hSpt16 or ETS-1 depletion rescues the replication defect of a VSV^M51R mutant

We previously showed that hSpt16 RNAi significantly enhances the replication of VSV in both A549 cells and primary human fibroblasts, suggesting that FACT contributes to VSV restriction [9]. However, we wanted to ask if ETS-1 was also restrictive to VSV replication because it was possible that FACT-mediated VSV restriction is independent of ETS-1 and the FEAR pathway. VSV^∆M51 or VSV^M51R strains are thought to exhibit replication defects due to their inability to block host antiviral gene expression [16,17,20], therefore we wondered whether these replication defects may be related to an inability of these mutants to antagonize the FEAR pathway.

To begin to address these questions, we assessed the replication of recombinant VSV strains encoding eGFP and either wild-type (VSV-eGFP) [30] or M51R mutant M (VSV^M51R-eGFP) proteins [30] in A549 cells transfected with small interfering RNAs (siRNAs) targeting hSpt16 or ETS-1 [9]. We found hSpt16 or ETS-1 RNAi to significantly enhance VSV-eGFP replication compared to scrambled (control) RNAi conditions, suggesting the FEAR pathway indeed restricts VSV. The VSV^M51R-eGFP strain exhibited a replication defect compared to VSV-eGFP under control RNAi conditions but strikingly, under either hSpt16 or ETS-1 RNAi conditions, this mutant replicated to titers indistinguishable from VSV-eGFP (Fig 1A and 1B). These results suggest that the FEAR pathway contributes to VSV restriction, however VSV^M51R-eGFP is more sensitive to restriction by this pathway.

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Fig 1. The FEAR pathway contributes to VSV restriction in human cells and is strongly activated by VSV^M51R-eGFP.

(A-B) VSV-eGFP (WT) and VSV^M51R-eGFP (M51R) titers 24 h post-infection (hpi) (MOI = 0.001) in A549 cells transfected with indicated RNAi treatments using two independent siRNAs for either hSpt16 (A) or ETS-1 (B) knockdown. Scram., scrambled siRNA. Statistical significance in A and B was determined by unpaired two-tailed Student’s t-tests between indicated treatments. (C) Immunoblot (IB) of endogenous ETS-1 in A549 whole cell extract (WCE) after infection with WT or M51R (MOI = 10). (D) Densitometric quantification of ETS-1 from multiple IB experiments as in C. Results of unpaired two-tailed Student’s t-test between ETS-1 levels in mock WCE and infected WCE are shown above each bar graph. (E) IB of U2OS cell WCE transfected with empty vector (EV) or ETS-1-HA constructs 48 h post-transfection. (F) Representative fluorescence microscopy images of U2OS cells transfected as in E and then infected with WT for 16 h. NucBlue was used to mark cell nuclei and to account for cell number. Scale bar = 500μm. (G) Quantification of total GFP signal/field normalized to NucBlue signal for experiments from F. Data are means ± SD; n = 3. Statistical significance was determined by unpaired two-tailed Student’s t-test between indicated treatments. * = P < 0.05; ** = P < 0.01; ns = not significant.

https://doi.org/10.1371/journal.ppat.1014430.g001

Wild-type VSV, but not VSV^M51R, inhibits ETS-1 expression to evade restriction

The results above were reminiscent of our VV studies where we found the ΔA51R strain to exhibit a replication defect that could be rescued by RNAi-mediated depletion of FACT subunits or ETS-1 [9]. We also found this ΔA51R strain to more strongly induce ETS-1 expression than wild-type VV during infection of human cells [9]. Thus, we next used infection time courses and immunoblots (IBs) to determine if ETS-1 levels differ between VSV-eGFP and VSV^M51R-eGFP infections in A549 cells. Infections were performed under high multiplicity of infection (MOI) conditions to ensure that all cells were infected, thereby allowing the assessment of a single round of infection. Interestingly, while ETS-1 protein levels were not significantly different between mock- and VSV-eGFP-infected cell lysates, VSV^M51R-eGFP-infected lysates showed significantly elevated ETS-1 by 4 h post-infection (hpi) (Fig 1C and 1D). VSV N was used as a marker for infection. These results indicate that infection with the VSV^M51R-eGFP mutant induces ETS-1 expression while wild-type (VSV-eGFP) infection does not.

Notably, hSpt16 or ETS-1 depletion still enhanced VSV-eGFP replication by approximately ~1 log (Fig 1A and 1B), despite the absence of detectable ETS-1 induction under the high MOI (10) infection conditions (Fig 1C and 1D) used for immunoblot analysis. One possible explanation is that the viral growth assays were performed under low MOI (0.001), creating conditions in which VSV-eGFP is unable to completely suppress the FEAR pathway, perhaps due to lower expression of its viral antagonist. Thus, VSV-eGFP benefits from prior depletion of Spt16 or ETS-1.

We hypothesized that wild-type VSV suppresses ETS-1 expression to prevent activation of antiviral gene expression programs that require elevated ETS-1 levels. Therefore, we next asked if ETS-1 overexpression prior to infection could suppress VSV-eGFP replication. To do this, we overexpressed an HA-tagged human ETS-1 construct (ETS-1-HA) in cells for 48 h (Fig 1E) and then challenged cells with VSV-eGFP. Using fluorescence microscopy, we quantified GFP signals as a readout for infection and normalized these signals to total cell number by labeling cell nuclei with NucBlue. Compared to cultures transfected with empty vector (control), ETS-1-HA-transfected cells displayed significantly reduced GFP signal (Fig 1F and 1G). Collectively, these data suggest that prior overexpression of ETS-1 in cells limits wild-type VSV-eGFP infection. However, our time course data (Fig 1C and 1D) suggests VSV-eGFP normally evades this restriction by preventing ETS-1 expression throughout infection. In contrast, VSV^M51R-eGFP fails to prevent cells from inducing ETS-1 during infection (Fig 1C and 1D).

