eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition

RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

mutants as controls (Fig. 1a). Here, we also included rRVFV expressing NSs of the PTV phlebovirus strains Adames (PTV-A; virulent) or Balliet (PTV-B; avirulent), but these could not be followed up later due their transcription shutoff activity (see below). The replicative capacity of the various RVFV recombinants was first compared in PKR knockdown versus PKR-expressing control cell lines. As expected from the PKRdestroying activity of RVFV NSs (17,20), recombinant wild-type RVFV (rRVFV) grew to similar titers in both cell lines, whereas recombinant (rRVFVΔNSs) or natural (RVFV clone 13) NSs deletion mutants exhibited reduced replication in control cells but reached levels comparable to those of rRVFV in PKR knockdown cells (Fig. 1b). The NSs proteins of SFSV as well as of PTV-A also enabled similar growth efficiencies in both cell lines, whereas PTV-B NSs had no such activity. Of note, clone 13 and PTV-B are natural isolates from febrile humans (21,22), and the NSs protein of PTV-B (but not clone 13) retains type-I interferon (IFN) antagonist function in murine cells (23). We also infected HEK293 FLP-IN cells in which the expression of PKR (or green fluorescent protein [GFP] as control) was induced by doxycycline treatment. Also, both NSs-deficient control viruses as well as the PTV-B NSs recombinant were highly and specifically sensitive to PKR, whereas recombinants containing the NSs genes of SFSV or PTV-A showed only a minor reduction in titers under conditions of PKR induction, similarly to recombinant wildtype RVFV (Fig. 1c). Thus, PKR depletion and overexpression experiments suggest that the NSs proteins of SFSV (and PTV-A) confer PKR resistance.
PKR escape activity that does not affect PKR signaling. We then investigated the influence of the phleboviral NSs proteins on the PKR signaling pathway. Infection with the recombinant viruses (Fig. 2a) confirmed that the NSs of RVFV, but not of SFSV or PTV, reduces PKR levels (17,18,20). Consequently, autophosphorylated PKR, an indicator of PKR activity, was undetectable in the presence of RVFV NSs. Curiously, however, when cells were infected with viruses expressing SFSV or PTV NSs, phosphorylation of Phlebovirus NSs Protects eIF2B from Phospho-eIF2␣ ® both PKR and its substrate eIF2␣ was upregulated similar to that in the NSs deletion virus rRVFVΔNSs (Fig. 2a). Time course analyses showed that PKR and eIF2␣ phosphorylation persisted despite the presence of SFSV or PTV NSs (see Fig. S1 in the supplemental material). In line with this, parental SFSV also triggered PKR and eIF2␣ phosphorylation, unlike the PKR-destroying RVFV (Fig. 2b). Thus, on one hand, the NSs proteins of SFSV and PTV are required to counter the antiviral activity of PKR ( Fig. 1), indicating a PKR escape phenotype. On the other hand, however, these NSs proteins seem to act in a manner that is different from that of prototypical PKR antagonists, as they do not interfere with the PKR-eIF2␣ phosphorylation axis.
