSm protein, a 78-kDa glycoprotein and two major viral structural glycoproteins, Gn and Gc [15,16]. L mRNA encodes L protein, a viral RNA-dependent RNA polymerase. Both N and L proteins are required for viral RNA synthesis, while Gn and Gc function as envelope proteins [14]. NSm and NSs, both nonstructural proteins, are dispensable for viral replication in cell cultures [17,18,19,20], but are involved in viral pathogenesis [21,22,23].
RVFV NSs protein is not essential for virus replication in cell cultures [19,20], yet plays a critical role in viral virulence [21]. A naturally occurring RVFV mutant Clone 13, which lacks approximately 70% of the NSs gene [20], is highly attenuated in mouse, and, when reassortant viruses between wt RVFV and Clone 13 were characterized, the NSs was revealed as a major determinant of viral virulence in the mouse model [21]. The NSs localizes in the nucleus and cytoplasm in both RVFV-infected cells and NSs-expressing cells; further, the nuclear NSs, but not the cytoplasmic NSs, forms a unique filamentous structure [24]. The NSs suppresses the transcription of host mRNAs by interacting with the p44 subunit of TFIIH, an essential transcriptional factor for cellular RNA polymerase II [25]. Furthermore, the RVFV NSs binds to Sin3A-Associated Protein 30 (SAP30), which is important for maintaining the repressor complex containing histone deacetylase 3 on the interferon (IFN)-b promoter, and suppresses the IFN-b promoter activation early in infection [26]. Accordingly, the NSs protein in the nucleus of infected cells most probably exerts these host transcriptional-suppressive activities, including that of IFN production inhibition, and contributes to viral virulence [21]. In contrast, the biological function of cytoplasmic NSs is largely unknown. RVFV NSs expression promotes RVFV minigenome RNA synthesis driven by N and L protein in expression studies [27]. Because RNA synthesis of bunyaviruses occurs in the cytoplasm, NSs protein in cytoplasm may promote the minigenome RNA synthesis by unknown mechanism.
To establish that RVFV NSs exhibits a novel function, we hypothesized that, when combined, RVFV replication and NSsinduced host transcription suppression likely induces a cellular environment that is unsuitable for viral replication. Thus, to secure efficient RVFV replication, the NSs protein, in turn, alters this putative, virally unfriendly cellular environment to one that supports efficient viral replication. To test this possibility, we examined the replication of RVFV lacking the NSs gene, a mutant which was generated by employing a reverse genetics system [19], in the presence of a host transcription inhibitor, e.g., actinomycin D (ActD) or a-amanitin; these drugs were selected because they mimicked the host transcriptional suppressive activities of the NSs. Consistent with our supposition, RVFV lacking the NSs, but not RVFV, failed to replicate efficiently in ActD-treated cells. We noted that double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2a phosphorylation suppressed the translation of RVFV lacking NSs in the presence of ActD. Further studies uncovered that RVFV NSs promoted PKR downregulation as early as 4 hours post-infection, and prevented eIF2a phosphorylation, which secured efficient viral translation. We speculate that this novel function of RVFV is important for counteracting the antiviral activities of PKR and allowing efficient virus replication and survival in infected hosts.

RVFV lacking the NSs undergoes poor viral protein synthesis and viral replication in ActD-treated cells
To explore a novel biological function of RVFV NSs protein, in addition to its host transcriptional shutoff activity, we investigated the effect of ActD, a host transcriptional inhibitor, on the replication of MP-12 lacking the NSs gene in IFN-incompetent VeroE6 cells. VeroE6 cells were mock-infected or infected with MP-12 or rMP12-rLuc, which expressed Renilla luciferase (rLuc) in place of the NSs ( Figure 1A) at a multiplicity of infection (moi) of 3. After 1 h virus adsorption, cells were incubated in the absence or presence of 5 mg/ml of ActD. Supernatants were harvested at 16 hours post-infection (h.p.i.), and virus titers were measured by plaque assay. ActD treatment had little effect on MP-12 titers, yet it significantly reduced the titer of rMP12-rLuc ( Figure 1B), which suggested that the RVFV NSs was important for an efficient virus replication in the presence of ActD.
To understand how the NSs protein exerted an efficient viral replication in the presence of ActD, we analyzed the status of host and viral translation. VeroE6 cells were mock-infected or independently infected with MP-12, rMP12-rLuc and rMP12-C13type, the latter of which lacks approximately 70% of the NSs ORF ( Figure 1A), at an moi of 3. Cells were radiolabelled with [ 35 S] Methionine/Cysteine from 15 to 16 h.p.i. Cell extracts were prepared at 16 h.p.i., and the samples were applied to SDS-PAGE ( Figure 1C, top panel). In the absence of ActD, the host protein synthesis of rMP12-rLuc-infected cells ( Figure 1C, lane 5) and rMP12-C13type-infected cells ( Figure 1C, lane 7) was more efficient than that of MP-12-infected cells ( Figure 1C, lane 3). It is possible that NSs-mediated transcriptional suppression that occurred only in MP-12-infected cells, but not in cells infected with viruses lacking NSs, caused a reduction of host mRNA abundance, leading to the less efficient translation of host mRNAs in MP-12-infected cells. Efficient N protein synthesis occurred in cells infected with all three viruses in the absence of ActD, a finding which was consistent with our previous studies [19], while for some unknown reasons the accumulation of N proteins of rMP12-rLuc and rMP12-C13type was slightly higher than that of MP-12. ActD treatment resulted in a strong inhibition of both host proteins and N protein synthesis in rMP12-rLuc-infected cells ( Figure 1C, lane 6) and rMP12-C13type-infected cells ( Figure 1C, lane 8). In contrast, ActD treatment did not inhibit N protein translation in MP-12-infected cells; rather, it moderately inhibited host protein synthesis in MP-12-infected cells ( Figure 1C, lane 4). A similar ActD-induced, moderate host protein synthesis inhibi-

Author Summary
The mosquito-borne bunyavirus Rift Valley fever virus (RVFV) devastates both humans and domestic animals; it causes abortions in ruminants and complications such as hemorrhage, encephalitis, or retinal vasculitis in humans. A major RVFV virulence factor, NSs, disables host cell mRNA synthesis. Here we describe our new evidence that showed NSs working in a second way; in addition to inhibiting host cell transcription, NSs kept translation active in infected cells. It is well-established that activated protein kinase PKR phosphorylates a translation factor, eIF2a, and then this phosphorylated eIF2a suppresses translation. We found that NSs decreased PKR abundance and prevented eIF2a phosphorylation in infected cells, allowing efficient viral translation and replication. In contrast, when cells were infected with an RVFV mutant lacking NSs in the presence of transcriptional inhibitors that mimic the transcription inhibition function of NSs, the PKR reduction did not occur and phoshorylated eIF2a was accumulated, resulting in the inhibition of virus gene expression and replication. Thus, NSs functions in two ways to help RVFV replicate in mammalian hosts: its newly identified PKR downregulation function secures efficient viral translation, and its host transcription inhibition function suppresses the expression of host innate antiviral functions.
tion also occurred in mock-infected cells ( Figure 1C, lane 2). Western blot analysis of cell extracts at 16 h.p.i. clearly showed that ActD treatment strongly inhibited N protein accumulation in rMP12-rLuc-infected cells and rMP12-C13type-infected cells, but not in MP-12-infected cells ( Figure 1C, bottom panels). In summary, ActD treatment had little effect on MP-12 replication, whereas it strongly inhibited the expression of both host and viral proteins in the cells infected with MP-12 lacking the NSs gene, which resulted in poor virus production.

