MLN4924 inhibits RVFV infection

To explore the mechanism of NSs mediated PKR proteasomal degradation, we first examined how small molecule inhibitors targeting different stages of the ubiquitin (UB)-proteasome pathway (UPP), including MG132 (proteasome inhibitor) and MLN4924 (CRL E3 ligase inhibitor), regulated RVFV infection. Both MG132 and MLN4924 inhibited RVFV infection, but to gain mechanistic insights into this inhibition, we focused on MLN4924 since it targets a specific class of E3 ligases unlike MG132, which globally inhibits the UPP. As shown in Fig 1A, MLN4924 at 1 μM inhibits the viral antigen expression of both the virulent strain ZH501 and the vaccine strain MP-12 based on immunofluorescence analysis (IFA) of HeLa cells. The latter were infected with the corresponding viruses for 24h at an MOI (multiplicity of infection) = 1. The potency of MLN4924 was determined by a 10-point concentration response curve in RVFV-infected HeLa cells (Fig 1B and 1C). Viral infections were measured by high-content image based analysis (HCA) using previously described methods [16,17]. Briefly, images of immunofluorescently labeled viral antigen-expressing cells were acquired by automated high-speed microscopy and subjected to image analysis software to enumerate the viral antigen expressing cells. The assay conditions were optimized such that greater than 50% of cells expressed viral antigen after multiple rounds of infection at a minimal MOI (further details in Methods section). HeLa cells treated with increasing concentrations of MLN4924 were infected with either ZH501 (Fig 1B) or MP-12 (Fig 1C) in a 96-well plate at an MOI = 1. After 24h, cells were subjected to IFA of glycoprotein (G) or nucleocapsid (N) protein expression. Viral infection was measured by HCA of N or G expression and was also compared to viral titers determined by plaque assay of the cell supernatants (Fig 1B and 1C). As shown in Fig 1B and 1C, irrespective of the method used to measure viral infection, MLN4924 showed a dose dependent inhibition of RVFV infection. Furthermore IC₅₀ values (of 10 nM—150 nM) fall within the range of values expected for MLN4924’s activity on CRL in cell culture assays [4] and there was no significant decrease in cell number when compared to mock-treated cells for the duration of the infection (24h). The IC₅₀ values by plaque assay were lower than the IC₅₀ values by HCA due to the differences in the parameters measured, but nevertheless followed the same trend of inhibition. For example when the percentage of G expressing cells decreased by two fold, the corresponding virus titers dropped by a log value (Fig 1B and 1C). For rest of the study, HCA was used for measuring RVFV infection. Under the conditions of infection described for HCA, RVFV infection showed a linear increase in percentage of antigen expressing cells with increasing MOI (S1A and S1B Fig). A similar trend in the increase of viral titers was observed when viral infection was measured by plaque assay; however, HCA is relatively more sensitive to small changes in MOI and is a more quantitative and robust assay compared to the plaque assay (S1A and S1B Fig).

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Fig 1. MLN4924 inhibits RVFV infection.

(A) IFA of RVFV N or G expression (green) in HeLa cells (cytoplasm in red) that were infected with ZH501 (MOI = 1, 24h) or MP-12 (MOI = 1, 24h) and were either treated with control DMSO (0.5%) or MLN4924 (1 μM) from 1h prior to start of infection to the end of the infection. (B-C) Dose response curve analyses of MLN4924’s antiviral activity against ZH501 (B) and MP-12 (C) virus infection in HeLa cells. Virus infections were measured by HCA of G or N expressing cells or by plaque assay. The values in the brackets next to the name of the dose response curve indicates the average infection in mock (DMSO) treated cells. The infection rates of MLN4924 treated cells were normalized with the corresponding values derived from DMSO treated controls, which were considered as 100% and expressed as the mean ± SD. The final values were expressed as the mean ± SD. (D) Dose response curve analyses of the MLN4924’s antiviral activity against RVFV ZH501 in different cell lines. Infections were measured by HCA of N expressing cells. The values in the brackets next to the names of each cell line, indicate the average infection rate of virus infected cells that were mock (DMSO) treated. Data were normalized as in B-C (E-F) MLN4924 activity against different viruses: VEEV (MOI = 0.1, 24h), MARV (MOI = 3, 48h) and LASV (MOI = 1, 48h) when compared to RVFV ZH501 and RVFV MP-12 infections in HeLa cells. The virus infections were measured by HCA of viral antigen expressing cells (E) or by plaque assay (F): the relative infection in E was calculated by normalizing infection rates of compound treated cells with mock treated cells. The values in the brackets below the virus names indicate the average infection rate in mock (DMSO) treated cells.

https://doi.org/10.1371/journal.ppat.1005437.g001

The MLN4924 mediated RVFV infection inhibition was also observed in multiple primary and cancer cell lines of human or mouse origin (Fig 1D). MLN4924 antiviral activity was specific to RVFV and was not observed with other RNA viruses including Venezuelan equine encephalomyelitis virus (VEEV), Lake Victoria Marburg virus (MARV), or Lassa virus (LASV) within the families Togaviridae, Filoviridae, and Arenaviridae, respectively, when viral infections were measured either by HCA (Fig 1E) or plaque assay (Fig 1F).

MLN4924 inhibits CRL activity by blocking conjugation of NEDD8 to the Cullin subunit. Further genetic evidence of NEDDylation’s role in RVFV infection was determined by overexpression of a point mutant, UBC12 (C111S), in the E2 NEDD8-conjugating enzyme, which has a dominant-negative effect on NEDD8 conjugation [18]. UBC12 (C111S) expression, and not the empty vector, inhibited RVFV infection specifically thus providing genetic proof for the role of NEDDylation (S1C Fig).

We next examined the stage of the viral life cycle that is critically dependent on CRLs by time of compound addition (ToA) studies (S1D Fig) and by viral RNA and gene expression kinetics during one viral replication cycle, which is ~12h (S1E–S1G Fig). HeLa cells were infected with a high MOI (MOI = 10) to achieve an infection rate >80% during one cycle of viral replication. Infection rates in ToA were evaluated by HCA of G expression in non-permeabilized cells in order to detect virus egress. ToA studies showed that MLN4924 treatment could be postponed until 5h post infection (PI [S1D Fig]). The kinetics of early N and late G gene expression in permeabilized cells showed that late gene expression was severely inhibited, while early gene expression was normal until 6h PI (S1E Fig). The inhibition in viral RNA expression levels also began to appear from 5-7h PI (S1F Fig) and compound addition could be postponed until 5h PI (S1G Fig). Collectively, these data suggest that MLN4924 does not target viral entry, rather it inhibits viral RNA and gene expression beginning at 5-6h PI as late gene expression was more severely inhibited than the early gene.