SFSV NSs enhances eIF2-dependent translation. Transfection of DNA plasmids is known to activate PKR (24). Hence, we aimed to assess the impact of SFSV NSs on cellular mRNA translation by using a transiently transfected luciferase reporter system. PTV NSs could not be included in these and further experiments as it impairs general host transcription (18) (data not shown). The luciferase reporter system encodes a bicistronic mRNA in which translation of the upstream firefly luciferase open reading frame (ORF) is canonically initiated from the 5= cap (and hence requires eIF2), whereas translation of the downstream Renilla luciferase ORF is initiated from the cricket paralysis virus IRES (IRES CrPV ) that does not require any eIFs (25). Cotransfection of SFSV NSs with the bicistronic reporter construct amplified firefly luciferase activity, i.e., eIF-dependent gene expression, in a dose-dependent manner (Fig. 3a), whereas it had no detectable effect on eIF-independent Renilla luciferase activity (Fig. 3b). Of note, the boost of firefly luciferase activity was observable only for C-terminally epitope-tagged NSs (SFSV NSs-3ϫFLAG), but not for the N-terminally tagged variant (3ϫFLAG-SFSV NSs) or the inert negative control (3ϫFLAG-ΔMx) (Fig. 3a). Both the C-terminally and the N-terminally tagged NSs proteins were, however, expressed at comparable levels ( Fig. 3c) and exhibited the previously reported (19) inhibitory activity toward transcriptional induction of type I interferon (see Fig. S2a and c). Consistent with the results from the bicistronic luciferase assay, C-terminally tagged SFSV NSs also boosted an eIFdependent SV40 luciferase reporter (Fig. S2b). Furthermore, puromycin labeling of de novo synthesized proteins showed that mRNA translation was comparable to that in noninfected cells during wt SFSV infection, whereas infection with the PKR-activating NSs mutant clone 13 (Fig. S1) led to a shutdown of protein synthesis (Fig. 3d). Thus, unlike other previously characterized viral PKR antagonists, SFSV NSs enables both viral replication and canonical eIF-dependent mRNA translation without blocking PKR activation or affecting the phosphorylation state of eIF2␣. SFSV NSs interacts with the eIF2B complex. SFSV NSs seems to protect protein synthesis by neutralizing antiviral PKR action downstream of eIF2␣. Ser51 phosphorylation of eIF2␣ is known to convert the eIF2␣␤␥ complex from a substrate into an inhibitor of its guanine nucleotide exchange factor eIF2B (26). Strikingly, our previous mass spectrometry-based approach to map the host interactome of SFSV NSs returned all five subunits of the eIF2B complex as the highest scoring candidate interactors (see Fig. S3) (27). To verify the interaction, we overexpressed the eIF2B subunits from cDNA plasmids together with C-terminally 3ϫFLAG-tagged SFSV NSs and performed immunoprecipitations for NSs or eIF2B with antibodies to epitope tags. Immunoprecipitation of FLAG-tagged NSs, but not the FLAG-tagged control protein (ΔMx), coprecipitated all five eIF2B subunits in a highly reproducible manner (Fig. 4a). Vice versa, when using an expression construct for epitope-tagged eIF2B-mCitrine-hemagglutinin (HA), we were Influence of SFSV NSs on a bicistronic reporter system with an upstream canonical (i.e., eIF-dependent) firefly luciferase ORF and a downstream Renilla luciferase ORF initiated from the eIF-independent IRES CrPV . HEK293 cells were transfected with the bicistronic reporter plasmid and expression plasmids for SFSV NSs containing either a C-or N-terminal 3ϫFLAG tag (SFSV NSs-3ϫFLAG and 3ϫFLAG-NSs SFSV, respectively) or for an irrelevant negative control (3ϫFLAG-ΔMx). Lysates were assayed for eIF2-dependent firefly (Fluc) (a) and IRES-dependent Renilla luciferase (Rluc) (b) activities (arbitrary units [AU]). Shown are data from 1 of 3 independent experiments. Each of these was conducted with three technical (i.e., parallel) replicates. Bars show means Ϯ SDs. ****, P Յ 0.0001; ***, P Ͻ 0.001 (one-way ANOVA, corrected for multiple comparisons using Dunnett test). For the Rluc data, none of the tests reached the significance level. (c) C-and N-terminally tagged SFSV NSs (SFSV NSs-3ϫFLAG and 3ϫFLAG-NSs SFSV, respectively) were transiently expressed in HEK293 cells, and expression levels were analyzed by immunoblotting (representative of 3 experiments). Phlebovirus NSs Protects eIF2B from Phospho-eIF2␣ ® able to pull down the entire eIF2B complex via the mCitrine tag and also SFSV NSs (Fig. 4b). As a specificity control, we expressed untagged eIF2B instead of eIF2B-mCitrine-HA and observed no such precipitations, as expected. Furthermore, SFSV NSs also enabled specific coprecipitation of endogenous eIF2B from both A549 and HEK293 cells ( Fig. S4a and data not shown), and superinfection with a PKR-activating NSsdeficient RVFV did not affect its interaction with eIF2B (Fig. S4b). Finally, only the translation-rescuing SFSV NSs with the C-terminal FLAG tag (SFSV NSs-3ϫFLAG) but not Overexpression of eIF2B subunits along with C-terminally 3ϫFLAG-tagged NSs (SFSV NSs-3ϫFLAG). Immunoprecipitation was performed with anti-FLAG antibody. (b) mCitrine-HA-tagged eIF2B (eIF2B-mCitrine-HA) was expressed along with eIF2B subunits ␣, ␤, ␥, and ␦ as well as SFSV NSs-3ϫFLAG. Untagged eIF2B served as control for unspecific binding. The eIF2B complex was precipitated using an mCitrine-binding matrix. (c) Overexpression of untagged eIF2B subunits along with C-or N-terminally 3ϫFLAG-tagged NSs (SFSV NSs-3ϫFLAG and 3ϫFLAG-NSs SFSV, respectively). Immunoprecipitation was performed with anti-FLAG antibody. (d) Immunoprecipitated proteins from the experiment shown in panel c after prolonged exposure. Shown data are representatives of 3 (a, c, and d) or 5 (b) experiments. *, light chain of the IP antibody. the inactive N-terminally tagged version (3ϫFLAG-SFSV NSs) interacted with eIF2B ( Fig. 4c and d). This result correlates with the functional data from the bicistronic reporter system (Fig. 3), suggesting that SFSV NSs boosts eIF-dependent translation and enables PKR-sensitive virus replication by acting directly on eIF2B.