VSV^M51R infection induces ETS-1 expression independently of the IFN response

We showed ΔA51R VV strains to induce ETS-1 expression in the absence of an intact IFN response [9]. Therefore, we wanted to determine if ETS-1 induction by VSV^M51R-eGFP infection was also independent of the IFN response. Interferon regulatory factor 3 (IRF3) is critical for IFN pathway activation during VSV infection of non-immune cell types [31]. Therefore, we first examined ETS-1 induction in IRF3 knockout A549 cells. No significant differences in ETS-1 induction were found between wild-type and IRF3 knockout cells during VSV^M51R-eGFP infection (S1A and S1B Fig). We also quantified ETS-1 expression levels in A549 cells lacking STAT1, another key component of the IFN signaling pathway that functions downstream of the IFN-α/β receptor [32,33]. ETS-1 induction by VSV^M51R-eGFP infection was also unchanged in STAT1 knockout cells compared to control cells (S1C and S1D Fig). Collectively, these data indicate that both DNA viruses (VV) and RNA viruses (VSV) activate ETS-1 expression independently of the IFN response.

Wild-type VSV, but not VSV^M51R, promotes hSpt16^SUMO depletion to prevent ETS-1 induction during infection

During FEAR pathway activation, FACT complexes comprised of hSpt16^SUMO and SSRP1 are required to induce downstream ETS-1 expression [9]. Therefore, we hypothesized that VSV-eGFP may suppress ETS-1 expression by altering FACT subunit levels. Using high MOI infection time courses and IBs, we determined if hSpt16^SUMO or SSRP1 levels differ between VSV-eGFP and VSV^M51R-eGFP infections. It is important to note that both hSpt16^SUMO and SUMOless hSpt16 forms can be resolved from one another by SDS-PAGE and simultaneously detected via IB with hSpt16 antibodies (Abs) [9]. Strikingly, our IBs revealed a clear and significant reduction of hSpt16^SUMO (upper band in Fig 2A) levels in VSV-eGFP-infected lysates compared to mock-infected lysates by 4 hpi, and hSpt16^SUMO levels became virtually undetectable by 8 hpi. In contrast, hSpt16^SUMO levels remained unchanged throughout the entire VSV^M51R-eGFP infection time course (Fig 2A and 2B). Interestingly, SUMOless hSpt16 (lower band in Fig 2A) or SSRP1 levels did not significantly change during infection with either virus (Fig 2A, 2C and 2D), suggesting that VSV infection leads to the specific depletion of hSpt16^SUMO subunits. When comparing the effect of MOI on hSpt16^SUMO depletion during VSV-eGFP infection, we noticed that complete depletion of hSpt16^SUMO levels occurred earlier (by 4 hpi) when using higher MOIs (e.g., 10 or 30) versus during lower MOI (e.g., 3) infections. However, VSV^M51R-eGFP could not deplete hSpt16^SUMO at any MOI or time point (Figs 2A and S2). Together, these findings suggest that VSV-eGFP depletes hSpt16^SUMO during infection, and that the loss of hSpt16^SUMO correlates with the absence of ETS-1 induction.

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Fig 2. VSV, but not VSV^M51R, promotes hSpt16^SUMO depletion to prevent ETS-1 induction.

(A) IB of FACT subunit levels in WCE from A549 cells infected with VSV-eGFP (WT) or VSV^M51R-eGFP (M51R) for indicated hpi (MOI = 10). (B-D) Densitometric quantification of IB experiments in A for hSpt16^SUMO (B) SUMOless hSpt16 (C) or SSRP1 (D) levels. Data in (B-D) are means ± SD; n = 3. Statistical significance was determined by unpaired two-tailed Student’s t-test comparing protein levels in mock WCE to infected WCE, shown above each bar graph. (E-F) IB of immunoprecipitated SUMO-1- (E) or SUMO-2/3- (F) conjugated protein fractions in WCE from mock-, WT-, or M51R-infected A549 cells (MOI = 3) 18 hpi. Cont., control IP. RanGAP1 is a known SUMOylated protein and used as a control for enrichment of SUMOylated fractions [34]. GFP is a marker for infection. (G) IB of endogenous ETS-1 and hSpt16 levels and in WCE of scrambled or hSpt16 siRNA-treated A549 cells 8 hpi with indicated strains (MOI = 10). (H) Densitometric quantification of IB experiments in G for ETS-1. Data are means ± SD; n = 3. Statistical significance was determined by unpaired two-tailed Student’s t-test between indicated treatments. ** = P < 0.01, or ns = not significant.

https://doi.org/10.1371/journal.ppat.1014430.g002

Next, we wanted to determine if the phenotypes we observed using the A549 cell line would also be observed in primary human cells. Thus, we used neonatal human dermal fibroblasts (NHDF), which we have previously shown to restrict viral replication in a hSpt16-dependent manner [9]. Consistent with a role for the FEAR pathway in restricting viral replication in these cells, hSpt16 or ETS-1 RNAi promoted VSV replication. Moreover, the replication defect of VSV^M51R-eGFP was fully complemented during conditions of hSpt16 or ETS-1 knockdown (S3A and S3B Fig). In addition, as with A549 cells, ETS-1 levels were strongly induced by VSV^M51R-eGFP, but not by VSV-eGFP, in NHDF cells (S3C and S3D Fig). Furthermore, only VSV-eGFP promoted hSpt16^SUMO depletion in these primary cells (S3C and S3E Fig). As with A549 cells, we did not observe infection induced changes in SUMOless hSpt16 or SSRP1 levels compared to mock-infected lysates (S3C, S3F and S3G Fig). Collectively, these data suggest that the FEAR pathway also restricts VSV in primary cells and that VSV-eGFP can also deplete hSpt16^SUMO and block ETS-1 induction in these cells.