MLN4924 causes NSs-dependent early PKR activation in RVFV-infected cells

Since MNL4924 regulated CRL E3 ligase activity, we hypothesized that MLN4924 treatment of RVFV infected cells might block NSs-mediated PKR proteasomal degradation resulting in accumulation of activated and phosphorylated PKR (p-PKR), which then might phosphorylate eIF2-α (p-eIF2-α) to suppress further viral protein synthesis. Thus, we examined the kinetics of PKR, p-PKR and p-eIF2-α expression during the course of a single cycle of infection (i.e., 12 h) in DMSO (vehicle control) or MLN4924 treated HeLa cells (Fig 2A). Due to a lack of suitable antibodies to the NSs protein, recombinant MP-12 (rMP-12) virus containing the NSs gene engineered with a V5 tag at the C terminus i.e., rMP-12-NSs-V5, was used in place of wildtype MP-12. As shown in Fig 2A, HeLa cells infected with rMP-12-NSs-V5 and treated with MLN4924 failed to clear PKR and led to a robust and simultaneous induction of p-PKR and p-eIF2-α beginning at 6h PI. Induction of p-PKR coincided with NSs expression. As expected, phosphorylation of eIF2-α suppressed further increases in NSs levels or synthesis of the late viral G protein, whose expression normally follows NSs. MLN4924 treatment also restored p62 expression levels, which under normal infection conditions would be targeted by NSs for proteasomal degradation [19]. As a control for MLN4924 activity, the lysates were also probed with anti-NEDD8 antibodies to determine the status of cullin NEDDylation. As expected, control treated cells showed a strong signal at a molecular weight that corresponded to that of cullin proteins, while no bands were detected with the MLN4924 treated lysates (Fig 2A). Collectively, these data show that MLN4924 prevented PKR degradation and instead activated PKR.

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Fig 2. MLN4924 regulates NSs-dependent early PKR activation in RVFV-infected cells.

(A) Western blot analysis demonstrating MLN4924 regulation of PKR activation and inhibition of viral gene expression during the course of a single cycle of viral replication (~ 12h). HeLa cells treated with control (DMSO) or MLN4924 (1 μM) were either mock infected or infected with rMP12-NSs-V5. At the indicated time points, cell lysates were harvested and analyzed for gene expression by Western blot analysis. (B) NSs regulates MLN4924 mediated PKR activation during RVFV infection. HeLa cells expressing control non-targeting siRNA or PKR siRNA were treated with DMSO (vehicle control) or MLN4924 (1 μM) and infected with either wildtype viruses including RVFV ZH501 or rMP-12 or NSs deficient viruses including clone 13 or rMP-12ΔNSs::Luci at an MOI = 10 for 12h. The infection rates were determined by enumerating G expressing cells (rMP-12 and rMP-12ΔNSs::Luci) or N expressing cells (RVFV ZH501 and clone13) by HCA. The data derived from MLN4924 treated cells was normalized with the corresponding DMSO treated controls (average infection rates are indicated under the bar graph), which were considered as 100% and expressed as the mean ± SD. The inset shows a decrease in PKR expression levels in PKR siRNA transfected cells by Western blot analysis. (C) Western blot analysis demonstrating the regulation of the PKR activation pathway and viral gene expression kinetics by NSs deficient virus (rMP-12ΔNSs::Luci virus) during single cycle of virus infection. (D) Western blot analysis which shows that MLN4924 treatment of HeLa cells that were transiently transfected with plasmid DNA expressing the NSs gene was sufficient to activate PKR.

https://doi.org/10.1371/journal.ppat.1005437.g002

The above data suggested that NSs probably blocked PKR activation by depleting PKR from cells. If that was the case, NSs deficient virus might induce PKR activation. Furthermore, if MLN4924’s antiviral activity was primarily due to PKR activation, loss of PKR expression should restore RVFV infection. Both these hypotheses were tested by measuring infection rates of wildtype or NSs-deficient RVFV mutants treated with DMSO (vehicle control) or MLN4924 under normal or low PKR siRNA knockdown conditions during one cycle of virus replication (Fig 2B). Two different NSs deficient mutants were used: Clone-13, which is a naturally attenuated isolate of RVFV encoding a non-functional NSs gene due to a large in-frame deletion, and rMP-12ΔNSs::Luci virus, in which the NSs gene of MP-12 is replaced with a luciferase (Luci) gene by reverse genetics [20]. We confirmed that PKR expression levels were indeed reduced in HeLa cells transfected with siRNA targeting PKR (inset in Fig 2B). MLN4924 treatment inhibited MP-12 and ZH501 infection and as predicted this inhibition was completely rescued in cells with low levels of PKR expression (Fig 2B). Therefore PKR activation was responsible for MLN4924 antiviral activity. But surprisingly, NSs deficient viruses (clone 13 and rMP-12ΔNSs::Luci) were insensitive to MLN4924 treatment. In addition, replication of these viruses did not increase under low PKR expression levels. One explanation was that NSs was essential for early PKR activation; alternatively, it was also possible that the NSs deficient viruses had delayed replication kinetics that may have further delayed PKR activation.

To address these questions, we examined the kinetics of viral gene expression and the PKR activation pathway by Western blot analysis during one cycle of virus replication with the NSs deficient virus (rMP-12ΔNSs::Luci) under mock or MLN4924 treatment conditions (Fig 2C). This virus showed the same kinetics of G expression as wildtype rMP-12, with an increase in G expression becoming apparent by 8 h PI in both viruses (compare G expression kinetics in Fig 2A and 2C). These data suggested that at least during the early stages of the first cycle of viral replication there is no significant difference in the replication kinetics between NSs deficient virus when compared to rMP-12 virus. In agreement with previous observations (Fig 2B), the NSs deficient virus was insensitive to MLN4924 treatment as no decrease in G expression levels during MLN4924 treatment was observed. Moreover, MLN4924 did not induce early PKR activation. However, the NSs deficient virus did induce late PKR activation at 10–12 h PI, by which time most of the viral proteins were expressed and the virus egress had already been initiated. Thus, NSs is essential for early PKR activation upon MLN4924 treatment in RVFV infected cells.

We next examined if NSs is sufficient for PKR activation by transient transfection of NSs into HeLa cells that were treated with DMSO (vehicle control) or MLN4924 (Fig 2D). Western blot analysis showed that NSs expression alone was sufficient to induce PKR activation (based on increase in p-PKR and p-eIF2-α expression levels) by MLN4924 treatment. Collectively, these data suggest that NSs has dual antagonistic functions, promoting PKR degradation, but also activating PKR under conditions when NSs-mediated PKR degradation is blocked. We next sought out to identify the specific CRL that regulated PKR degradation by NSs.