Further attempts at identifying the eIF2B subunit(s) targeted by SFSV NSs using Far-Western blotting (employing cells that transiently expressed NSs-3ϫFLAG as bait and individual, bacterially expressed nondenatured eIF2B subunits as prey) did not reveal any interaction (data not shown). Moreover, bacterially produced SFSV NSs lost its ability to interact with cellular eIF2B in coimmunoprecipitation experiments, precluding binding studies with NSs produced in an eIF2B-free background (data not shown).
Established mechanisms of cellular eIF2B modulation are not applicable to SFSV NSs. Phosphorylation of eIF2␣ is not only mediated by PKR but also by other kinases of the so-called integrated stress response (ISR) (28). Besides virus infection, the ISR can also be activated by compounds such as arsenite. Due to its low cellular levels and tight regulation, eIF2B is considered the central hub of translation regulation by the ISR (26). Cancer cells, for example, elevate eIF2B expression to satisfy their demand for increased protein synthesis (29). Moreover, the stabilization of the decameric form of eIF2B (consisting of two copies of each of the five subunits), a process facilitated by the small molecule ISRIB (integrated stress response inhibitor), enables translation despite the presence of phospho-eIF2␣ (30)(31)(32). However, when testing for these mechanisms, we did not find SFSV NSs to elevate eIF2B levels under ectopic expression or infection ( Fig. 5a and b and data not shown). Moreover, after sucrose gradient ultracentrifugation of cell lysates, a shift of eIF2B␦ and eIF2B toward high-density fractions that is indicative of decamer formation was only detected for the ISRIB control and not for NSs-expressing SFSV or the ISR-activating rRVFVΔNSs::Kat virus ( Fig. 5c and data not shown). Thus, despite strongly binding to eIF2B, SFSV NSs seems not to act by any of the established phospho-eIF2␣ bypass mechanisms.
SFSV NSs does not interfere with binding of phospho-eIF2␣ to eIF2B. Comparative cryo-electron microscopy (cryo-EM) analyses have recently elucidated that non- rRVFVΔNSs::Kat served as negative control for infection-induced but NSs-independent effects on eIF2B stoichiometry, whereas ISRIB was included as positive control for eIF2B decamerization. Cell lysates were fractionated via 5% to 20% sucrose gradients, and fractions were analyzed for a shift of eIF2B subunits toward fractions of higher density by immunoblotting. Shown data are representatives of 2 (a) or 3 (b and c) experiments.