To determine if loss of hSpt16^SUMO levels during VSV-eGFP infection was due to a global down-regulation of intracellular SUMOylation, we used Abs against SUMO-1 or SUMO-2/3 to immunoprecipitate (IP) SUMOylated protein fractions from mock-, VSV-eGFP-, or VSV^M51R-eGFP-infected lysates [9]. As expected, hSpt16^SUMO was specifically depleted in VSV-eGFP-infected lysates, while present in mock- and VSV^M51R-eGFP-infected lysates. Consistent with our prior study [9], hSpt16^SUMO bands were observed in either SUMO-1 or SUMO-2/3 IPs, indicating that hSpt16 can be SUMOylated with SUMO-1 or SUMO-2/3 proteins. Importantly, no overt reductions in either total SUMOylation banding patterns or levels of SUMOylated-RanGAP1 (an abundant, SUMOylated protein [34]) were observed in VSV-eGFP versus VSV^M51R-eGFP infections (Fig 2E and 2F). These data suggest that VSV-eGFP promotes depletion of hSpt16 SUMOylated with either SUMO-1 or SUMO-2/3, and that the specific loss of hSpt16^SUMO levels during VSV-eGFP infection is not due to a generalized decrease in intracellular SUMOylation.

Finally, because hSpt16^SUMO is required for ETS-1 induction during VV infection [9], we wanted to determine if hSpt16 knockdown prior to infection would inhibit ETS-1 induction by VSV^M51R-eGFP. Consistent with our VV studies, hSpt16 RNAi abrogated ETS-1 induction after VSV^M51R-eGFP infection (Fig 2G and 2H). Collectively, these data indicate that the inability of VSV^M51R-eGFP to deplete hSpt16^SUMO results in FEAR pathway activation, robust ETS-1 expression, and subsequent restriction of viral replication.

VSV M promotes depletion of cytosolic hSpt16^SUMO subunits independently of other VSV proteins

Given that the only difference between VSV-eGFP and VSV^M51R-eGFP is the M51R substitution in VSV M, we hypothesized that this viral factor was responsible for hSpt16^SUMO depletion. Consistent with this, a VSV^∆M51 strain expressing GFP (VSV^∆M51-GFP [35]) was also unable to deplete hSpt16^SUMO levels and strongly induced ETS-1 expression when compared to its wild-type VSV-GFP control strain (S4 Fig). However, we still conducted a screen of 4 of the 5 VSV-encoded proteins to determine if any of these viral factors could reduce hSpt16^SUMO levels when expressed from plasmids in uninfected human cells. The VSV L protein, encoding the viral RNA polymerase, was excluded from this screen as it seemed unlikely to be involved in immune evasion. Consistent with our hypothesis, only expression of Flag-tagged VSV M (VSV M-Flag) resulted in hSpt16^SUMO depletion (Fig 3A). Expression of either VSV M^M51R-Flag or VSV M^∆M51-Flag mutant constructs failed to deplete hSpt16^SUMO levels (Fig 3B and 3C). Moreover, these did not alter SUMOless hSpt16 levels (Fig 3B and 3D), consistent with our infection results. These data indicate that wild-type VSV M, but not M^M51R or M^ΔM51, can promote hSpt16^SUMO depletion in the absence of other VSV proteins.

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Fig 3. VSV M promotes hSpt16^SUMO depletion independently of other VSV proteins and blocks ETS-1 expression.

(A) IB of endogenous hSpt16 in 293T whole cell extract (WCE) transfected with indicated Flag-tagged VSV protein expression constructs for 24 h. Empty vector, EV; Matrix, M; Nucleocapsid, N; Glycoprotein, G; Phosphoprotein, P. Asterisks indicate probable VSV M degradation products. (B) IB of endogenous hSpt16 in 293T WCE transfected with indicated VSV M expression constructs 24 h post-transfection. EV and Flag-GFP constructs were negative controls. (C-D) Densitometric quantification of IB experiments in B for hSpt16^SUMO (C) and SUMOless hSpt16 (D). Data are means ± SD; n = 3. Statistical significance was determined by unpaired two-tailed Student’s t-test comparing protein levels in EV to indicated transfection treatments, shown above each bar graph. ** = P < 0.01, ns = not significant. (E) IB of endogenous ETS-1 in U2OS WCE after transfection with indicated expression constructs for 24 h and then infected with VSV^M51R-eGFP (M51R) (MOI = 10) for 8 h. (F) Densitometric quantification of IB experiments in E for ETS-1. (G) IB of endogenous ETS-1 in U2OS WCE after transfection with indicated expression constructs for 24 h and then infected with ΔA51R VV strain (MOI = 10) for 8 h. (H) Densitometric quantification of IB experiments in G for ETS-1. In F and H, data are means ± SD; n = 3. Statistical significance was determined by unpaired two-tailed Student’s t-test between indicated treatments. ** = P < 0.01, ns = not significant.

https://doi.org/10.1371/journal.ppat.1014430.g003

Given that VV A51R blocks the FEAR pathway by binding to cytosolic hSpt16^SUMO subunits [9], we next asked if VSV M also targets cytosolic hSpt16^SUMO subunits. To do this, we co-expressed VSV M-Flag constructs with either a HA-tagged wild-type (HA-hSpt16) or nuclear localization sequence (NLS)-deleted (HA-hSpt16^∆NLS) hSpt16 construct we reported previously [9]. Importantly, we confirmed that HA-hSpt16 displayed both cytosolic and nuclear localization while HA-hSpt16^∆NLS was restricted to the cytosol, as expected (S5A Fig). Upon co-transfection with VSV M-Flag, the SUMOylated forms of both HA-hSpt16 and HA-hSpt16^∆NLS were depleted (S5B Fig), indicating VSV M can promote depletion of cytosolic hSpt16^SUMO subunits.