Cullin1 binds to NSs most efficiently and regulates PKR degradation

We hypothesized that NSs targeted PKR degradation by assembling CRL E3 ligase. Thus, we evaluated the binding of NSs to various cullin family members by co-immunoprecipitation (co-IP) assay (Fig 3A). Lysates from 293T cells that were transfected with vector alone or myc-tagged CUL1-3,-4A,-4B, -5 and -7 or HA-tagged CUL9 expression vectors were combined individually with rMP-12-NSs-V5 infected cell lysates and immunoprecipitated with anti-myc or anti-HA antibodies to determine NSs binding to the corresponding CRL. NSs bound to CUL1, but not with the other cullin family members (CUL2,-3,-4A,-4B, -5, -7 or -9). Furthermore, overexpression of dominant negative CUL1, but not other cullin family members, activated PKR in RVFV-infected cells (S2 Fig), thus providing functional significance to the NSs-CUL1 interaction.

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Fig 3. NSs binds to CUL1 most efficiently and regulates PKR degradation.

(A) Co-immunoprecipitation (Co-IP) of the NSs protein shows that CUL1 bound most efficiently to NSs among the 8 cullin family members, including CUL1 to 3, CUL4A-4B, CUL5, CUL7 or CUL9. Lysates of 293T cells that were transfected to express the vector alone or myc-tagged CUL1, -2, -3, -4A, -4B, -5 or -7 or HA tagged CUL9 proteins were combined with rMP-12-NSs-V5 infected cell lysates and immunoprecipitated with the anti-myc or anti-HA antibodies. The bound proteins were detected by Western blot analysis as shown in the figure. Lysate controls represent 5% of the lysate used in co-IP. (B) NSs protein binding to various CUL1 mutant proteins that were transiently expressed in 293T was determined as described above in A. αA represents IP using all three: HA, myc and Flag antibodies in the control (C) Tentative model of PKR degradation by SCF complex: CUL1 forms a molecular scaffold to organize the SCF by forming two distinct functional modules. The C terminal domain (CTD) of CUL1 (green) binds to RBX1 (R, grey), that recruits ubiquitin (U, brown) loading E2 enzyme (light green) for catalysis while the N terminus binds to SKP1 (S, grey), which recruits substrate in this case NSs (red)-PKR (blue),) via substrate recognizing F-box protein (yellow). PKR is presumably poly-ubiquitinated (UUU, brown) by the E2 enzyme and subjected to proteasome degradation. CUL1 is conjugated with NEDD8 (purple) and is essential for SCF E3 ligase activity.

https://doi.org/10.1371/journal.ppat.1005437.g003

CUL1 forms a molecular scaffold to organize CRL1 by forming two distinct modules. The carboxyl (C) terminal domain binds to RBX1, which recruits the ubiquitin-loaded E2 enzymes for catalysis while the amino (N) terminus of CUL1 binds to the adaptor protein SKP1, which recruits the substrate via the substrate recognizing F-box protein (Fig 3C). Using previously characterized CUL1 mutants (see Methods section) that selectively lost their ability to interact with SKP1 or RBX1; we tested for NSs binding by co-IP assay. As shown in Fig 3B, NSs bound to wt-CUL1 (lane 1) and to the CUL1 mutants that have retained their capacity to interact with SKP1 (lanes 2, 5, 6, 7 and 8); in contrast, NSs binding to CUL1 was severely reduced with the mutants CUL1 ΔN53 and CUL1 Y42A/M43A, which cannot bind SKP1 (lanes 3 and 4). Taken together, these data suggest that NSs binds to the CRL1/SCF complex via the classical substrate binding motif in the CUL1 subunit, and thus this interaction is most likely facilitated by an F-box (see model in Fig 3C).

F-box protein FBXW11 binds to NSs and regulates RVFV infection

We speculated that if NSs-PKR were recruited by an F-box protein, then depletion of the specific F-box protein would prevent PKR degradation, activate PKR, and inhibit RVFV infection. Thus, we used an siRNA screening assay targeting 70 out of the 72 F-box genes characterized in the human genome to determine the specific F-box gene(s) involved in RVFV infection [21,22]. CDRT1 and FBXO49 were not classified as F-box genes during the time this study was initiated and therefore were not included in our siRNA screening assay (refer to gene list in S3 Fig). For this assay, a pool of 4 different siRNAs was used to knockdown each of the 70 F-box genes in triplicate. As shown in Fig 4A, siRNA knockdown of four genes resulted in 30–50% inhibition of ZH501 infection in HeLa cells (marked in orange). Further validation of the hits was achieved by de-convoluting the pool of four siRNAs into individual siRNAs and testing their antiviral activity, cellular cytotoxicity and their ability to decrease target mRNA levels. A hit was considered genuine if two or more siRNAs showed antiviral activity without cytotoxic effects (<15% decrease in cell number). Among the four hits, siRNA’s targeting FBXW11 alone passed these criteria (Fig 4B and 4C). As shown in Fig 4B, 3 out of the 4 individual siRNAs targeting FBXW11 showed greater than 70% knockdown efficiency in mRNA levels and also inhibited viral infection of both MP-12 and ZH501 strains by greater than 50%. The fourth siRNA was able to knockdown FBXW11 mRNA by approximately 70%, however, it did not inhibit viral infection. These data suggest that a higher level (greater than 70%) of FBXW11 mRNA knockdown is required to inhibit RVFV infection. These data were also validated with three additional siRNAs targeting different regions of the mRNA (S4A Fig). Furthermore, antiviral activity due to loss of FBXW11 expression was specific to RVFV infection and was effective in multiple cell lines including 293T and HSAEC (human small airway epithelial cells) primary cells, thus following the same pattern of antiviral activity displayed by MLN4924 treatment (S4B Fig).

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Fig 4. F-box protein FBXW11 binds to NSs and regulates RVFV.