NSs protein expression promotes viral replication in rMP12-rLuc-infected cells in the presence of ActD
To further confirm that the NSs exerted an efficient viral replication in the cells that underwent ActD-induced host transcriptional suppression, 293 cells, which showed higher RNA transfection efficiencies than did VeroE6 cells (data not shown), were infected with rMP12-rLuc at an moi of 2. After virus adsorption, infected cells were mock-transfected or independently transfected with in vitro-synthesized RNA transcripts encoding chloramphenicol acetyltransferase (CAT), MP-12 NSs, or wt RVFV ZH501 NSs. Then, the cells were mock-treated or treated with ActD. Analysis of rLuc activities at 16 h.p.i. demonstrated that NSs expression had little effect on rLuc activities in the absence of ActD (Figure 2A). In the presence of ActD, rLuc activities were clearly higher in the cells transfected with MP-12 NSs RNA transcripts or ZH501 NSs RNA transcripts than in those transfected with CAT RNA transcripts or in the mocktransfected samples (Figure 2A). In NSs RNA-transfected cells, similar levels of rMP-12-rLuc titers were observed in both the ActD-treated and mock-treated samples, whereas the rMP12-rLuc virus titers in mock-transfected cells and CAT RNA-transfected cells were significantly lower in the presence of ActD compared to the ActD-untreated cells ( Figure 2B). Western blot analysis showed that NSs proteins were indeed expressed in the cells transfected with RNA transcripts encoding MP-12 NSs or wt RVFV ZH501 NSs ( Figure 2C, lanes 3, 4, 7, and 8). Also NSs protein expression increased the accumulation of rMP12-rLuc N protein in the presence of ActD ( Figure 2C, lanes 7 and 8). These data demonstrated that NSs exerted efficient rMP12-rLuc replication in the presence of ActD. We noted that ActD treatment modestly reduced the accumulation of the CAT protein ( Figure 2C, lane 6). Probably large amounts of CAT RNA transcripts that were introduced into the cells partly overcame the translational suppressive effects that were induced by the combination of rMP-12-rLuc replication and ActD treatment.
To test the possibility that ActD treatment alone suppressed translation and RVFV NSs counteracted it, 293 cells were mocktreated or treated with 5 mg/ml of ActD. We examined the resulting polysome profiles at 16 h post-ActD treatment ( Figure  S1A). Because ActD treatment at 5 mg/ml inhibits the transcription mediated by RNA polymerases I, II and III [28], we expected a reduction in the abundance of cellular mRNAs, tRNAs, and ribosomal RNAs, leading to reduced abundances of polysomes. ActD treatment indeed resulted in a reduced abundance of polysomes, whereas it did not substantially alter the polysome pattern, a finding which suggested to us that ActD treatment did not abolish translational activities. To test the translational competence of the cells treated with transcriptional inhibitors, we transfected 293 cells with in vitro-synthesized RNA transcripts encoding rLuc gene. Cells were mock-treated or treated with ActD or a-amanitin, an RNA polymerase II inhibitor [29]. The rLuc activities at 16 h post-transfection were slightly increased in the cells treated with ActD or a-amanitin ( Figure S1B), demonstrating active host translation activities in the presence of either ActD or a-amanitin. These data led to the suggestion that by combining the replication of RVFV lacking the NSs and treatment of ActD, a cellular condition that is unfavorable for translation could be induced and that NSs expression somehow altered the cellular environment from one that was translationally inactive to one translationally active.

MP-12 replication promotes translation of rLuc mRNA of rMP12-rLuc
To establish that NSs exerts an efficient viral translation in the presence of a transcription inhibitor, we tested whether coinfection of MP-12 and rMP12-rLuc increases the translation of rLuc mRNA of rMP12-rLuc in the presence of ActD. VeroE6 cells were mock-infected or co-infected with rMP12-rLuc and MP-12; rMP12-rLuc was infected at an moi. of 2, while MP-12 was infected at moi. of 0.1 or 1, as indicated in Figure 3. The cell extracts were harvested at 16 h.p.i., and the rLuc activities ( Figure 3A) and accumulation of viral RNAs ( Figure 3B) were examined. As expected, ActD-treatment reduced the rLuc  activities in cells infected with rMP12-rLuc alone ( Figure 3A). Coinfection of MP-12 resulted in the reduction of both rLuc activities ( Figure 3A) and the amounts of rLuc mRNA ( Figure 3B, lane 4) in the absence of ActD. In the presence of ActD, MP-12 co-infection also reduced the rLuc mRNA abundance ( Figure 3B, lane 9), whereas it increased rLuc activities ( Figure 3A). The results suggested that NSs protein expressed from MP-12 S-segment promoted an efficient translation of the rLuc mRNA of rMP12-rLuc in the presence of ActD. We also noted that MP-12 coinfection did not reduce the abundance of the viral-sense rMP-12-rLuc S segment in the presence of ActD, and yet it caused the reduction of rLuc mRNA abundance ( Figure 3B, lane 9), which we believe implies that NSs expression and ActD treatment generated a cellular environment that was more favorable for rMP-12-rLuc RNA replication than for transcription.