VSV M proteins from the Indiana and New Jersey strains deplete hSpt16^SUMO and block infection-induced ETS-1 expression

We next examined if plasmid-based expression of VSV M-Flag was sufficient to block ETS-1 induction by either VSV^M51R-eGFP (Fig 3E and 3F) or ΔA51R VV (Fig 3G and 3H) infection. Prior expression of VSV M-Flag, but not VSV M^M51R-Flag, was able to suppress ETS-1 induction by either of mutant virus (Fig 3E-3H). Given that the experiments above utilized the VSV M protein derived from the Indiana serotype (VSV-IN), we asked whether M from the New Jersey serotype (VSV-NJ) [36] [which shares ~59.8% amino acid sequence identity with VSV-IN (S6A Fig)] depleted hSpt16^SUMO. Like VSV-IN M, the M protein from VSV-NJ also depleted hSpt16^SUMO and inhibited ETS-1 induction when expressed transiently in cells (S6B–S6D Fig). The ability of VSV M proteins to block ΔA51R-induced ETS-1 expression demonstrates that these viral factors are bona fide FEAR pathway antagonists that function independently of other VSV proteins. Collectively, our prior [9] and current findings suggest that VV and VSV have independently evolved unique strategies to inhibit FACT-dependent ETS-1 expression by either tethering hSpt16^SUMO to microtubules (VV A51R) or by promoting hSpt16^SUMO degradation (VSV M), illustrating that FEAR pathway suppression is important for both DNA and RNA viruses.

Rae1-Nup98 interaction with VSV M does not contribute to hSpt16^SUMO depletion

VSV M is thought to inhibit host gene expression by interacting with Rae1, a component of the cellular Rae1-Nup98 protein complex that exports mRNA from the nucleus [17], although the exact mechanism is controversial [17,22]. In one proposed model, VSV M binds Rae1 to inhibit Rae1-Nup98-mediated mRNA export from the nucleus while an alternative model suggests VSV M usurps Rae1-Nup98 complexes to inhibit cellular gene transcription [17,22]. Notably, VSV M^M51R or M^ΔM51 mutants cannot interact with Rae1 [17]. Despite the unresolved nature of VSV M-Rae1 interaction models, we wanted to determine if Rae1 or Nup98 depletion would affect hSpt16^SUMO levels. Thus, we tested if Rae1 or Nup98 RNAi altered hSpt16^SUMO levels in mock-infected cells or if their depletion inhibited the ability of VSV-eGFP to reduce hSpt16^SUMO levels during infection. In either case, we observed no effect of Rae1 or Nup98 depletion on hSpt16^SUMO levels (S7A and S7B Fig). This suggests that VSV M antagonism of the FEAR pathway cannot be explained by its interaction with Rae1-Nup98 complexes.

VSV M-mediated hSpt16^SUMO depletion requires the ubiquitin-proteasome system (UPS)

Most proteins in the cell are turned over by the UPS [37]. Therefore, we first wanted to determine if VSV M-mediated hSpt16^SUMO depletion required ubiquitination. We expressed VSV M-Flag in human cells in the presence or absence of a ubiquitin activating enzyme inhibitor, TAK-243 [38], which globally blocks ubiquitination. We found TAK-243 treatment to prevent VSV M-mediated depletion of hSpt16^SUMO, suggesting ubiquitination is required for hSpt16^SUMO loss (Fig 4A and 4B). We then determined whether VSV M promotes proteasome-dependent degradation of hSpt16^SUMO by chemically inhibiting the proteasome using bortezomib or MG-132. hSpt16^SUMO depletion was blocked in the presence of either proteasome inhibitor (Fig 4C–4F), suggesting VSV M promotes the proteasome-dependent degradation of hSpt16^SUMO.

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Fig 4. VSV M, but not VSV M^M51R, promotes hSpt16 ubiquitination, leading to the proteasomal-degradation of hSpt16^SUMO.

(A) IB of endogenous hSpt16 in U2OS whole cell extract (WCE) after transfection with empty vector (EV) or VSV M-Flag. Where indicated, 1 μM TAK-243 was added at 6 h post-transfection. Vehicle (DMSO) treatments served as negative controls. WCE were prepared 24 h post-transfection. (B) Densitometric quantification of IB experiments in A for hSpt16^SUMO. (C) IB of endogenous hSpt16 in U2OS WCE after transfection with EV or VSV M-Flag. Where indicated, 25 nM bortezomib was added at 6 h post-transfection. Vehicle (DMSO) treatments served as negative controls. WCE were prepared 24 h post-transfection. (D) Densitometric quantification of IB experiments in C for hSpt16^SUMO. (E) IB of endogenous hSpt16 in U2OS WCE after transfection with EV or VSV M-Flag. Where indicated, 40 μM MG-132 was added at 6 h post-transfection. Vehicle (DMSO) treatments served as negative controls. WCE were prepared 24 h post-transfection. (F) Densitometric quantification of IB experiments in E for hSpt16^SUMO. (G) IB of TUBE pulldowns from 293T cells transfected with EV, VSV M or M^M51R-Flag for 24 h. Bortezomib was added to all cultures at 6 h. (H) IB of TUBE pulldowns from mock-, VSV-eGFP (WT) or VSV^M51R-eGFP (M51R) infected A549 cells for 24 h (MOI = 10). Bortezomib was added to all cultures at 6 hpi. In B, D and F, data are means ± SD; n = 3. Statistical significance was determined by unpaired two-tailed Student’s t-test between indicated treatments. ** = P < 0.01, ns = not significant.