(A) siRNA screening assay targeting 70 out of 72 human F-box genes to determine RVFV infection inhibitors. HeLa cells were transfected with non-targeting control siRNA or with a pool of four different siRNA targeting each of the 70 F-box genes for 48h followed by incubation with ZH501 (MOI = 1) for 24h. Percentage of infected cells was determined by HCA of N expressing cells. Each data point in panels A to D is an average of three replicates ±SD and is representative of 2 independent experiments. (B) Validation of FBXW11 as a true hit from the siRNA screening: Each siRNA from the pool of 4 siRNAs was individually tested for its ability to inhibit RVFV (ZH501 or MP-12) infection and reduce FBXW11 mRNA levels. Infection was quantified by HCA of viral antigen expressing cells as described in A, while mRNA levels were determined by real time PCR. The relative infection or mRNA levels were calculated by normalizing with the values derived from controls cells that were transfected with non-targeting siRNA and infected with the corresponding viruses. The infection rates of control siRNA treated cells are indicated in the brackets next to the virus names. (C) FBXW11 is the major homologue of βTrCP gene regulating RVFV infection, but its activity is enhanced by BTRC. Same as in B except siRNA targeting BTRC was used either singly or combined with FBXW11 siRNA. (D) Real-Time PCR analysis of BTRC and FBXW11 mRNA levels in HeLa cells that were transfected with the siRNAs are indicated on the X-axis. Relative mRNA levels were derived by normalizing mRNA levels in cells transfected with non-targeting siRNA. (E) Western blot analysis shows that βTrCP siRNA knockdown induces PKR activation and inhibition of viral gene expression similar to MLN4924 treated cells. HeLa cells were transfected with different siRNAs as indicated for 48h and then either mock infected or infected with rMP-12-NSs-V5 (MOI = 10, 8h), and were either treated with DMSO or MLN4924 at 2h, PI. Cells lysates were harvested at 8h, PI for Western blot analysis. (F) Co-immunoprecipitation (co-IP) assay is consistent with the WD40 domain regulating βTrCP binding to NSs: Top panel, depicts the domain structure of the BTRC and FBXW11 protein which contains D or the Dimerization domain, F or F-box motif, and the seven WD40 repeats. In the bottom panel is data from co-IP assay showing the binding of NSs or CUL1 to the various deletion mutants of BTRC and FBXW11. 293T cell lysates overexpressing myc-CUL1 and the vector alone or flag tagged wildtype βTrCP or deletion mutants of βTrCP were combined with rMP-12-NSs-V5 (MOI = 10, 8h) infected cell lysates and immunoprecipitated with anti-Flag antibody. The bound proteins were detected by Western blot analysis as described in the figure. Lysate controls represent 5% of extract used for co-IP.

https://doi.org/10.1371/journal.ppat.1005437.g004

FBXW11 and BTRC are two distinct homologues of the βTrCP gene and many studies suggest that they are functionally redundant [23]. Thus we determined, whether BTRC partially compensated for loss of FBXW11 expression in RVFV infection. As shown in Fig 4C, combined knockdown of both BTRC and FBXW11 was more potent in suppressing RVFV infection than knockdown of FBXW11 alone. Surprisingly loss of BTRC expression had no effect on RVFV infection and was compensated by FBXW11. The observed antiviral effects were not due to cytotoxicity or non-specific effects of siRNA knockdown (Fig 4D). Thus, FBXW11 plays a dominant role among the two homologues in RVFV infection.

We then tested if knockdown of βTrCP inhibited RVFV infection through the same mechanism as global inactivation of CRL by MLN4924 treatment. This included loss of PKR degradation, PKR activation that resulted in increased expression levels of p-PKR and p-eIF2-α, followed by inhibition of viral gene expression within 8 h of RVFV infection. This pattern of antiviral gene expression was evaluated by Western blot analysis. As shown in Fig 4E, loss of FBXW11 expression alone (lane 5) or FBXW11 and BTRC combined together (lane 7) showed the same gene expression pattern as inactivating CRLs by MLN4924 treatment (lane 3). However the phenotype of FBXW11 knockdown was milder when compared to combined knockdown of BTRC and FBXW11. FBXW11 played a dominant role and loss of FBXW11 expression recapitulated loss of global activity by CRLs during RVFV infection.

Based on these data, we predicted that FBXW11 might be the key factor that recruited NSs-PKR to the SCF complex, and thus examined the NSs-FBXW11 interaction. As shown in Fig 4F (top panel), the βTrCP protein contains the N terminal “D” or protein dimerization domain and the F-box motif that regulates recruitment to the SCF complex via binding to SKP1. The seven WD40 repeat motifs in the C terminus together form a beta-propeller like structure and regulate substrate binding. We characterized the NSs-βTrCP interaction by co-IP assay. As shown in Fig 4F (bottom panel), NSs binds to both homologues, BTRC and FBXW11, of βTrCP. Furthermore, WD40 repeats play an essential role in this interaction. As a control, the CUL1 binding to βTrCP mutants was analyzed. As reported previously, F-box mutants (ΔF and ΔNF deletion mutants) reduced the CUL1-βTrCP interaction. Overall, it appears that NSs targets PKR degradation by assembling SCF^FBXW11.

SCF^FBXW11 regulates NSs-mediated PKR degradation

If SCF^FBXW11 targets PKR degradation, then loss of expression of key components of this complex should inhibit RVFV infection by the same mechanism as global inactivation of CRL by MLN4924 treatment. We therefore examined the status of the PKR activation pathway and viral gene expression by Western blot analysis of mock or rMP-12-NSs-V5 infected HeLa cells in which the individual components of the SCF^FBXW11 were knocked down by siRNA, either singly or in combination. As shown in Fig 5A, knockdown of FBXW11 in combination with CUL1 and SKP1 (lane 4) or with CUL1 alone (lane 5) resulted in the same gene expression signature as MLN4924-treated cells (lane 7). Knockdown of FBXW11 (lane 3) had a milder effect; whereas, knockdown of CUL1 (lane 1) or SKP1 (lane 2) alone had no effect and was identical to control siRNA treated cells. SiRNA knockdown reduces, but does not completely eliminate, the protein in the cells. Therefore, low residual expression of CUL1 or SKP1, in the presence of endogenous expression levels of FBXW11 was probably sufficient to rapidly degrade PKR. Overall, these data suggest that SCF^FBXW11 is the primary complex that regulates PKR degradation, and failure to destroy PKR leads to a concomitant increase in p-PKR and p-eIF2-α levels, which suppresses viral protein synthesis.

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Fig 5. SCF ^FBXW11-NSs is the E3 ligase that targets PKR degradation during RVFV infection.

(A) Western blot analysis to determine the PKR activation and viral gene expression inhibition of rMP-12-NSs-V5 (MOI = 10 for 8h) infected HeLa cells in which the expression of the individual components of the SCF^FBXW11 E3 ligases were either knocked down singly or in combination with siRNA. (B) Evaluating SCF^FBXW11 regulation of RVFV infection under normal or PKR depleted conditions. The inset on the right is a Western blot showing PKR levels in the HeLa cell line that stably expresses doxycycline inducible PKR shRNA under untreated or doxycyline treated conditions. HeLa cells that were either treated with control DMSO or doxycycline were transfected with siRNA targeting the individual components of SCF^FBXW11, singly or in combination as indicated in the figure for 48h followed by infection with RVFV ZH501 (MOI = 1 for 24h). The percentage of infected cells was determined by HCA analysis of RVFV N expressing cells. Each data point is an average of 3 replicates ±SD. (C) Same as in B except that RVFV Clone 13 (MOI = 3, 24h) was used in infection. (D) Co-immunoprecipitation assay showing that PKR is recruited to the SCF^FBXW11 complex by NSs. 293T cell lysates transfected with empty vector or expression plasmids of myc-CUL1 or HA-FBXW11 or myc-CUL1 and HA-FBXW11 (as indicated in the figure) were combined with mock infected or rMP-12-NSs-V5 (MOI = 10 for 8h) infected cell lysates and immunoprecipitated with anti-myc or anti-HA, or a combination of both. Western blot of lysates represents 5% of the total cellular extracts. (E) Model of NSs recruiting PKR to the SCF via NSs binding to FBXW11. The symbols R, S, and U represent RBX1, SKP1 and Ubiquitin respectively.