The status of eIF2a phosphorylation in infected cells
The dsRNAs generated during viral replication activate PKR, which in turn phosphorylates eIF2a [30]. Also 59-triphosphated single-stranded RNAs activate PKR [31]. The eIF2a is a component of eIF2, which binds to GTP and Met-tRNA to deliver the Met-tRNA to the start codon in capped mRNA, forming a 43S pre-initiation complex [32]. Upon the binding of the 60S ribosomal subunits to the 43S preinitiation complex, eIF2-GDP is released from the ribosome and undergoes a GTP exchange reaction catalyzed by binding with eIF2B, and the resultant eIF2-GTP is recycled for the next round of translation initiation. The phosphorylated eIF2a binds to eIF2B with a high affinity and prevents eIF2B to be used for the subsequent eIF2-GDP-to-eIF2-GTP exchange reaction, leading to the suppression of translation initiation. Hence, the phosphorylation status of eIF2a plays a critical role in translational control [32].
We suspected that rMP12-rLuc replication in the presence of ActD may generate dsRNAs and/or 59-triphosphated singlestranded RNAs, which activate PKR, resulting in the phosphorylation of eIF2a. When we analyzed the level of eIF2a phosphorylation in VeroE6 cells infected with rMP12-rLuc in the absence of transcription inhibitor, we found low levels of eIF2a phosphorylation from 8 to 24 h.p.i. and an efficient accumulation of N protein at 8 h.p.i. and onward ( Figure 4A-4C). In contrast, when we treated rMP12-rLuc-infected cells with ActD or aamanitin, either compound induced an efficient accumulation of phosphorylated eIF2a from 8 to 16 h.p.i., with a concomitant poor N protein accumulation ( Figure 4A-4C) and virus replication suppression ( Figure 4D). The mechanism of reduction in the abundance of the phosphorylated eIF2a at 24 h.p.i. in the presence of ActD or a-amanitin ( Figure 4, lanes 13 and 19) is unknown. When we treated rMP12-rLuc-infected cells with different concentrations of ActD or a-amanitin, we found that the inhibition of phosphorylated eIF2a accumulation was dependent on the concentrations used ( Figure S2). Also a strong correlation was seen between an increase in eIF2a phosphorylation and the decrease in both N protein accumulation and infectious virus production ( Figure S2). In contrast, no significant accumulation of phosphorylated eIF2a occurred in MP-12infected VeroE6 cells in the presence of ActD ( Figure 4A-4C). As expected, ActD treatment had little effect on MP-12 replication ( Figure 4D). These data strongly suggested that the presence of highly phosphorylated eIF2a levels suppressed the translation of viral mRNAs, leading to inefficient virus production, and that the NSs protein somehow suppressed eIF2a phosphorylation, and facilitated efficient viral mRNA translation under the conditions of host transcriptional shutoff.
Because activated caspases 3, 7 and 8 can cleave PKR, releasing the biologically active C-terminus kinase domain from the Nterminus inhibitory domain, resulting in eIF2a phosphorylation [33], we subsequently tested whether the accumulation of phosphorylated eIF2a following the combined activity of viral replication and ActD-treatment was due to induction of apoptosis [34]. VeroE6 cells, either infected with rMP12-rLuc or mockinfected, received the pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp(OMe) fluoromethylketone (Z-VADfmk), in the presence of ActD or a-amanitin ( Figure S3). Judging from the resulting inhibition of cleaved caspase-3 accumulation in these cells, the Z-VADfmk treatment indeed inhibited apoptosis, whereas it had little effect on the eIF2a phosphorylation status and infectious virus yield. These data suggested to us that the accumulation of phosphorylated eIF2a in cells supporting rMP12-rLuc replication in the presence of ActD or a-amanitin was caspase-independent.

PKR exerts a critical role in eIF2a phosphorylation in infected cells
Four different kinases, including PKR, PKR-like ER kinase (PERK), heme-regulated inhibitor and the general control, nondepressible-2 (GCN2), are known to phosphorylate eIF2a [35]. To determine the role of PKR in the accumulation of phosphorylated eIF2a in rMP12-rLuc-infected cells treated with ActD or aamanitin, we generated a recombinant MP-12, rMP12-PKRDE7, which expresses a dominant-negative form of PKR, PKRDE7 [36] carrying the N-terminal Flag epitope tag, in place of the NSs ( Figure 5A). If the replication of MP-12 lacking the NSs gene in cells subjected to host transcriptional suppression activates PKR, which in turn phosphorylates eIF2a, then the virally-encoded PKRDE7 in rMP12-PKRDE7-infected cells would interfere with the PKR function, resulting in the inhibition of PKR-mediated eIF2a phosphorylation and thereby leading to efficient viral translation and virus production. VeroE6 cells were mock-infected or independently infected with rMP12-rLuc, rMP12-PKRDE7 and MP-12 at an moi of 3. After the removal of the inocula, cells were treated with ActD or a-amanitin, and cell extracts were harvested at 16 h.p.i. As expected, rMP12-rLuc replication in the presence of ActD or a-amanitin induced eIF2a phosphorylation, resulted in reduced virus replication ( Figure 5B-5D). In contrast, efficient N protein accumulation and efficient virus replication, with no significant accumulation of phosphorylated eIF2a occurred in both MP-12-infected cells and rMP12-PKRDE7infected cells in the presence of ActD-or a-amanitin ( Figure 5B-5D). This finding strongly suggested that PKR is important for eIF2a phosphorylation in cells infected with the RVFV lacking the NSs in the presence of transcription inhibitors.
To further confirm these data, viral protein accumulation and replication were analyzed in wt mouse embryonic fibroblast (MEF) cells and in Pkr 0/0 MEF cells lacking a functional PKR expression [37]. MP-12 efficiently replicated in both wt MEF and Pkr 0/0 MEF cells, and ActD treatment had little effect on N protein accumulation and virus replication ( Figure 5E, top and middle panels). rMP12-rLuc replication was not as efficient as MP-12 in wt MEF cells in the absence of ActD for an as yet unidentified reason ( Figure 5E, middle panel). In ActD-treated wt MEF cells, both rMP-12-C13type and rMP12-rLuc failed to efficiently accumulate N proteins, and rMP-12-rLuc replicated poorly, whereas rMP-12-rLuc underwent efficient N protein accumulation and viral replication in ActD-treated Pkr 0/0 MEF cells ( Figure 5E, top and middle panels). Furthermore, accumulation of phosphorylated eIF2a did not occur in Pkr 0/0 MEF cells that were infected with rMP12-rLuc in the presence of transcriptional inhibitors