https://doi.org/10.1371/journal.ppat.1014430.g004

VSV M, but not VSV M^M51R, promotes ubiquitination of hSpt16 proteins

Given that VSV M promotes proteasome-dependent degradation of hSpt16^SUMO, we asked whether hSpt16 was differentially ubiquitinated during transient expression of VSV M or M^M51R. Importantly, we included bortezomib in our cultures to prevent hSpt16^SUMO degradation during VSV M expression. Cells were harvested under denaturing buffer conditions then incubated with Tandem Ubiquitin Binding Entities (TUBEs), which are beads conjugated to pan-ubiquitin-binding domains designed to enrich for ubiquitinated proteins [39]. The ubiquitination pattern of hSpt16 was then assessed via IB. Although we did not observe overt differences in ubiquitination patterns in our lysate inputs across transfection treatments, after TUBE-based enrichment of ubiquitinated protein fractions, there was a clear increase in high molecular weight hSpt16 protein species in wild-type VSV M-Flag-transfected lysates, indicative of ubiquitinated forms of hSpt16. In contrast, these higher molecular weight hSpt16 species were largely absent in empty vector or VSV M^M51R-Flag transfected lysates (Fig 4G). These data suggest that wild-type VSV M promotes hyper-ubiquitination of hSpt16 in the absence of other VSV proteins. Similar hSpt16 hyper-ubiquitination patterns were observed during VSV-eGFP, but not VSV M^M51R-eGFP, infection suggesting VSV M also promotes hSpt16 ubiquitination in the context of infection (Fig 4H).

VSV M directly interacts with hSpt16 proteins

Since VSV M promotes hSpt16 ubiquitination, yet only induces hSpt16^SUMO (but not SUMOless hSpt16) degradation, we hypothesized that VSV M, but not the M^M51R mutant, interacts with hSpt16^SUMO for targeted degradation. To test this hypothesis, we performed coimmunoprecipitation (Co-IP) assays using Flag Abs from lysates transiently expressing VSV M- or M^M51R-Flag in the presence of bortezomib. Surprisingly, we observed endogenous hSpt16^SUMO and SUMOless hSpt16 to both Co-IP with VSV M (Fig 5A). This was unexpected because VSV M only promotes the degradation of hSpt16^SUMO, yet these data suggest that VSV M interacts with both hSpt16 and hSpt16^SUMO proteins. Furthermore, VSV M^M51R also co-immunoprecipitated with both forms of hSpt16 (Fig 5A). This was also unexpected since VSV M^M51R is unable to promote hSpt16^SUMO degradation (Fig 3B and 3C). To validate these results in the context of infection, we performed additional Co-IPs from VSV-eGFP- or VSV^M51R-eGFP-infected lysates using VSV M Abs. Similar to when VSV M-Flag was expressed in uninfected cells, we found both hSpt16 forms to Co-IP with VSV M and VSV M^M51R in infected cells (Fig 5B). We performed reciprocal Co-IPs using Abs against endogenous hSpt16 and again observed VSV M and M^M51R to Co-IP with both forms of hSpt16 during infection (Fig 5C). Together these data suggest that VSV M proteins interact with both SUMOylated and SUMOless hSpt16 proteins.

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Fig 5. VSV M interacts with hSpt16.

(A) Representative IB of VSV M or M^M51R-Flag proteins and hSpt16 after immunoprecipitation with Flag or isotype control immunoglobulin (IgG) Abs from transfected 293T whole cell extract (WCE) 24 h post-transfection. Bortezomib was added to all cultures at 6 h post-transfection. (B) Representative IB of VSV-eGFP (WT) or VSV^M51R-eGFP (M51R) M proteins and hSpt16 after immunoprecipitation with VSV M or control IgG Abs from infected A549 lysates 24 hpi (MOI = 10). Bortezomib was added to all cultures at 6 hpi. (C) Representative IB of hSpt16 and WT or M51R M proteins after immunoprecipitation with hSpt16 or control IgG Abs from WT or M51R infected A549 lysates 24 hpi (MOI = 10). Bortezomib was added to all cultures at 6 hpi. (D) Representative IB of in vitro GST pulldown of purified Flag-hSpt16 proteins incubated with purified GST-His, GST-VSV M-His, or GST-VSV M^M51R-His proteins using glutathione beads.

https://doi.org/10.1371/journal.ppat.1014430.g005

Next, we wanted to determine if the VSV M and hSpt16/hSpt16^SUMO interaction is direct. To do this, we used in vitro pulldown experiments with purified Flag-tagged hSpt16/hSpt16^SUMO [9] and purified VSV M proteins encoding an N-terminal GST tag and a C-terminal His tag (GST-VSV M-His). GST encoding a C-terminal His tag (GST-His) was used as a negative control protein. We incubated purified Flag-hSpt16/hSpt16^SUMO with either GST-His, GST-VSV M-His, or GST-VSV M^M51R-His, then pulled down GST-fused proteins using glutathione beads. GST-His failed to pull down either Flag-hSpt16 or hSpt16^SUMO, however, both GST-VSV M-His and -M^M51R-His interacted with both forms of Flag-hSpt16 (Fig 5D), consistent with our Co-IP data (Fig 5A–5C). Collectively, this suggests that VSV M and M^M51R directly bind to both SUMOless and SUMOylated hSpt16, and that the M51R mutation does not affect hSpt16 binding.

The N-terminus of VSV M is required for interaction with hSpt16 proteins and FEAR pathway suppression

Since VSV M^M51R does not affect Spt16 binding, yet fails to promote hSpt16^SUMO degradation, we hypothesized that residues beyond M51 are required for hSpt16 interaction and subsequent hSpt16^SUMO degradation. To identify VSV M regions potentially involved in hSpt16 binding and hSpt16^SUMO degradation, we generated a series of constructs with increasing N-terminal lengths of VSV M fused to GFP-Flag (Fig 6A). We then assessed the ability of these constructs to promote hSpt16^SUMO degradation and suppress ETS-1 induction. Notably, VSV M fragments containing the first 75 residues retained the ability to promote hSpt16^SUMO depletion (Fig 6B and 6C) and suppress ETS-1 induction (Fig 6B and 6D). In contrast, constructs expressing only the first 25 or 50 residues of VSV M failed to promote hSpt16^SUMO degradation (Fig 6B and 6C) and did not suppress ETS-1 induction (Fig 6B and 6D). These data suggest that the N-terminal 75 amino acids of VSV M are sufficient for FEAR pathway suppression.