Phlebovirus NSs Protects eIF2B from Phospho-eIF2␣ ® phosphorylated and phosphorylated eIF2 bind to distinct sites on eIF2B (33)(34)(35)(36). Binding of nonphosphorylated eIF2␣ triggers the so-called productive mode of eIF2B in which eIF2·GTP is recycled and translation of mRNAs enabled. In contrast, phospho-eIF2␣ is structurally rearranged and consequently excluded from the productive binding site of eIF2B. Instead, it associates with eIF2B in a nonproductive binding mode, through which access of nonphosphorylated eIF2 to eIF2B is blocked and hence eIF2B activity abrogated, halting translation. To test the influence of SFSV NSs on the binding of phospho-eIF2␣ to eIF2B, we performed cofractionation as well as coimmunoprecipitation experiments. In the cofractionation experiments, lysates from cells that were treated with the ISR activator arsenite were separated by sucrose gradient ultracentrifugation, and the fractions were analyzed by immunoblotting. In lysates from control cells, the phospho-eIF2␣ peak was found to overlap that for fractions containing eIF2B, suggesting complex formation ( Fig. 6a and Fig. S5). However, expression of SFSV NSs did not shift the phospho-eIF2␣ peak toward lower-density fractions, as would have been expected if it interfered with the binding of phospho-eIF2␣ to eIF2B. Rather, phospho-eIF2␣ was exclusively recovered in the fractions containing eIF2B, similar to the situation in untransfected or control (ΔMx) transfected cells (Fig. 6a).  Fig. S5 in the supplemental material for results with the unstimulated cells). Sucrose gradient ultracentrifugation was performed, and fractions were analyzed by immunoblotting for phospho-eIF2␣ with eIF2B. *, gel artifact; **, remaining signal for SFSV NSs-3ϫFLAG (lower band) from previous FLAG antibody staining due to incomplete stripping of the anti-FLAG antibody. (b and c) Coprecipitation experiments. HEK293 cells were transfected with expression plasmids, treated with arsenite to induce eIF2␣ phosphorylation, and lysed, and protein complexes were precipitated via specific tags and analyzed by immunoblotting. (b) eIF2B pulldown. eIF2B-mCitrine, SFSV NSs, and ΔMx were expressed, and eIF2B was precipitated via the mCitrine tag. *, cleavage or partial degradation product of eIF2B. (c) NSs pulldown. SFSV NSs-Myc-SBP and eGFP-SBP were expressed, and NSs was precipitated via the SBP tag. Shown data are representatives of 2 (a), 5 (b), or 3 (c) experiments.
For the coimmunoprecipitations, two different approaches were chosen. First, we coexpressed the eIF2B-mCitrine-HA-containing eIF2B complex with SFSV NSs, stimulated eIF2␣ phosphorylation with arsenite, and subsequently immunoprecipitated eIF2B via the mCitrine moiety. Phospho-eIF2␣ specifically and reproducibly coprecipitated with eIF2B as expected (Fig. 6b). eIF2␣ phosphorylation had no effect on the amount of SFSV NSs binding to eIF2B. Vice versa, the presence of SFSV NSs also did not reduce the signal of coprecipitated phospho-eIF2␣. To further support these findings, we directly investigated whether phospho-eIF2␣ is attached to NSs-precipitated eIF2B. To this aim, we constructed an expression plasmid for NSs (NSs-Myc-streptavidinbinding peptide [SBP]) that was C-terminally tagged with Myc (for immunoblot detection) and streptavidin-binding peptide (for precipitation with streptavidin-coated beads). eGFP-SBP served as negative control. Cells were transfected with these cDNA constructs, the ISR was stimulated with arsenite, and the lysates were subjected to streptavidin-mediated precipitation. As observed previously with the NSs-3ϫFLAG construct, NSs-Myc-SBP coprecipitated the endogenous eIF2B (represented by the eIF2B subunit), whereas eGFP-SBP did not (Fig. 6c). Importantly, endogenous phospho-eIF2␣ also specifically coprecipitated along with the NSs-eIF2B complex, demonstrating that NSs does not impede the binding of phospho-eIF2␣ to eIF2B. Thus, the results from the fractionation and coprecipitation experiments argue for a model in which SFSV NSs is binding to the eIF2B-phospho-eIF2 complex. Because, on the other hand, SFSV NSs enables virus replication despite the presence of phospho-PKR and phospho-eIF2␣, our data suggest that it renders eIF2B resistant to inhibition by bound phospho-eIF2.