Testing the possible NSs-mediated suppression of PKR phosphorylation
To know how the NSs suppressed the eIF2a phosphorylation activity of the PKR function, a dsRNA-binding assay was performed to test the possibility that the NSs binds to dsRNA, sequesters dsRNA from PKR, and interferes with the dsRNAmediated PKR activation. 293 cells were mock-infected or infected with rMP12-NSs-Flag carrying Flag-tagged NSs, rMP12-rLuc-Flag carrying Flag-tagged rLuc ( Figure 6A) or rMP12-PKRDE7 ( Figure 5A). In a separate experiment, 293 cells were transfected with in vitro-synthesized RNA transcripts encoding NSs. Lysates were prepared at 16 h.p.i. or 16 h post-transfection, and incubated with poly I:C beads (dsRNA) or poly (C) beads (ssRNA). Then the dsRNA-bound complexes were analyzed by a Western blot in which we used an anti-Flag antibody or anti-NSs antibody ( Figure 6B). As expected, dsRNA bound to PKRDE7 [36], whereas it poorly bound to the NSs from rMP12-NSs-Flaginfected cells and that from the NSs-expressing cells ( Figure 6B), which suggested that NSs did not suppress PKR activation by its binding to dsRNA.
Because activated PKR undergoes a structural alteration and autophosphorylation [30], we tested whether NSs prevented PKR autophosphorylation. 293 cells were infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRDE7, and mock-treated or immediately treated with ActD. Cell lysates were harvested at 16 h.p.i., and PKR was immunoprecipitated by anti-human PKR antibody, and the PKR bound to the protein A beads was subjected to an immunoprecipitation (IP)-kinase assay by using [c-32 P]ATP. We used 293 cells for this assay, because anti-human PKR antibody efficiently immunoprecipitated human PKR in 293 cells, but not non-human primate PKR in VeroE6 cells (data not shown). PKR is induced by IFN-a/b treatment [38] and the abundance of PKR could affect the results of the IP-kinase assay. We suspected that replication of MP-12 mutants lacking NSs may induce IFN-b production, leading to PKR induction [38], whereas ActD treatment prevented the IFN-b production [39]. Indeed, IFN-b mRNA accumulation occurred at 8 h.p.i. in rMP-12-rLucinfected cells in the absence of ActD, but not in the presence of ActD ( Figure 6C), a finding which suggested that ActD treatment inhibited the transcriptional induction of PKR which was induced by the type I IFN in infected 293 cells. We noted an efficient accumulation of rMP12-rLuc N mRNA at 8 h.p.i. in the presence of ActD ( Figure 6C). Because rMP-12-rLuc replication in the presence of ActD did not induce an accumulation of phosphorylated eIF2a early in the course of the infection ( Figure 4A), this efficient rMP12-rLuc replication probably occurred prior to 8 h.p.i. in the presence of ActD. As shown in Figure 6D, immunoprecipitated PKR from rMP12-rLuc-Flag-infected cells was phosphorylated both in the presence and absence of ActD, which led us to suggest that rMP12-rLuc-Flag replication activated PKR. In contrast, PKR phosphorylation did not occur in mockinfected cells or cells infected with rMP12-NSs-Flag or rMP12-PKRDE7 ( Figure 6D). Most probably, a dominant-negative PKRDE7 suppressed PKR activation and prevented PKR phosphorylation [36]. To determine why we failed to detect the presence of PKR autophosporylation in rMP12-NSs-Flag-infected cells, we examined the amounts of the immunoprecipitated PKR in these samples ( Figure 6D, bottom). Strikingly, substantial reductions in the abundance of cytoplasmic PKR occurred only in the cells supporting rMP12-NSs-Flag replication both in the presence and absence of ActD.

RVFV NSs promotes PKR downregulation
We subsequently tested the possibility that the NSs downregulated PKR expression or sequestered PKR into the nuclear compartment, leading to the suppression of eIF2a phosphorylation. 293 cells were mock-infected or infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRDE7, treated with ActD, and cytoplasmic and nuclear fractions of cell extracts were prepared at 16 h.p.i. Western blot analyses showed the presence of RVFV NSs both in the cytoplasmic and nuclear fractions, while rLuc and PKRDE7 signals were observed only in the cytoplasmic fraction. A substantial reduction in PKR abundance occurred in both the cytoplasmic and nuclear fractions of cells infected with rMP-12-NSs-Flag, but not in mock-infected cells and in those infected with rMP12-rLuc-Flag or rMP12-PKRDE7 ( Figure 7A), demonstrating that NSs induced the PKR downregulation in the infected cells. The PKR downregulation also occurred in MP-12infected VeroE6 cells and in MRC-5 cells that were infected with the wt ZH501 strain of RVFV (data not shown).
To determine whether NSs expression alone induces PKR downregulation, 293 cells were mock-infected or infected with rMP12-rLuc, immediately transfected with in vitro-synthesized RNA transcripts encoding rLuc or NSs, and treated with ActD ( Figure 7B, left panel). Cells were harvested at 16 h posttransfection. The clear reduction in the PKR abundance occurred in mock-infected cells expressing NSs, but not in those expressing rLuc ( Figure 7B, left panel), demonstrating that NSs protein alone exerted the PKR downregulation.
We subsequently examined the requirement of the ActD treatment for the PKR downregulation. 293 cells were transfected with in vitro-synthesized RNA transcripts encoding NSs or rLuc in the absence of ActD. Analysis of cell extracts harvested at 8 h posttransfection showed the PKR downregulation in the NSsexpressing cells ( Figure 7B, right panel), demonstrating that the NSs-mediated PKR downregulation occurred in the absence of ActD.
To further understand the mechanism of the NSs-mediated PKR downregulation, we examined whether NSs expression promoted degradation of PKR mRNA. 293 cells were mocktransfected or transfected with in vitro-synthesized RNA transcripts encoding NSs or rLuc. Then the cells were mock-treated or treated with ActD. Total RNAs were harvested at 8 h posttransfection and the expression levels of PKR mRNA were examined by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis ( Figure 7C). The relative expression levels of PKR mRNA were significantly increased in cells that were transfected with rLuc RNA transcripts or NSs RNA transcripts both in the absence and presence of ActD, which demonstrated to us that NSs expression did not promote the degradation of PKR mRNA. Efficient ActD-mediated suppression of IFN-b mRNA accumulation in rMP-12-rLuc-infected 293 cells ( Figure 6C) led us to suggest that unexpected increases in the abundance of PKR mRNA in the RNA-transfected 293 cells in the presence of ActD were IFN-b-independent. We suspect that the RNA transcripts that were taken up by the cells induced robust PKR mRNA synthesis prior to ActD-or NSs-mediated general transcription suppression.
Since NSs expression did not decrease the abundance of PKR mRNA ( Figure 7C), we next tested whether putative NSsmediated translational inhibition leads to a reduction in the abundance of PKR. To this end, 293 cells were mock-infected, infected with MP-12, or transfected with in vitro-synthesized RNA transcripts encoding NSs. Then cells were incubated with 100 mg/ ml of puromycin to completely shut off cellular translation or puromycin untreated, and cell extracts were harvested at 16 h.p.i. or post-transfection. As expected, puromycin treatment completely abolished the synthesis of N and NSs proteins in MP-12-infected cells ( Figure 7D, lane 4) and that of NSs in cells transfected with the RNA transcripts encoding NSs ( Figure 7D, lane 6). We found that treatment of 293 cells with puromycin for 16 h decreased the abundance of PKR only slightly ( Figure 7D, lane 2). Accordingly, it is highly unlikely that putative NSs-induced translational  We next performed pulse-chase experiments to know whether NSs expression promoted PKR degradation. Because immunoprecipitation experiments using various anti-PKR antibodies failed to convincingly demonstrate a radiolabelled endogenous PKR signal from extracts of 293 cells (data not shown), we examined the effect of NSs expression on the stability of an expressed mutant PKR, PKRK296R, lacking kinase activity [40] and carrying a Nterminal myc tag; expression of wild-type PKR was not used due to its strong host translation suppression effects (data not shown). 293 cells were mock-transfected or transfected with pcDNA3.1-Myc-PKRK296R, a plasmid encoding PKRK296R under cytomegalovirus promoter and radiolabelled with [ 35 S] methionine/cycteine between 14 and 16 h post-DNA transfection. After pulse-radiolabelling cell extracts were prepared from some samples. In other samples, cells were transfected with in vitrosynthesized RNA transcripts encoding rLuc or NSs, and cell extracts were harvested at 8 h post-RNA transfection. Then the cell extracts were subjected to radioimmunoprecipitation analysis using anti-myc antibody, which immunoprecipitated the pulseradiolabelled, myc-tagged PKR ( Figure 7E, lane 2). After 8 h chase, the amount of the radiolabelled myc-tagged PKR was clearly reduced in the cells expressing NSs ( Figure 7E, lane 4), but not those expressing rLuc ( Figure 7E, lane 3). Instead, the amount of radiolabelled myc-tagged PKR was increased slightly in rLucexpressing cells, presumably due to radiolabelling of continuously synthesized myc-tagged PKR by residual [ 35 S] methionine/ cysteine.
The complete shutoff of cellular translation by puromycin for 16 h did not result in the loss of endogenous PKR ( Figure 7D), whereas as early as 8 h post-transfection of RNA transcripts encoding NSs, the abundance of PKR decreased substantially ( Figure 7B) without reducing the abundance of PKR mRNA ( Figure 7C). Furthermore, NSs expression reduced the abundance of myc-tagged PKR that had been radiolabelled prior to NSs expression ( Figure 7E). These data strongly suggested that NSs induced the downregulation of PKR at a post-transcriptional level and pointed to a possibility that the NSs promoted PKR degradation.
We next tested whether RVFV NSs downregulated PKR by promoting PKR degradation through a ubiquitin-proteasome pathway. 293 cells were infected with rMP12-rLuc or MP-12, and were immediately treated with proteasome inhibitor MG132 or lactacystin. Cells were harvested at 8 h.p.i. and analyzed in Western blotting. As expected, rMP12-rLuc replication did not induce PKR downregulation ( Figure 7F, lane 1), while MP-12 replication induced the reduction of PKR abundance ( Figure 7F,  lanes 4). It was evident that the treatment of MP-12-infected cells with those proteasome inhibitors suppressed NSs-induced PKR downregulation ( Figure 7F, lanes 5 and 6), suggesting that NSs promoted PKR downregulation by the degradation through the proteasome pathway.
To know how quickly PKR downregulation occurred in MP-12infected cells, whole-cell extracts were prepared at 2, 4, 6 and 8 h.p.i. from MP-12-infected 293 cells and the amounts of NSs and PKR were determined ( Figure 7G). In the absence of proteasome inhibitor, a substantial reduction of PKR abundance occurred as early as 4 h.p.i., where the abundance of NSs ( Figure 7G, lane 2) was not as great as that at 8 h.p.i. (Figure 7G, lane 4). In the presence of MG132, the abundance of PKR decreased only slightly, and NSs accumulation was also somewhat less efficient. A similar trend for less efficient NSs accumulation in the presence of proteasome inhibitors was also shown in the data for Figure 7F. Our observation of less efficient NSs accumulation in MG132-treated cells was consistent with the report that MG132 induces eIF2a phosphorylation through GCN2 activation and translational suppression [42]. The abundance of NSs in MG132treated cells at 8 h.p.i. and that in untreated cells at 4 h.p.i. was similar, and yet PKR abundance in the former cells was clearly higher than that in the latter cells. Taken together, these data strongly suggested that RVFV NSs promoted PKR degradation through the proteasome-dependent pathway.