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Fig 6. N-termini of VSV M mediates hSpt16 interaction and promotes hSpt16^SUMO degradation.

(A) Diagram of N-terminal VSV M-GFP-Flag fusion constructs. (B) IB of U2OS whole cell extract (WCE) 24 h post-transfection with indicated VSV M-GFP-Flag constructs infected with VV ∆A51R (VV^∆A51R) to assess ETS-1 induction. I3L is used as a marker for VV infection. (C-D) Densitometric quantification of hSpt16^SUMO (C) or ETS-1 (D) from multiple IB experiments as in B. (E) Diagram of differentially expressed VSV M proteins from VSV M mRNA. Full length a.a. 1-229, M1; a.a. 33-229, M2; a.a. 51-229, M3 showing alternative translation start sites. (F) IB of endogenous hSpt16 in 293T lysates transfected with indicated VSV M expression constructs from E 24 h post-transfection then infected with VV ∆A51R (MOI = 10) for 8 h. EV constructs served as negative controls. (G-H) Densitometric quantification of IB experiments in F for hSpt16^SUMO (G) and ETS-1 (H). (I) Representative IB of VSV M1- or M2-Flag proteins and hSpt16 after immunoprecipitation with Flag or isotype control immunoglobulin (IgG) Abs from transfected 293T lysates 24 h post-transfection. Bortezomib was added to all cultures at 6 h post-transfection. (J) Alphafold3-generated structure of full-length (a.a. 1-229) VSV M (strain Indiana). M1, M33 and M51 are shown in red and represents the first methionine of M1, M2, and M3 forms of VSV M, respectively. Alpha-helix 1 (α1) noted in green. KKIL motif highlighted in orange. (K) Representative IB of endogenous hSpt16 and ETS-1 in U2OS WCE after transfection with indicated expression constructs for 24 h and then infected with ΔA51R VV strain (MOI = 10) for 8 h. (L-M) Densitometric quantification of IB experiments in K for hSpt16^SUMO (L) and ETS-1 (M). (N) Representative IB of VSV M or KKIL^Ala-Flag proteins and hSpt16 after immunoprecipitation with Flag or isotype control immunoglobulin (IgG) Abs from transfected 293T lysates 24 h post-transfection. Bortezomib was added to all cultures at 6 h post-transfection. For data in C-D, G-H, and L-M, data are means ± SD; n = 3 and statistical significance was determined by unpaired two-tailed Student’s t-test between indicated treatments. ** = P < 0.01, ns = not significant.

https://doi.org/10.1371/journal.ppat.1014430.g006

Consequently, we next examined N-terminal deletions of VSV M. For these studies, we took advantage of the fact that the VSV M gene actually encodes for three independent proteins; M1 (full length), M2, and M3, the latter two encoding shorter N-termini. These three VSV M proteins are expressed in the same frame but start at alternative initiation sites producing three M proteins with differing N-termini [14,40] (Fig 6E). We therefore individually expressed each of these VSV M variants and observed that only full length (M1) promoted hSpt16^SUMO degradation (Fig 6F and 6G) and inhibited ETS-1 induction (Fig 6F and 6H). Given that M1 and M2 N-termini differ by only 32 a.a. residues, we asked whether they might differ in their ability to interact with hSpt16 proteins. As expected, VSV M1 interacted with hSpt16/hSpt16^SUMO. However, VSV M2 failed to interact with either form of hSpt16 (Fig 6I), suggesting the first 32 residues of VSV M, are critical for hSpt16 protein interaction, hSpt16^SUMO depletion, and FEAR pathway suppression.

Collectively, our data suggest that VSV M residues 1–75 are sufficient for promoting hSpt16^SUMO degradation indicating a critical role for the N-terminus of VSV M in FEAR pathway antagonism. Moreover, our finding that the VSV M51 residue is required for hSpt16^SUMO degradation, but not for hSpt16 protein binding, indicates that hSpt16 binding on its own is not sufficient to promote hSpt16^SUMO degradation, and that other regions of the N-terminus are involved in hSpt16 binding. Consistent with this, the M2 protein, which is missing residues 1–32 (but still retains M51), could neither bind hSpt16 proteins or promote hSpt16^SUMO degradation. These data suggest that hSpt16 binding, as well as an additional function mediated by the M51 residue, are both required for hSpt16^SUMO degradation.

VSV M encodes an N-terminal hSpt16-binding motif important for FEAR pathway suppression