https://doi.org/10.1371/journal.ppat.1005437.g005

If this is the case, then depleting PKR in the cell should rescue the viral inhibition resulting from knockdown of the SCF^FBXW11 complex. We approached this question by using a HeLa cell line that stably expresses a doxycycline-inducible PKR shRNA. We first confirmed by Western blot that doxycycline treatment decreased PKR expression in these cells (Fig 5B, inset). Next we examined the effects of knocking down SKP1, CUL1, FBXW11 alone or in combination, in the presence or absence of PKR, on infection levels of RVFV ZH501 in HeLa cells. As shown in Fig 5B, under normal PKR expression conditions, at saturating infection rates, depletion of CUL1 had no effect on RVFV infection, while SKP1or FBXW11 depletion resulted in greater than 40% or 80% reduction in infection. Moreover, knockdown of FBXW11 in combination with CUL1 and SKP1, individually or in combination, resulted in nearly complete inhibition of RVFV infection, similar to that observed when cells were treated with MLN4924. The infection inhibition by loss of key components of SCF^FBXW11 was completely reversed by loss of PKR expression as expected. These data support the idea that SCF^FBXW11 is the primary complex that regulates PKR degradation, and failure to destroy PKR acts to suppress viral protein synthesis and thus viral infection.

To further examine the role of NSs in the degradation of PKR via SCF^FBXW11 and in the activation of PKR, we repeated the experiment using RVFV Clone13, which encodes a non-functional NSs gene. As shown in Fig 5C, knockdown of any of the components of the SCF^FBXW11 complex did not inhibit viral infection, which was similar to that observed with MLN4924 treatment. These data strongly suggest NSs regulated PKR activation in conditions when PKR degradation was blocked by inactivation of SCF^FBXW11.

We speculated that PKR is recruited to the SCF^FBXW11 complex for degradation by the NSs protein. Thus, we examined the binding between PKR and the SCF^FBXW11 complex in the presence and absence of NSs protein. As shown in Fig 5D, PKR bound to CUL1 or FBXW11 in the presence of NSs (lanes 1 and 3), but no binding of PKR to CUL1 or FBXW11 was detected in the absence of NSs (lanes 2 and 4). Furthermore, FBXW11 appears to serve as the NSs-PKR adaptor of the SCF complex and a limiting factor for this interaction as well since in the absence of exogenously expressed FBXW11, there was substantially lower amounts of NSs and no PKR detected in the CUL1-bound fraction (lanes 5 and 6). However, similar levels of NSs or PKR were bound to FBXW11, even in the absence of exogenous CUL1 expression (lanes 7 and 8). Taken together, these data provide strong evidence that NSs-FBXW11 serves as the PKR receptor of the SCF^FBXW11 complex as shown in the model presented in Fig 5E. Furthermore, the protein binding data, combined with siRNA knockdown results, suggests that FBXW11 serves as the limiting factor in the assembly of SCF^FBXW11 complex.

Degron sequence of NSs regulates NSs-FBXW11 interaction

Numerous studies have shown that βTrCP recognizes a 6–9 linear amino acid sequence also known as degron (or destruction) sequence that starts with two acidic amino acid residues followed by a small amino acid (usually glycine) and another acid moiety in the last position. These acidic amino acids and glycine were shown to play an essential role in βTrCP-substrate interaction. We found one degron-like sequence, DDGFVE₂₆₃, in the C-terminal acid-rich region of the NSs protein (Fig 6A). Using a reverse genetics system, we generated several NSs degron mutants and subsequently rescued the mutant viruses. We speculated that rMP-12 encoding NSs mutants that cannot bind to FBXW11 will be non-infectious in HeLa cells due to PKR activity (Fig 4E). We also included in this analysis the rMP-12-NSs-R173A-V5 mutant virus, which was previously described as incapable of binding to PKR [15]. As shown in Fig 6B, NSs-GE and NSs-DGE mutants in lanes 5 and 6, respectively, showed the same gene expression profile as global inactivation of CRLs (lane 3) with potent inhibition of late viral gene expression. On the other hand, the NSs-D259A mutant was similar to wildtype virus. This mutation was probably compensated by the surrounding acidic amino acid residues. Most importantly, NSs-R173A induced PKR activity, as reported previously [19], but was not as pronounced as NSs-GE or DGE mutants and did not inhibit viral gene expression. We next examined if the GE and DGE mutants indeed inhibited infection due to PKR activation by testing virus infectivity in PKR knockdown cells.

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Fig 6. Characterization of the degron/destruction sequence that is essential for NSs-FBXW11 interaction to promote PKR degradation.

(A) Description of the NSs degron mutants. The six linear amino acid sequence predicted as the degron sequence of the NSs gene is underlined and the alanine substitution mutations are marked in red. The number in the subscript denotes the amino acid residue number of the NSs protein. (B) Western blot analysis to determine the PKR activation and viral gene expression status in HeLa cells that were infected with rMP-12 encoding wildtype NSs gene or the NSs mutants as indicated in the figure at MOI = 10 for 8h. (C) IFA of NSs (green) filament formation in the nucleus of HeLa cells that were infected with rMP-12 virus expressing wildtype NSs gene or any of the various NSs mutants as indicated in the figure at MOI = 1 for 24h under normal or PKR depleted conditions. The inset shows the magnified image of white box marked in Doxycycline treated cells. Nuclei are stained blue with Hoechst (33342). The panel on the right side is the Western blot showing PKR levels in the HeLa cell line that stable express doxycycline inducible PKR shRNA under untreated or doxycycline treated conditions. (D) Same as C, but the percentage of infected cells was determined by HCA analysis of G expressing cells. Each data point is an average of 3 replicates ±SD. **** indicates a P < 0.0001. (E) Western blot analysis of p-PKR and PKR expression kinetics in HeLa cells that were infected with rMP-12 virus expressing wildtype NSs gene or any of the NSs mutants as indicated in the figure. GAPDH was used as the loading control. (F) Co-immunoprecipitation assay demonstrating binding between NSs mutants and FBXW11 or CUL1. 293T cell lysates transfected with Flag-FBXW11 and myc-CUL1 expression plasmids were combined with mock infected or rMP-12 encoding wildtype or mutant NSs-V5 (MOI = 10 for 8h) under normal or PKR depleted conditions as described in the figure. Lysates were immunoprecipitated with anti-V5 antibodies to test NSs binding to FBXW11, CUL1 and PKR. Western blot of lysates represents 5% of the total cell extracts.