DISCUSSION
Phleboviruses are gaining increased attention as agents of emerging zoonoses (4, 7, 37). As RNA viruses infecting vertebrates, they have to cope with the broadly antiviral PKR system, and the virulent phleboviruses RVFV and TOSV rapidly destroy PKR itself as a countermeasure (17). SFSV, one of the most widespread phleboviruses, is a more moderate pathogen and, as we show here, inhibits the PKR-mediated translation block by targeting eIF2B. All phleboviruses investigated so far also inhibit induction of the antiviral interferons (IFN-␣/␤) (7,38,39). Interestingly, virulent RVFV and TOSV do this by NSs-mediated destruction of key host factors, whereas SFSV NSs obstructs the DNA-binding domain of transcription factor IRF3, blocking its access to the IFN promoter (19). Thus, there seems to be a correlation between the mode of NSs action against host defenses (rapid and enzymatic versus slowly building up and stoichiometric) and the respective virulence level of the particular phlebovirus, observed not only in humans but also in an outbred mouse model (23).
Viruses have evolved a plethora of measures to avoid translation inhibition by the antiviral PKR system ( Fig. 7a and b). The destruction of PKR by RVFV and TOSV NSs proteins was mentioned above. At the upper end of the signaling chain, PKR-triggering dsRNA is sequestered by the influenza A NS1 protein and the vaccinia virus E3L, among others (40). dsRNA-induced PKR autophosphorylation is inhibited by the Kaposi's sarcoma herpesvirus vIRF2 (41), vaccinia virus K3L acts as a PKR pseudosubstrate (42), and herpes simplex virus ICP34.5 recruits a protein phosphatase to revert eIF2␣ phosphorylation (43). Besides these examples, all viral strategies reported so far act on the level of PKR, affect phosphorylation of its substrate eIF2␣ (2, 3), or circumvent eIF-dependent translation with a special IRES that directly binds to the 40S ribosome (25). Although, mechanistically, viruses may not necessarily target PKR itself, they nonetheless neutralize its negative effect on viral replication by a variety of other means, i.e., biologically, they encode a PKR escape activity. As shown by our knockdown and overexpression experiments, the NSs of SFSV only offers a growth advantage when PKR is present, i.e., phenotypically, it is a PKR antagonist. By targeting eIF2B, however, it employs a distinct and apparently novel PKR escape mechanism (Fig. 7c). There are several modes of cellular eIF2B regulation that can relieve the negative influence of phosphorylated eIF2␣, most prominently, upregulation of eIF2B subunits and eIF2B decamerization, but none of these apply to SFSV NSs and NSs did not displace phospho-eIF2␣ from eIF2B. Thus, NSs can enable eIF2-dependent protein synthesis in the presence of activated PKR by a novel mechanism that protects eIF2B from the nonproductive mode that is normally imposed by phospho-eIF2␣.
It remains to be shown where on the eIF2B complex NSs is binding and how NSs manages to force phospho-eIF2-bound eIF2B into the productive mode. One possibility would be that SFSV NSs enables binding of phospho-eIF2 to the productive site that is normally occupied by nonphosphorylated eIF2. However, since phospho-eIF2 is excluded from the productive binding site due to electrostatic repulsion by the eIF2B␤ subunit (35), this possibility appears unlikely. Alternatively, due to its complex architecture, eIF2B offers potential for manifold posttranslational modifications. For instance, Ser539 of the catalytic eIF2B subunit is a regulatory site for enzymatic eIF2B activity (44). Although we did not observe differential eIF2B Ser539 phosphorylation in the presence of SFSV NSs when using commercial antibodies (data not shown), other posttranslational modifications cannot be ruled out. Moreover, in addition to its known binding sites for phosphorylated and nonphosphorylated eIF2, GDP, and GTP, eIF2B possesses a significant amount of further surface area without an assigned physiological function. It could thus be envisioned that NSs acts as mimic of an as-yet-unidentified host protein with eIF2B regulatory activity. Further elucidation of the supercomplex of eIF2B, phosphorylated eIF2␣, and SFSV NSs might aid the ongoing efforts to understand the intricate regulation of eIF2B.