Biological significance of NSs-mediated PKR downregulation
The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene initially in type I IFN-incompetent VeroE6 cells [43,44] in the presence of ActD or a-amanitin, which served as a surrogate of the host mRNA synthesis suppression function of the NSs. The NSs protein was essential for efficient virus replication in the presence of ActD or a-amanitin. We found that the replication of RVFV lacking the NSs gene in the presence of a transcription inhibitor induced an accumulation of phosphorylated eIF2a, The accumulation of phosphorylated eIF2a in the presence of transcriptional inhibitors, did not occur in VeroE6 cells that were infected with RVFV expressing a dominant-negative form of PKR (PKRDE7) and in MEF cells lacking functional PKR ( Figure 5). These findings suggested to us that PKR played a major role in increasing phosphorylated eIF2a; however, it is currently unclear whether other kinases, such as PERK or GCN2, have a possible role in eIF2a phosphorylation in rMP-12-rLuc-infected cells in the presence of transcriptional inhibitors. The endogenous PKRmediated eIF2a phosphorylation suppressed translation of the RVFV lacking the NSs gene, resulting in poor virus replication. Our data further suggested that the NSs promoted PKR degradation most probably through a proteasome-dependent pathway and prevented eIF2a phosphorylation, leading to efficient viral translation.  (8 h). Radiolabelled myc-tagged PKR was immunoprecipitated by anti-myc monoclonal antibody, analyzed by SDS-PAGE with 7.5% poryacrylamide gel, and visualized by autoradiography. (F) 293 cells were infected with rMP12-rLuc or MP-12 at an moi of 3, and, then, treated with 10 mM of MG132 (MG) or 50 mM of lactacystin (LA) or they were mock-treated (2). Whole-cell lysates were collected at 8 h.p.i., and the abundance of PKR, NSs and b-actin were examined by Western blot analysis. (G) 293 cells were mock-infected (Mock) or infected with MP-12 (MP-12) at an moi of 3, and treated with MG132 (MG132) at 10 mM or they were untreated (no drug used). Whole-cell lysates were collected at 2, 4, 6, and 8 h.p.i. Anti-PKR antibody, anti-NSs antibody, and anti-b-actin antibody were used to detect PKR, NSs, and b-actin, respectively (F and G). Data are representative of two to three independent experiments (A-G). doi:10.1371/journal.ppat.1000287.g007 The past studies [25,26,45] and the data shown in this study illustrate that two distinct biological activities of the RVFV NSs protein worked together to secure efficient RVFV replication. Namely, nuclear NSs protein inhibits transcription of host mRNAs, including the IFN mRNAs; this activity is critical for efficient RVFV replication in IFN-competent systems [25,45]. However, a combination of RVFV replication and NSs-mediated host mRNA transcriptional suppression possibly induces PKR activation and subsequent eIF2a phosphorylation as a combination of RVFV replication and ActD or a-amanitin treatment induced it. The NSs protein, in turn, promotes PKR downregulation as early as 4 h.p.i., and prevents eIF2a phosphorylation to secure the translation of viral mRNAs and efficient virus replication. We think that both of these two NSs functions are tightly related and protect efficient viral replication by suppressing host antiviral responses. In RVFV-infected cells, the NSs establishes general transcriptional suppression at a later stage of infection (after 8 h.p.i.) [25], while NSs also suppresses specific IFN-b mRNA transcription at early stages of infection (about 3 h.p.i.) by maintaining repressor complex including SAP30 on IFN-b promoter [26,45]. PKR downregulation early in infection is probably important for maintaining efficient viral translation in combination with the suppression of host IFN responses.
We suspect that the NSs-mediated PKR downregulation activity is important for RVFV replication and survival in infected mammalian hosts. RVFV-infection in rhesus monkeys showed that type I IFN is detectable around 1 day post-infection in both clinically ill surviving monkeys and lethally infected monkeys, and one dead monkey even kept high titer of IFN (120 to 480 U/ml) from 1 day post-infection [46]. In fact, the best correlation with outcome was early detection of IFN and not necessarily the height of the response. NSs suppresses IFN-b mRNA transcription early in the course of infection in cultured cells [26,45]. Our present data suggest that the early downregulation of PKR in RVFVinfected cells might contribute to the inhibition of type I IFN induction, because PKR is known to serve as a pathogenrecognition receptor [47]. Furthermore, some of the host pathogen-recognition receptors, e.g., toll-like receptors 3 and 7, in uninfected cells located near the RVFV-infected cells, recognize virus-specific signals, e.g., viral RNAs in the virus particles or viral dsRNAs in the cell debris from infected cells, leading to type I IFN production. Because the transcriptional promoter of the PKR gene contains an IFN-stimulated response element, and IFN stimulation induces PKR mRNA transcription [38], PKR abundance is likely increased in many uninfected cells of infected animals; RVFV needs to replicate in such cells to survive in infected hosts. The reduction in PKR abundance occurred as early as 4 h.p.i. ( Figure 7G) and the NSs protein is produced very early in RVFV infection [48]. Immediate NSs synthesis and subsequent NSsinduced PKR downregulation in the cells, some of which have increased PKR abundance, could rapidly disarm the PKRmediated, antiviral functions and contribute to RVFV replication and survival in animal hosts.
Possible mechanisms of the accumulation of phosphorylated eIF2a in ActD-treated, rMP12-rLucinfected cells ActD treatment of uninfected cells resulted in a significant reduction of the polysome fraction, but it did not abolish the translational activities ( Figure S1). rMP12-rLuc replication in the cells treated with ActD or a-amanitin promoted the accumulation of the phosphorylated eIF2a, resulting in translational suppression of viral proteins (Figure 4). The increased amounts of phosphorylated eIF2a clearly correlated with the concentration of ActD or a-amanitin in rMP12-rLuc-infected cells ( Figure S2). In contrast, rMP12-rLuc replication in the absence of transcriptional suppressor induced only low levels of eIF2a phosphorylation (Figure 4). Yet, the phosphorylation of PKR occurred in cells supporting the replication of RVFV lacking NSs both in the absence and presence of ActD ( Figure 6D). Thus, a significant accumulation of phosphorylated eIF2a occurred only in cells in which replication of RVFV lacking NSs was combined with the treatment with transcriptional inhibitors.
Several different mechanisms are conceivable for the accumulation of phosphorylated eIF2a in rMP12-rLuc-infected in cells treated with ActD or a-amanitin (Figure 4). One possible mechanism relates to the eIF2a dephosphorylation step. Phosphorylation of eIF2a at Serine 51 induces a rapid synthesis of activating transcription factor (ATF)-4 mRNA, which can be translated in the presence of phosphorylated eIF2a [49]. Expressed ATF4 then induces GADD34 mRNA transcription [50] and GADD34 protein interacts with type 1 protein serine/ threonine phosphatase, PP1, and this complex dephosphorylates eIF2a to resume the cellular translation [51]. rMP12-rLuc replication in transcriptionally active cells probably induced PKR activation and eIF2a phosphorylation, the latter of which then induced GADD34 upregulation and subsequent eIF2a dephosphorylation allowing efficient viral translation. rMP12-rLuc replication in cells treated with ActD induced PKR activation, the extent of which was similar to that of infected, ActD-untreated cells ( Figure 6D), whereas the transcription inhibitors would prevent GADD34 upregulation and subsequent eIF2a dephosphorylation, causing an accumulation of phosphorylated eIF2a, which inhibited viral translation. Other possible mechanisms relate to the failure to suppress the PKR function. Cells infected with adenovirus, human immunodeficiency virus-1, or herpes simplex virus undergo a dramatic increase in the abundance of Alu RNA, which carries a repetitive element of ,300-nt in length that is transcribed by RNA polymerase III [52,53,54,55]. Alu RNA forms a stable complex with PKR, and antagonizes the PKR activation [56]. If rMP12-rLuc replication induces Alu RNA accumulation to prevent PKR activation, then the drug-induced transcriptional suppression would inhibit Alu RNA accumulation, preventing Alu RNA-mediated PKR inactivation. Another possibility is that rMP12-rLuc replication transcriptionally induces host mRNAs, and some of their gene products have PKR inhibition activities. An example of this possibility is that influenza virus replication induces P58 IPK , an inhibitor of PKR [57]. ActD or a-amanitin treatment suppresses the expression of the putative PKR inhibitor, resulting in an accumulation of phosphorylated eIF2a. Alternatively, transcriptional suppression may result in reduced amounts of ribosomal proteins, such as L18, which binds to PKR and inhibits PKR activation [58,59]. These possibilities are not mutually exclusive, and the combination of these possibilities may contribute to the accumulation of phosphorylated eIF2a in the cells supporting replication of RVFV lacking the NSs in the presence of ActD or aamanitin.