https://doi.org/10.1371/journal.ppat.1005437.g006

A HeLa cell line that stably expresses doxycycline inducible PKR-shRNA was used to evaluate the replication of rMP-12 expressing NSs-GE or DGE mutants under normal or low PKR expression conditions. We first examined NSs gene expression by IFA at 24h PI. As shown in Fig 6C, NSs-GE or DGE mutant viruses showed severe inhibition in NSs gene expression, but in cells treated with doxycycline in which PKR expression is reduced, the NSs expression was restored. The NSs-GE and DGE mutants formed filamentous structures in the nucleus identical to the wildtype NSs (see inset). This pattern of NSs gene expression was also similar to MLN4924 treated and wildtype rMP-12 virus infected cells. In contrast the NSs-R173A mutant, formed mosaic structures in the nucleus, as previously reported [15], and did not show any difference in NSs gene expression under normal or PKR depleted conditions. These data suggested that NSs-GE or DGE mutations, unlike NSs-R173A, did not undergo a conformational change and retained most of the properties of the wildtype NSs in structure and function.

We also examined infection by these mutant viruses by enumerating the percentage of cells that expressed G on the cell surface (a reflection of successful virus egress) using HCA after multiple rounds of infection (Fig 6D). As expected, rMP-12-NSs-GE or DGE expressing mutant viruses showed potent inhibition of infection under normal PKR expression levels, but their infection was completely restored under low PKR expression levels similar to the observations made with MLN4924 treatment of wildtype rMP-12 infected cells. On the other hand, the R173A mutant replicated efficiently irrespective of PKR levels. We next examined the kinetics of PKR activation to understand the NSs mutant’s role in activating PKR.

As shown in Fig 6E, rMP12-NSs-GE or DGE mutants, similar to MLN4924 treated and wildtype virus infected cells, failed to promote PKR degradation and instead caused a steady increase in PKR activity based on p-PKR levels starting from 6 to 18h PI. Although the NSs-R173A mutant blocked PKR degradation, it induced low levels of PKR activation and for a short period of time (6-8h PI). The low p-PKR expression levels were probably not sufficient to severely inhibit NSs-R173A mutant replication. Furthermore, similar to earlier observations (Fig 2C) a functional NSs gene was essential for early PKR activation since induction of p-PKR activity was not observed until the very late stages of infection (10h PI) in the rMP-12ΔNSs::Luci infected cells. Taken together, these data suggest that NSs activates PKR when PKR degradation is blocked. In addition, NSs-R173A is not as effective as the wildtype NSs gene in activating PKR.

We next examined if NSs-GE or NSs-DGE mutant viruses indeed blocked NSs binding to FBXW11. Only under very low conditions of PKR expression did the NSs-GE or NSs-DGE mutants express virus abundantly (Fig 6C). Therefore, HeLa cells stably expressing doxycycline inducible PKR shRNA were used for co-IP assay. Lysates of 293T cells that were transfected with HA-FBXW11 and myc-CUL1 expression plasmids were combined with the lysates of HeLa cells infected with wildtype rMP-12 or rMP-12 mutant viruses expressing NSs -R173A, -D259A, -GE, or -DGE mutants that were either mock treated or treated with doxycycline for 48h prior to start of infection. Cell lysates were immunoprecipated with the V5 antibody to detect NSs binding to FBXW11 and CUL1 (Fig 6F). The NSs mutants, NSs-GE and NSs-DGE expressed abundantly only in HeLa cells that had low PKR expression and did not bind to FBXW11 or CUL1 protein, unlike the wildtype or NSs-D259A or NSs-R173A mutant proteins. Furthermore, NSs-R173A did not interact with PKR similar to previously reported observations [19], while NSs-DGE or NSS-GE mutants bound PKR to the same extent as wildtype NSs. Overall, these data suggest that “DDGFVE” is the degron sequence that regulates the NSs-FBXW11 interaction. NSs-GE and DGE mutants that bind to PKR, but cannot bind to FBXW11, failed to assemble SCF^FBXW11 to promote PKR degradation. Meanwhile, other unknown NSs activities in the cell (details in Discussion) stimulated PKR activity that induced phosphorylation of eIF2-α, leading to global translational suppression including inhibition of viral protein synthesis.

Cells and viruses

HeLa, 293T, HepG2, mouse embryonic fibroblasts (MEFs) and Vero E6 cells were obtained from the American Type Culture Collection (ATCC) and were maintained under humidified conditions at 37°C, 5% CO₂ in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS, Life Technologies). HSAECs (Lonza, USA) were maintained in Ham’s F12 medium supplemented with nonessential amino acids, pyruvate, β-mercaptoethanol and 10% fetal calf serum (FCS). RVFV strains ZH548/MP-12 (MP-12) and ZH501 were obtained from The Salk Institute’s Government Services Division and from Dr. Michael Turell (USAMRIID), respectively. MP-12 was derived from a virulent strain of RVFV (ZH548) and is attenuated in its virulence due to several nucleotide mutations in its genome, but encodes a functional NSs gene [13,14] and can be handled safely under biosafety level (BSL)-2 laboratory conditions. ZH501 is the wild-type strain of RVFV and is fully virulent, requiring use under BSL-3 laboratory conditions. Clone 13 virus was a gift from Dr. Friedemann Weber (Philipps-University Marburg, Germany). Clone 13 was isolated from the 74HB49 strain of RVFV from a human case and encodes a non-functional NSs gene due to a large internal frame deletion that removed 69% of the ORF. VEEV (1CSH3), LASV (Josiah) and MARV (Ci67) were obtained from the USAMRIID collection. All viruses were propagated in Vero E6 cells. Virus-containing supernatants were clarified by centrifugation at 12,000 x g for 30 min prior to storage at -80°C. All virus stock titers were determined by plaque assay on Vero E6 cells as previously described [37].

Virus infections

RVFV ZH501 and VEEV infections were performed in a BSL-3 lab, and MP-12 infections were performed in BSL-2 lab. Work with infectious filoviruses or arenaviruses were performed in BSL-4 labs. All infection assays that required enumeration of infected cells based on immunofluorescence assay (IFA) of viral antigen expressing cells were optimized as described previously [16,17]. The infection conditions were optimized such that at a minimal MOI, greater than 50% of cells expressed viral antigen after multiple rounds of infection. This is to ensure that irrespective of the stage of the viral life cycle targeted by the small molecule or siRNA, the infection rates after multiple rounds of infections are decreased. For all the assays described here, HeLa cells were permissive to infection and were seeded at 20,000 cells per well a day prior to infection in a 96-well plate (black well, clear and flat bottom from Greiner) and infected with virus at the required MOIs in a total volume of 100 μl. Inocula were removed 1h post virus incubation, unless stated otherwise, washed one time with 1x PBS, and replaced with an equal amount of fresh medium. Infection was allowed to proceed for the specified duration of time as indicated in the figure legends of each experiment, followed by fixation in 10% neutral buffered Formalin (Sigma) for one day for BSL-3/4 viruses or 15 min for BSL-2 viruses. Cells were then subjected to immunofluorescence assay (IFA) as described below.