MATERIALS AND METHODS
Cell culture. A549, BHK-21, HEK293, HEK293T, Vero B4, and Vero E6 cells were cultured in Dulbecco's minimal essential medium (DMEM) and CCM34 medium (DMEM with addition of 17.8 mg/liter L-alanine, 0.7 g/liter glycine, 75 mg/liter L-glutamic acid, 25 mg/liter L-proline, 0.1 mg/liter biotin, 25 mg/liter hypoxanthine, and 3.7 g/liter sodium bicarbonate) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 g/ml streptomycin. HeLa PKR knockdown and control cells, generated by stable transfection with the pSUPER vector encoding a short hairpin RNA against the PKR gene or with the empty pSUPER, respectively (kindly obtained from Charles Samuel, UC Santa Barbara) (45), were maintained in full DMEM or CCM34 additionally supplemented with 2 g/ml puromycin. HEK293 FLP-IN T Rex cells with inducible expression of PKR or GFP were as described previously (17) or obtained from Ju-Tao Guo (Baruch S. Blumberg Institute) (46) and maintained in full DMEM or CCM34 medium additionally supplemented with 50 g/ml hygromycin and 5 g/ml blasticidin, respectively. All cell lines were routinely tested for mycoplasma contamination.
For infection, viruses were diluted to the desired multiplicity of infection (MOI) in serum-free medium and incubated with the cells for 1 h at 37°C, after which, the inoculate was replaced by full cell culture medium.
Plasmids. Expression constructs for 3ϫFLAG-tagged NSs of RVFV and SFSV (GenBank EF201822.1) as well as 3ϫFLAG-ΔMx were described previously (19,51). C-terminally tagged SFSV NSs was generated by amplification of the ORF with specific primers, of which, the reverse primer contained the 3ϫFLAG or Myc-SBP tag sequence, restriction with BamHI and XhoI, and subsequent ligation-dependent cloning into pI.18 (kindly provided by Jim Robertson, National Institute for Biological Standards and Control, Hertfordshire, UK) and pcDNA3.1 (Invitrogen). Coding sequences for eIF2B subunits eIF2B1 (NG_015862.1), eIF2B2-B10 (NG_013333.1), eIF2B3 (NG_015864.1), and eIF2B4 (NG_009305.1) were amplified from pDONR223 constructs (kindly provided by BIOSS Centre for Biological Signaling Studies, University of Freiburg, Germany), inserted into pcDNA3.1/V5-His-TOPO via TA cloning, and finally subcloned into pcDNA3 via the HindIII and NotI restriction sites. The eIF2B5 ORF (NM_003907.2) was amplified and subcloned from pRevTRE2-hIF2B-GFP (52) (kind gift of Dirk Görlich, Max Planck Institute for Biophysical Chemistry, Germany) and additionally supplemented with an mCitrine-HA cassette using the NotI and ApaI sites. Primer and insert sequences are available upon request. All expression constructs were confirmed by Sanger sequencing with primers covering the respective inserts and multiple-cloning sites.
Translation reporter pFR_CrPV_xb, constructed by Philipp Sharp (53), was obtained from Addgene (plasmid 11509). Subsequently, the HSV-TK promoter was replaced with an SV40 promoter by directional cloning using the BglII and HindIII restriction sites. Firefly reporter construct p-125Luc was kindly donated by Takashi Fujita (54); pGL3-Control and pRL-SV40 were purchased from Promega.
Replication assays. HeLa cells were seeded into 6-well plates and infected with recombinant viruses (MOI, 0.01). HEK293 FLP-IN T Rex cells were seeded into 6-well plates, induced with 2 g/ml doxycycline (Sigma) for 24 h, and infected with recombinant viruses (MOI 0.01) under continuing induction. Culture supernatants were harvested 24 h postinfection (hpi), and virus titers were determined by immunoplaque assay as follows: BHK-21 cells were infected with serial dilutions of cell culture supernatant and incubated under an overlay of 0.6% Avicel (FMC BioPolymer) (50) for 24 h prior to fixation and permeabilization. After staining with polyclonal mouse ascites fluid raised against recombinant RVFV N (kindly provided by Michèle Bouloy, Institut Pasteur Paris, France) (55) and horseradish peroxidase (HRP)-coupled secondary antibody, plaques were visualized using TrueBlue peroxidase substrate (KPL) and titers were calculated.
Immunoblot analysis. Protein samples were run on 12% or 15% acrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) via semidry blotting. After blocking in Tris-buffered saline (TBS) with 5% bovine serum albumin (BSA) or milk powder, primary antibody staining was performed for 1 h at room temperature or overnight at 4°C. Membranes were washed in TBS-0.1% Tween 20, stained with secondary antibodies for 45 min, and washed again in TBS-0.1% Tween 20 and once in TBS. Finally, membranes were developed with a SuperSignal West Femto kit (Pierce), and bands were visualized using a ChemiDoc imaging system and Image Lab software (Bio-Rad). For kinetic analysis after infection with the recombinant virus panel, proteins were blotted onto nitrocellulose membranes (Whatman Protran) and stained as described above, and bands were detected using an Odyssey imaging system (LI-COR).