Possible mechanisms of the NSs-mediated PKR downregulation
Although many viruses suppress PKR function using various strategies [32], poliovirus is known to promote PKR degradation in infected cells [60,61]; poliovirus RNA and viral protein components are required for this activity [61], and both eIF2a and PKR are highly activated in poliovirus-infected cells before PKR downregulation occurs [60]. In contrast to poliovirus, only RVFV NSs protein was probably required for PKR downregu-lation since the downregulation of PKR occurred as early as 4 h.p.i. in RVFV-infected cells ( Figure 7G), whereas phosphorylated eIF2a accumulation occurred around 8 h.p.i. in rMP-12-rLuc-infected cells in the presence of ActD (Figure 4). Furthermore, NSs downregulated PKRK296R, a non-phosphorylatable mutant PKR ( Figure 7E). These data suggest that the phosphorylation of PKR was not essential for the NSs-mediated PKR downregulation.
A substantial reduction in the abundance of PKR occurred in both the cytoplasmic and nuclear fractions of MP-12-infected cells, but not in the mock-infected cells and in those infected with MP12 lacking NSs (Figure 7). Expression of NSs alone resulted in a reduction in the amount of PKR ( Figure 7B). These data established that NSs protein induced PKR downregulation. The NSs-induced PKR downregulation occurred as early as 4 h.p.i. of MP-12 ( Figure 7G). We also demonstrated the presence of PKR mRNA in NSs-expressing cells ( Figure 7C) and that ActD treatment had little effect on the NSs-induced PKR downregulation ( Figure 7B). We failed to detect PKR at 16 h.p.i. of MP-12infected 293 cells, and yet we easily detected the PKR after a 16 hlong incubation of 293 cells with puromycin ( Figure 7D). Accordingly, the putative NSs-induced translational inhibition was not a main reason for the reduction in the abundance of PKR. Furthermore, pulse-chase experiments showed that NSs expression reduced the abundance of radiolabelled myc-tagged PKR ( Figure 7E). All of these data strongly suggested that the NSs induced PKR downregulation at post-transcriptional levels.
It was reported that treatment of a macrophage cell line with IFN-c induced PKR degradation [62]. Because NSs induced PKR degradation in ActD-treated cells and ActD treatment completely inhibited the IFN-c mRNA accumulation ( Figure 6C), it is highly unlikely that IFN-c was involved in the NSs-induced PKR downregulation. PKC activation also potentially induces PKR downregulation [41]. Treatment of MP-12-infected cells with a PKC inhibitor, GÖ 6983, did not inhibit NSs-mediated PKR downregulation ( Figure 7D), which implied that PKC was not involved in it. Experiments using a proteasome inhibitor suggested that the NSs promoted PKR degradation through a proteasome pathway ( Figure 7F). MG132 can induce GCN activation and translational suppression [42]. In fact, treatment of MP-12infected cells with MG132 and lactacystin moderately reduced the accumulation of NSs ( Figure 7F and 7G). Accordingly, a possibility still exists that the MG132-induced moderate translational inhibition affected the NSs-induced PKR degradation in MG132-treated cells. For example, if the NSs-induced PKR downregulation requires an unstable host protein, then the MG132-induced moderate translational suppression would prevent accumulation of this putative host protein, resulting in inhibition of the NSs-induced PKR downregulation. Obviously further studies are required to elucidate the mechanism of PKR downregulation mediated by RVFV NSs.