Immunofluorescence assay

IFA of viral antigen expression was carried out as described previously [17]. If permeabilization was required then Formalin-fixed cells were treated with 0.1% (v/v) Triton-X 100 for 15 min at room temperature prior to blocking. Otherwise Formalin fixed cells were directly blocked and incubated with primary antibody (at 1:1000 dilution) prepared in blocking buffer containing 3% BSA/PBS for 1h at room temperature (RT). Then cells were washed three times in PBS followed by incubation with the corresponding fluorescent secondary antibody for 1h at RT. Cells were washed in PBS to remove excess antibody. During the last wash, cell nuclei and cytoplasm labelling reagents, which include Hoechst 33342 (Life Technologies) and HCS Deep Red (Life Technologies), respectively, were added to the wash solution at a 1:10,000 dilution. The following antibodies, 4D4, R3-1D8-1-1a, 1A4A-1, 6D8-1, MBG II 9G4-1, L52-161-6 (from USAMRIID hybridoma bank) were used to detect RVFV G, RVFV N, VEE envelope 2 (E2) protein, MARV GP and Lassa virus GP, respectively.

For measuring RVFV infections (MOI = 1, 24 h) that involved multiple rounds of infection, 4D4 antibody that detects RVFV Gn was used with MP-12 virus. Since the 4D4 antibody does not detect Gn expression of ZH501, N expression was used for measuring infection by ZH501 and clone 13 specifically for most of the study except for Figs 1B and S1A where Gc antibody (5G3) was used. Since the Gc antibody was only available in limited amounts, it was used only for these figures to show that under the conditions of infections used N or G expression did not significantly affect the interpretation of the data. G was used to represent Gn expression throughout the manuscript except in Figs 1B and S1A where Gc was used. As described in the introduction, Gn and Gc form heteromers and are produced by proteolytic processing of the precursor GPC. Therefore Gn or Gc detection for these two figures did not change the interpretation of the data. For NSs-V5 tagged protein and Flag-FBXW11, anti-V5 and anti-Flag (Sigma) antibodies were used. The following secondary antibodies were used: Alexa 488-conjugated goat anti-mouse secondary antibody, Alexa 568-conjugated goat anti-rabbit antibody or Alexa 647-conjugated goat anti-rabbit (Life Technologies) at 1:1000 dilutions.

High content image analysis

Confocal images were collected using a Leica TCS-SP5 confocal/multiphoton microscope with a 40x oil objective. High-content quantitative imaging data were acquired and analyzed on an Opera confocal reader (model 3842 [Quadruple Excitation High Sensitivity] or model 5025; PerkinElmer) at two exposures using a 10x air objective. High-content image (HCI)-based analysis (HCA) was accomplished within the Opera or Columbus environment using standard Acapella scripts as described previously [17,38]. Briefly, images of Hoechst stained nuclei were used to draw nuclear boundaries and to count cells, while images of Cell Mask Deep Red stained cells were used for drawing cell boundaries. Images of viral antigen expression were used for enumerating percentage of infected cells.

Concentration response curve analysis

A 10-point dose-response curve (1/3 fold serial dilution from 30 μM) assay was used for dose response curve analyses. Each concentration of the hit compound was tested in triplicate in a 96-well plate format. Cells were treated with the indicated concentrations of MLN4924 for 1h prior to incubating cells with MP-12 or ZH501 at MOI = 1. One hour post virus incubation, cells were washed and replaced with compound containing media. Twenty-four hours post-infection, cells were fixed and subjected to IFA followed by HCA to evaluate the percentage of RVFV infection by enumerating the viral antigen expressing cells. Relative infection inhibition was determined by the ratio of the percentage of infected cells in compound treated cells with mock treated (0.5% DMSO) and RVFV infected cells. Data were analyzed using the non-linear regression formula: log (inhibitor) vs. response–variable slope (4 parameters) in GraphPad Prism 6. The IC₅₀, defined as the effective concentration resulting in a 50% inhibition of infection, was used to evaluate compound activity. The relative cell number was determined by normalizing the cell number of compound treated + virus-infected cells with mock treated (0.5% DMSO) + virus-infected cells.

Co-immunoprecipitiation (co-IP) assay and antibodies

Cells infected with rMP-12 (MOI = 10 for 8 h) encoding wildtype NSs-V5 or its mutants served as sources of NSs protein. These lysates were combined with cell lysates expressing the protein of interest. Transient overexpression by plasmid DNA transfection was carried out using Lipofectamine 2000 (Life Technologies). For co-IP assay, cells in a 12 well dish were lysed in IP lysis buffer (Life Technologies, cat # 87787) supplemented with complete protease inhibitor cocktail (Roche) at 4°C for 20 min and clarified at 14,000 x g for 15 min at 4°C. Ten percent of the cleared lysate was kept aside as a lysate control, while the remaining lysate was incubated with 2 μg of the indicated antibody and 25 μl of washed protein A/G plus agarose beads and allowed to rotate mildly for 3h at 4°C. The beads were then washed three times in lysis buffer and one last wash with high salt PBS containing 300 mM NaCl. The bound proteins were eluted by boiling in Laemmli sample buffer, and separated by SDS-PAGE, followed by transfer to a polyvinylidene (PVDF) membrane. The blots were incubated for 1h at RT or overnight at 4°C with the indicated primary antibodies. After three washes, the blots were incubated with the appropriate alkaline phosphatase (AP)-conjugated secondary antibodies (GE Healthcare) according to the manufacturer's recommendations. The blots were washed and developed by a Western blotting detection system (GE Healthcare). For direct protein analyses, cells in 12-well dishes were lysed in 100 μl of 1X Laemmli sample loading buffer and boiled for 5 min at 95°C. Equal amounts of samples were used for Western blot analysis as described above. The following antibodies were used for Western blot analysis or immunoprecipitation (when required): T446 phosphorylated PKR (Abcam), PKR (BD Transduction), S52 phosphorylated EIF2α (Cell Signaling), EIF2α (Cell Signaling), TFIIH-p62 subunit (Santa Cruz), NEDD8 (Cell Signaling), CUL1 (Santa Cruz), SKP1 (Cell Signaling) and alkaline phosphatase (AP) conjugated V5 antibody (Life Technologies). The antibodies to HA, V5, Flag and Myc were from Sigma.