Primary antibodies were used as follows: ␤-actin Bicistronic translation and interferon reporter assay. HEK293 cells seeded into 96-well plates were transfected the following day with a bicistronic firefly and Renilla luciferase reporter construct (40 ng) as well as expression constructs for NSs proteins or the control protein ΔMx (0.1 ng, 1 ng, and 10 ng) via TransIT-LT1. Total transfected DNA was adjusted to equal amounts with the empty vector pI.18. Cells were processed 48 h after transfection, and luciferase activities were determined using the Dual Luciferase Reporter Assay system (Promega) according to the manufacturer's instructions and a LB 942 TriStar 2 multimode reader (Berthold Technologies). Means and standard deviations (SDs) were calculated from three technical replicates within each biological replicate.
For interferon reporter assays, cells were seeded and transfected as described above, using p125Luc and pRL-SV40 as reporter plasmids (40 ng each). Luciferase activities were determined 24 h after transfection, and means and SDs were calculated as described above.
Puromycin labeling. To monitor ongoing translation, medium was supplemented with 10 ng/ml puromycin and incubated for a further 10 to 30 min at 37°C prior to harvesting (56). Incorporation of puromycin was visualized via immunoblotting as described above.
Proteomics. As described previously (19,27,51), approximately 2 ϫ 10 8 HEK293T cells were infected with the recombinant RVFV strain expressing C-terminally TAP-tagged SFSV NSs (rZH548ΔNSs::NSsSFSV-CTAP) at an MOI of 5. The cells were washed with and scraped off in prechilled phosphate-buffered saline (PBS) 16 h postinfection (hpi). The cell pellet was snap-frozen in liquid nitrogen, lysed in TAP buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.2% NP-40, 5% glycerol) supplemented with protease and phosphatase inhibitors, snap-frozen again, and stored at Ϫ80°C until further processing. TAP purification was performed by sequential pulldowns using streptavidin-agarose and HA-agarose beads. Bound protein complexes were eventually eluted in Laemmli buffer and subjected to one-dimensional SDS-PAGE prior to trypsin digestion and peptide analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which was described in detail elsewhere (27). Network visualization of high-confidence interactors was generated using STRING database (57).
Coimmunoprecipitation. HEK293 cells seeded into 10-cm dishes were transfected with expression plasmids (2 g each per eIF2B subunit and NSs or control constructs) via the calcium phosphate method (58) and lysed 16 to 24 h after transfection or after additional infection with rZH548ΔNSs:: Ren (MOI of 5, 5 hpi). A549 cells were transfected with 4 g expression plasmids for SFSV NSs or ΔMx using Lipofectamine 2000 (Thermo Fisher). Cells were scraped into PBS and lysed in prechilled lysis buffer (50 mM Tris-HCl [pH 7.0], 150 mM NaCl, 1% IGEPAL-630, 1ϫ protease inhibitor cocktail, 1ϫ phosphatase inhibitor cocktail). Cell debris was removed (10,000 ϫ g, 10 min, 4°C), and supernatants were used for further processing. For immunoprecipitation via FLAG or HA, FLAG M2 or HA antibodies were coupled to magnetic beads (143-21D or 10004D; Invitrogen) overnight; for immunoprecipitation of SBP-tagged proteins, streptavidin-coated beads (11205; Invitrogen) were used. Coated beads were then processed according to the manufacturer's recommendations, equilibrated to lysis buffer, and incubated with cell lysate under rotation at 4°C for 4 h or overnight. After extensive washing, bound proteins were eluted by heating in Laemmli gel sample buffer at 94°C for 5 min. For immunoprecipitation via mCitrine, supernatants were applied to prewashed wells of a GFP-multiTrap (Chromotek) and incubated at 4°C for 60 to 90 min. Wells were then washed extensively with lysis buffer, and bound proteins were finally eluted with preheated Laemmli buffer (60°C) under strong agitation for 15 to 20 min.
Data availability. The data sets generated during the present study are available from the corresponding author on reasonable request.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.