Drug treatment
Cells were treated with transcriptional inhibitors, ActD (Sigma) (5 mg/ml) or a-amanitin (Sigma) (50 mg/ml) immediately after infection or transfection. To induce the inhibition of proteasome function, cells were immediately treated with MG132 (Sigma) at 10 mM or lactacystin (Sigma) at 50 mM after infection or transfection. To suppress PKC activity, cells were treated with a general PKC inhibitor, GÖ 6983 (Calbiochem) at 100 nM immediately after infection with rMP12-rLuc or MP-12. Cells were treated with puromycin (Cellgro) at 100 mg/ml immediately after infection or transfection to inhibit cellular translation.

Plasmid constructions
Standard molecular biological techniques, including a PCRbased mutagenesis method, were used for plasmid constructions. PCR fragments encoding PKRDE7 ORF with an N-terminal Flag-tag sequence were cloned between the Hpa I and Spe I sites of pProT7-S(+) plasmid [19], designated as pProT7-S(+)-PKRDE7. PCR fragments encoding the rLuc ORF or NSs with a C-terminal Flag-tag sequence were cloned between the Hpa I and Spe I sites of the pProT7-S(+) plasmid, designated as pProT7-S(+)-rLuc-Flag or pProT7-S(+)-NSs-Flag, respectively. All of the constructs were confirmed to have the expected sequences. The PCR product of the entire human PKR ORF carrying a point mutation at K296R and the N-terminal myc tag was cloned between KpnI and XhoI of pcDNA3.1myc-His (Invitrogen), resulted in pcDNA3.1-Myc-PKR296R.

RNA transfection
RVFV MP-12 or ZH501 NSs ORF, or CAT ORF were cloned downstream of the T7 promoter between the Kpn I and Xho I sites of the pcDNA3.1-myc-HisA (Invitrogen) plasmid. For rLuc RNA transcripts, the rLuc ORF in pRL-SV40 plasmid (Promega Corporation) was inserted downstream of the T7 promoter. Capped and polyadenylated RNA transcripts were synthesized in vitro by using mMESSAGE mMACHINE T7 Ultra (Ambion) according to the manufacturer's instructions [64]. One microgram of in vitrosynthesized RNA transcripts was transfected into 293 cells in a 12well plate with a TransIT-mRNA Transfection kit (Mirus Bio Corporation) according to the manufacturer's instructions.

Plaque assay
VeroE6 cells in 6-well plate were infected with a series of diluted virus samples in 400 ml. After 1 h adsorption at 37uC, we removed the inocula and added 2 ml of MEM containing 0.6% noble agar (Difco Laboratories), 5% FBS and 5% tryptose phosphate broth. The cells were incubated at 37uC for 3 days. Then, 2 ml of MEM containing 0.6% agar, 100 mg of neutral red (N2889, Sigma), 5% FBS and 5% tryptose phosphate broth were added into the wells, and incubated for 16 h. The virus titers were determined in triplicate.

Western blot analysis
Cells were lysed in sample buffer and boiled for 10 min. Equal amounts of samples were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electroblotted onto polyvinylidene difluoride membranes (immobilon P; Millipore). Western blot was performed as described previously [27]. The following primary antibodies were used: anti-RVFV [27]; anti-NSs [48]

Radiolabelling of proteins
For the radiolabelling of host and viral proteins in infected cells, VeroE6 cells were incubated at 14.5 h post-infection for 30 min at 37uC with medium made up with MEM lacking methionine, cystine, and L-glutamine (M2289, Sigma); 1% dialyzed FBS (Invitrogen); 20 mM L-glutamine; penicillin (100 U/ml) and streptomycin (100 mg/ml). Then, Trans[ 35 S]label metabolic reagent (MP biomedicals) was directly added into the medium (100 mCi/ml). After 1 h labelling, cells were washed with PBS once and lysed in sample buffer. Equal amounts of samples were subjected to SDS-PAGE in 10% polyacrylamide gel. The gel was dried and exposed to X-ray film (KODAK BioMax XAR) overnight at 280uC. For the radiolabelling of PKR, 293 cells were mock-treated or transfected with pcDNA3.1-Myc-PKRK296R, and labelled with Trans[ 35 S]label metabolic reagent between 14 and 16 h post-DNA transfection. In some samples, cell extracts were prepared with chase using a lysis buffer. In other samples, cells were transfected with in vitro-synthesized RNA transcripts encoding rLuc or MP-12 NSs. Then, cells were washed, and incubated for 8 h in the presence of 2 mM methionine/ cysteine. At 8 h post-RNA transfection, cell extracts prepared using lysis buffer, were employed for immunoprecipitation with anti-Myc antibody (Santa Cruz: sc-40), as described in an IPkinase assay. Immunoprecipitated samples were separated on a 7.5% poryacrylamide gel and visualized on an autoradiograph. dsRNA-binding assay 293 cells were mock-infected or infected with rMP12-rLuc-Flag, rMP12-NSs-Flag or rMP12-PKRDE7 at an moi of 3. Alternatively, 293 cells were transfected with in vitro-synthesized RNA transcripts encoding MP-12 NSs. The cytoplasmic lysate was harvested at 16 h.p.i. or 16 h post-transfection, and incubated with poly C beads or poly I:C beads on ice for 45 minutes. After washing beads with buffer-A for 4 times, the beads were mixed with 26 sample buffer, and bound proteins were analyzed with anti-Flag antibody (Sigma) on a Western blot.