Generation of doxycycline inducible PKR shRNA expressing stable cell line

HeLa cells were transduced with lentivirus expressing doxycycline (Sigma) inducible human PKR shRNA (cat # V2THS_170555, GE Healthcare) encoding the antisense sequence 5’-TTTATCTCTGATGTATCTG-3’ and selected in Puromycin (1 μg/ml, from Sigma) containing media. The HeLa stable cell line can be induced with doxycyline (1 μg/ml) to express PKR shRNA.

Mutations and virus rescue

The plasmid DNA for recombinant MP-12 (rMP-12) virus rescue including the S segment encoding plasmid DNA in which the NSs ORF was replaced by a luciferase gene was a kind gift from Dr. Shinji Makino (Univ. of Texas Medical Branch, US). Mutations in the NSs gene, including the addition of a V5 tag to the gene were introduced using Q5 Site-Directed Mutagenesis Kit (New England Biolabs) by following the manufacturer’s recommendations. The virus was rescued as described previously [20]. Briefly, subconfluent BSR-T7/5 cells were co-transfected with an S-genome RNA expression plasmid, such as pProT7-S(+) or its variants encoding NSs mutations and a mixture of pPro-T7-M(+), pPro-T7-L(+), pT7-IRES-vN, pCAGGS-vG, and pT7-IRES-vL using TransIT-LT1 (Mirus Bio Corporation). The culture medium was replaced with fresh medium 24h later. At 5 days post-transfection, the culture supernatants were collected, clarified and inoculated onto VeroE6 cells. The supernatant of infected VeroE6 cells at 2–3 days PI were aliquoted and stored at -80°C.

siRNA screening targeting human F-box genes

The 70 siRNAs targeting human F-box genes were cherry picked from the Dharmacon ON-TARGETplus SMARTpool siRNA Library—Human Ubiquitin Conjugation Subset 2 or 3 (list in the supplementary data S4 Fig). The smart pool contained 4 different siRNAs targeting individual gene and were screened against RVFV ZH501 in triplicate at a concentration of 15 nM in a 96-well plate format using optimized assay conditions as described previously [9]. Briefly, HeLa cells (12,500 cells per well) were reverse transfected using HiPerfect (Qiagen) transfection reagent (0.6 μl per well) in a total volume of 100 μl per well in 96-well plates following the manufacturer’s instructions. As a positive control, 3 wells of each plate were transfected with non-targeting siRNAs. Mock infected cells were treated as negative controls. On the following day cells were washed and replaced with fresh media. Forty-eight hours post transfection, cells were subjected to RVFV infection (MOI = 1, 24h). The percentage of infected cells was determined using HCA analysis of N expressing cells. Data were represented as the average ±SD.

Plasmid constructs and siRNAs

pcDNA3-FLAG-UBC12 (C111S) was a gift from Dr. Tetsu Kamitani [18]. The cullin expression plasmids, pcDNA3-myc3-CUL1,-2,-3, -4A,- 4B, -5, -7 or HA2-PARC/CUL9 were gifts from Dr. Yue Xiong [39–41] (Addgene # 19896, 19892, 19893, 19951, 19922, 20695, 19895 and 20937 respectively). The cullin mutant expression plasmids, pcDNA3-HA-CUL1 ΔC22, L756A/I757A, K720A, Y42A/M43A, Δ610–615 or ΔN53 (19942, 19938, 19939, 19940, 19941 and 19950 respectively) were gifts from Dr. Yue Xiong [42,43]. The dominant negative (Dn) cullin expression plasmids pcDNA3-CUL1-3, 4A, 4B and CUL5 were a gift from Dr. Wade Harper [21] (Addgene plasmid # 15818, 15819, 15820, 15821 and 15822 respectively). pcDNA3-HA-FBXW11 was a gift from Dr. Yi Sun [44]. pcDNA3- Flag-BTRC/FBXW11-Wt, ΔN, ΔF, ΔD, ΔNF and ΔWD40 constructs were a gift from Dr. Tomoki Chiba [45].

The binding properties of the CUL1 mutant proteins with SKP1 or RBX1 were as follows: the wildtype CUL1 contains 776 amino acid (aa) residues of which the N terminal 249 aa regulate its binding to SKP1, while the remaining 527 aa recruit RBX1 [21,42,43].The dominant negative Dn-CUL1 (truncated protein expressing the first 452 aa) or CUL1Δ610–615 cannot bind to RBX1, but retains interaction with SKP1. CUL1-ΔN53 or Y42A/M43A mutants, cannot bind to SKP1, but retain their binding to bind to RBX1. Lastly, the CUL1-ΔC22, L756A/I757A, or the NEDDylation defective mutant K720A can bind to both SKP1 and RBX1.

NSs-V5 gene expression plasmid was generated by sub-cloning the PCR amplified product from the pProT7-S plasmid expressing NSs-V5 gene into pcDNA3.1 vector. The siRNAs targeting BTRC (cat# S17109) was from Ambion while FBXW11 (cat # J-003490-07) CUL1 (cat # J-004086-08) and SKP1 (cat # J-003323-15-0002) were from GE Healthcare.

pcDNA3-FLAG-UBC12 (C111S) was a gift from Dr. Tetsu Kamitani (Wada, H., Yeh, E.T. and Kamitani, T., 2008, J Biol Chem, 275, 17008–15). Dominant negative (Dn) Cullin expression plasmids pcDNA3-CUL1-3, 4A, 4B and CUL5 were a gift from Dr. Wade Harper (Jin, J., Ang, X.L., Shirogane, T. and Wade Harper, J. 2005. Methods Enzymol, 399, 287–309.) (Addgene plasmid # 15818, 15819, 15820, 15821 and 15822 respectively). pcDNA3-HA-FBXW11 was a gift from Dr. Yi Sun (Zhao, Y., Xiong, X. and Sun, Y. 2011. Mol Cell, 44, 304–16).

The siRNA’s-1, -2 and -3 targeting FBXW11 were from Ambion with cat# S23485, S23486 and S23487 respectively.

Quantitative real-time PCR

Relative changes in RVFV RNA levels or mRNA levels of host genes were determined by qRT-PCR on an ABI Prism 7900HT sequence detection system using RNA UltraSense one-step kit and TaqMan probes (Life Technologies). Total RNA was extracted using the RNeasy Plus minikit (Qiagen). Fifty nanograms of RNA was used per qRT-PCR reaction. The target gene mRNA or viral RNA expression levels were normalized to the PPIB housekeeping gene. Relative expression levels were determined using the comparative threshold cycle (C_T) method [46]. The taqman probe sequences used for RVFV RNA have been described previously [47].

Statistical methods

The student Paired t test was used to determine P values. **** indicated P<0.0001.