After mapping the hSpt16^SUMO degradation activity to the first 75 N-terminal residues of VSV M, we next examined whether structural information could provide insight into this region. Classically, full length VSV M is described to be composed of a flexible N-terminal domain (residues 1–57) known as the N-Terminal Extension (NTE) followed by a globular C-terminal domain (S8A Fig) [36,41,42]. Although a full VSV M structure for the Indiana serotype has not been solved, the crystal structure of a VSV M fragment encompassing the globular domain (a.a. 44–229) bound to Rae1-Nup98 complexes has been reported (4OWR) [42] (S8B Fig). In this structure, M contacts Rae1-Nup98 complexes using three distinct regions that project from a “fist-like” globular domain: a finger (a.a. 49–61), a thumb (a.a. 213–223), and a “web” region (a.a. 142–144) (S8B Fig). Importantly, the finger motif, encompassing the M51 residue, makes critical contacts with residues in a Rae1 β-propeller [42], illustrating the importance of this region to host interactions. Unfortunately, this structure does not contain the additional N-terminal residues (a.a. 1–43) which we have shown are critical for hSpt16/hSpt16^SUMO protein binding (Fig 6I), hSpt16^SUMO degradation (Fig 6F and 6G), and suppression of ETS-1 expression (Fig 6F and 6H). Therefore, we utilized Alphafold3 [43] to ascertain the predicted structure of full length VSV M that would include the entire N-terminus of M1 (Fig 6J). We compared this predicted structure with the 4OWR crystal structure by overlaying them (S8C Fig) and conducting pairwise similarity tests using the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RSCB PDB) [44] to assign root mean square deviation (RMSD) values and template modeling (TM) scores. RMSD scores of <3 Å and TM scores >0.5 were considered to have similar overall folds while those producing RMSD values >3 Å and TM scores <0.5 were considered to be structurally unrelated [45]. Pairwise comparisons between 4OWR and our Alphafold3-predicted VSV M structure produced a RMSD of 1.46 Å and a TM score of 0.95, providing confidence in our Alphafold3-generated VSV M structure (S8D Fig). Interestingly, the AlphaFold3-generated structure predicted a small helical motif encompassing residues S3-K6 (Fig 6J). Since residues K5-L8 are conserved between the Indiana and New-Jersey serotypes [36], which both promote hSpt16^SUMO degradation (S6B and S6C Fig), we substituted this “KKIL” motif with alanine to determine if this motif was necessary for hSpt16^SUMO depletion and ETS-1 inhibition. Transient expression of VSV M-Flag encoding alanine in place of the KKIL motif failed to promote hSpt16^SUMO degradation (Fig 6K and 6L) or block ETS-1 expression (Fig 6K and 6M). This mutant also failed to interact with hSpt16 proteins (Fig 6N), indicating a role for this motif in VSV M-hSpt16 binding. Collectively, these data suggest that these N-terminal VSV M residues are necessary for VSV M-hSpt16 interaction.

We next asked whether M proteins from other vesiculoviruses that encode the N-terminal “KKIL” motif were capable of antagonizing the FEAR pathway. This included M proteins from Maraba virus (MARAV), Cocal virus (COCV), and Alagoas virus (VSAV) M proteins that share an overall amino acid identity with VSV M-IN of ~75–80% (S9A). To determine whether these proteins target the FEAR pathway, we transiently expressed Flag-tagged versions of these M proteins in human cells and assessed their ability to deplete hSpt16^SUMO and suppress ΔA51R-induced ETS-1 expression. Similar to VSV M, MARAV, COCV, and VSAV M proteins all promoted hSpt16^SUMO depletion (S9B and S9C Fig) and significantly reduced ETS-1 induction by ΔA51R infection (S9B and S9D Fig). Together, these data indicate that other closely-related rhabdoviruses encode M proteins that can also suppress the FEAR pathway.

FACT contributes to VSV host range restriction in Lymantria dispar insect cells

We originally became interested in understanding poxvirus A51R protein function after discovering that these proteins rescue the post-entry abortive infection of VSV in Lymantria dispar (gypsy moth or spongy moth)-derived LD652 cells [12]. With the knowledge that VV A51R binds to hSpt16^SUMO to antagonize its antiviral function [9] and that Spt16 proteins are well-conserved across invertebrate and vertebrate eukaryotes [9], we wondered if VV A51R-mediated rescue of VSV in LD652 cells related to its ability to antagonize insect Spt16 function.

After sequencing the L. dispar genome [46], we discovered that hSpt16 and L. dispar Spt16 (LdSpt16) proteins share an overall a.a. identity of ~60% [9]. However, because VV A51R only binds SUMOylated forms of hSpt16 [9], it was important to determine if LdSpt16 was also SUMOylated. Interestingly, IBs of LD652 cell lysates revealed two LdSpt16 forms as found with hSpt16 in human cells. Treatment of LD652 cultures with tannic acid, a global SUMOylation inhibitor [47], eliminated the upper LdSpt16 form on IBs, suggesting this band represents SUMOylated LdSpt16 (LdSpt16^SUMO) (Fig 7A). To determine if VV A51R interacts with LdSpt16^SUMO, we infected LD652 cells with a recombinant VV strain encoding Flag-tagged A51R (FA51R) under its natural A51R gene promoter (ΔA51R^FA51R) and then performed Co-IP [9]. LdSpt16^SUMO, but not SUMOless LdSpt16 proteins, specifically co-immunoprecipitated with FA51R in LD652 cell lysates (Fig 7B), suggesting VV A51R can indeed interact with SUMOylated LdSpt16 proteins. Next, we tested if A51R-LdSpt16^SUMO interactions were required for A51R-mediated rescue of VSV replication in LD652 cells. To do this, we transfected LD652 cells with a control expression vector encoding Flag-tagged GFP (FGFP) or vectors encoding wild-type FA51R, or a FA51R mutant that cannot bind to SUMOylated Spt16 proteins FA51R^158-162Ala [9]. 48 h post-transfection, we challenged cells with a recombinant VSV strain encoding firefly luciferase (VSV-LUC) [48]. We have previously used this strain and luciferase assays to sensitively and quantitatively measure VSV gene expression in these highly restrictive insect cells [12,49]. We also collected supernatants from these LD652 cultures to assess VSV replication by plaque assay [12]. While the wild-type FA51R construct was able to significantly enhance both VSV gene expression (Fig 7C) and replication (Fig 7D), the FA51R^158-162Ala mutant, was completely defective in this regard. These results suggest that VV A51R-mediated rescue of VSV requires A51R-LdSpt16^SUMO interaction.

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Fig 7. Abortive VSV replication in L. dispar cells can be complemented by VV A51R expression or by Spt16 RNAi.