Renilla luciferase assay
The luciferase assay was performed on a Renilla Luciferase Assay System (E2810, Promega Corporation) according to the manufacturer's instructions.

Real-time PCR
293 cells in 6-well plates were transfected with 2 mg of in vitrosynthesized RNA transcripts encoding rLuc or MP-12 NSs by TransIT mRNA transfection kit (Mirus). Cells were mock-treated or treated with ActD (5 mg/ml) immediately after RNA transfection. Total RNA were extracted by using an RNeasy Mini kit (Qiagen) at 16 h post-transfection. For each sample, we used 500 ng of RNA to synthesize 1 st strand cDNA by High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). Real-Time PCR was performed at the Real-Time PCR core facility, Sealy Center for Cancer Cell Biology, UTMB. We used an Applied Biosystems made-to-order 206 assay mix of primers and TaqMan MGB probes (FAM dye-labled) for our target gene PKR (Applied Biosystems: assay ID#: Hs01091592_m1) and predeveloped an 18S rRNA (FAM-dye labelled probe) TaqMan assay reagent (Applied Biosystems: 4352930E) for endogenous control. Separate tubes (singleplex) real-time PCR was performed with 40 ng cDNA for both target gene and endogenous control by using a Taqman Gene expression master mix (Applied Biosystems: 4370074). The cycling parameters for real-time PCR were: UNG activation at 50uC for 2 min, AmpliTaq activation at 95uC for 10 min, denaturation at 95uC for 15 sec, and annealing/extension at 60uC for 1 min (repeat 40 times) on ABI7000. Duplicate C T values were analyzed by the comparative C T (DD C T ) method, as described by the manufacturer (Applied Biosystems). The amount of target (2 2DDCT ) was obtained by normalized to endogenous reference (18S rRNA) and relative to a calibrator (one of the experimental samples). Figure S1 Effects of ActD treatment on host translation activities. (A) 293 cells cultured in 10-cm dishes were treated with 5 mg/ml of ActD (ActD) or were left untreated (Mock). Cells were harvested at 16 h post-ActD-treatment in 900 ml of Lysis buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 100 mM KCl; 1% Triton X-100; 100 mg/ml cycloheximide; and 0.5 mg/ml heparin) on ice for 5 min. The cytoplasmic lysates were collected after the removal of nucleus by centrifugation at 10,0006g for 5 min. The lysates were loaded onto a 10-50% linear sucrose gradient containing 50 mM Tris-HCl, pH 7.5; 5 mM MgCl2; 100 mM KCl; 0.5 mM dithiothreitol; 100 mg/ml cycloheximide; and 0.5 mg/ml heparin, and centrifuged at 38,000 rpm for 3 h at 4uC using a Beckman SW41 rotor. The gradients were pumped by syringe pump (Brandel) and analyzed by a density gradient fractionator (Brandel) connected to an ISCO UA-6 (ISCO Inc.) at the absorbance of 254 nm according to the manufacturer's instructions. The data were representative of two independent experiments. (B) 293 cells were transfected with in vitrosynthesized rLuc RNA transcripts and mock-treated (no drug) or immediately treated with 5 mg/ml of ActD (ActD) or 50 mg/ml of a-amanitin (Amanitin). Luciferase activities were measured at 16 h post-transfection. The data shown in the graphs (mean+/ 2standard deviation) were obtained from three independent experiments with p values by using Student's t-test (*: p,0.05). Found at: doi:10.1371/journal.ppat.1000287.s001 (4.02 MB TIF) Figure S2 Effects of different concentrations of ActD or aamanitin on eIF2a phosphorylation and N protein accumulation in rMP-12-infected cells. VeroE6 cells were infected with rMP12-rLuc at an moi of 3, and then treated with different concentrations of ActD or a-amanitin. Cell extracts and culture supernatants were harvested at 16 h.p.i. Note that ActD suppresses about 80% of RNA polymerase I activity at 0.04 mg/ml, and about 80% of RNA polymerase II and III at 4.0 mg/ml [28], while a-amanitin suppresses nearly 100% of RNA polymerase II and about 50% of RNA polymerase III at 50 mg/ml [64]. (A) Western blot analysis of N protein, phosphorylated eIF2a, total eIF2a and a-actin in each cell extract. (B) Top panels represent the relative abundance of phosphorylated eIF2a and total eIF2a. The relative abundance of phosphorylated eIF2a and total eIF2a in the untreated cells represents 100%. The middle panels and the bottom panels represent the abundance of N protein and the virus titers, respectively. The results were obtained from three independent experiments with p values by using Student's t-test (*: p,0.05). Found at: doi:10.1371/journal.ppat.1000287.s002 (4.33 MB TIF) Figure S3 Effects of pan-caspase inhibitor Z-VADfmk on PKRmediated eIF2a phosphorylation in infected cells. VeroE6 cells were mock-infected (Mock) infected with rMP12-rLuc (rMP12-rLuc) and then treated with 5 mg/ml ActD (Act) or 50 mg/ml of aamanitin (Ama) or mock-treated (M) in the presence or absence of 100 mM of Z-VADfmk. Cells and culture supernatants were harvested at 16 h.p.i. (A) Western blot analysis of eIF2a, phosphorylated eIF2a, cleaved caspase 3 using anti-Cleaved Caspase 3, Asp175, antibody (Cell Signaling Tech. #9661), and a-actin in the absence (Top) and presence (Bottom) of Z-VADfmk. The data are representative of three independent experiments. (B) The relative abundance of phosphorylated eIF2a and total eIF2a. The relative abundance of phosphorylated eIF2a and total eIF2a in the mock-infected, mock-treated cells represents 100%. The average and standard deviation from three independent experiments were shown (*: p,0.05 compared to mock-treated cells). (C) Virus titers of rMP12-rLuc from three independent experiments were shown (*: p,0.05 compared to mock-treated cells). Found at: doi:10.1371/journal.ppat.1000287.s003 (2.29 MB TIF)