(A) IB of L. dispar Spt16 (LdSpt16) in LD652 whole cell extract (WCE) in the absence or presence of tannic acid (TA) treatment. (B) Co-IP of endogenous LdSpt16 with FA51R in ΔA51R^FA51R-infected (MOI = 25) LD652 WCE. (C) Fold-change in luciferase (LUC) signal in LD652 cells transfected with indicated FA51R expression constructs for 24 h and then infected with VSV-LUC (MOI = 10) for 48 h. Fold-change in LUC is plotted as relative to LUC signal in Flag-GFP (FGFP) transfection treatments (negative control). Data are means ± SD; n = 3. (D) VSV-LUC titers from supernatants collected from C. Data are means ± SD; n = 3. (E) Fold-change in LUC signal after 72 h of transfection with dsRNAs (for RNAi) targeting either GFP (control) or LdSpt16 (Spt16-A/B) and infection with VSV-LUC (MOI = 10) for 48 h. Data are means ± SD; n = 4. (F) VSV-LUC titers from supernatants collected from E. Data are means ± SD; n = 4. (G) IB of LdSpt16 levels in LD652 WCE 48 h post-transfection with empty vector (EV) or increasing amounts of VSV M-Flag p166 expression vector. (H) Co-IP of endogenous LdSpt16 with FA51R or VSV M-Flag overexpressed in LD652 WCE. In C-F, statistical significance was determined by unpaired two-tailed Student’s t-test between indicated treatments. ** = P < 0.01, ns = not significant.

https://doi.org/10.1371/journal.ppat.1014430.g007

To directly test if LdSpt16 was involved in VSV restriction, we used in vitro-transcribed dsRNAs to target LdSpt16 by RNAi [12]. Compared to control RNAi targeting an irrelevant GFP mRNA sequence, VSV-LUC gene expression (Fig 7E) and replication (Fig 7F) were significantly enhanced in LD652 cell cultures transfected with two independent dsRNAs targeting LdSpt16 mRNA. These results suggested that the abortive infection of wild-type VSV in LD652 cells is at least in part dependent upon LdSpt16^SUMO-mediated restriction. Therefore, we hypothesized that wild-type VSV M may be defective in its ability to target LdSpt16^SUMO for degradation. Consistent with this, VSV M-Flag transfection into LD652 cells failed to promote LdSpt16^SUMO depletion at any expression level tested (Fig 7G). Furthermore, VSV M-Flag failed to Co-IP with LdSpt16 proteins, in contrast to Flag-A51R proteins that we included as a positive control (Fig 7H). These results suggest that VSV M can neither bind or promote degradation of LdSpt16^SUMO proteins. Thus, VSV host range restriction in LD652 cells may be due to an inability of VSV M to antagonize LdSpt16^SUMO antiviral function.

Cell lines and primary cultures

Mammalian cell lines were maintained at 37°C in 5% CO₂ atmosphere. A549, U2OS, 293T, and BHK-21 cells were cultured in DMEM supplemented with 10% FBS. BSC-40 cells were passaged in MEM containing 5% FBS. All media above additionally contained 1% non-essential amino acids, 1% L-glutamine, and 1% antibiotic/antimycotic. NHDF cells were passaged in fibroblast basal medium supplemented with low serum fibroblast growth kit. LD652 were cultured in a 1:1 mixture of Ex-Cell 420 and Graces insect medium supplemented with 10% FBS at 27°C under normal atmospheric conditions [12,68].

Viruses and virus titrations

All VSV strains were amplified using low MOI infections in BHK-21 cells. BSC-40 cells were used for VV stock preparation [12,69]. Unless otherwise noted, virus strains were titrated by plaque assay (VSV-LUC, ΔA51R^FA51R, ΔA51R) or fluorescent foci/plaque assay (VSV-eGFP, VSV^M51R-eGFP, VSV^∆M51-GFP) on A549, U2OS, or BSC-40 cell monolayers, with a 1.5% low-melting point agarose (Invitrogen) overlay used for VSV titrations [9]. Experimental viral infections were incubated for 1 h in serum free DMEM at 37°C before the inoculum was replaced with complete media for the remainder of the infection. Where indicated, 25 nM bortezomib was added 6 h post VSV-eGFP infection and was kept in the media until the end of the experiment. At indicated times post-infection, infected cell culture supernatants were collected, clarified by centrifugation, and clarified supernatant titers were determined by serial dilution followed by plaque assay or fluorescent foci/plaque assay [9].

Expression vectors

The human ETS-1-HA pEZ-M07 and the control expression vector were obtained from GeneCopoeia. VSV P-Flag, VSV N-Flag, VSV M(NJ)-Flag, VSV M(IN)-Flag, MARAV M-Flag, COCV M-Flag, VSAV M-Flag, and M51R/ΔM51 mutant derivatives were constructed by gene synthesis (Gene Universal or TWIST BioScience) with inclusion of 5’-SacII and 3’-PacI sites for cloning into SacII/PacI-digested pcDNA3 [9]. VSV M2, M3, and KKIL^Ala variants were constructed by PCR amplification with inclusion of SacII and PacI sites for cloning into SacII/PacI-digested pcDNA3 [9]. VSV M-Flag was also cloned into the insect p166 expression vector [12] using SacII/PacI sites. A.a. 1–229, -150, -100, -75, -50, and -25 VSV M-N-terminal GFP-Flag constructs were PCR amplified with the inclusion of 5’-BamHI and 3’-SacII sites for cloning into BamHI/SacII-digested GFP-Flag pcDNA3 [9]. His, VSV M-His or M^M51R-His was cloned into a pGEX vector containing a N-terminal GST using 5’-BamHI and 3’-SalI sites. VSV G-Flag was PCR amplified with inclusion of SacII and PacI sites for cloning into SacII/PacI-digested pcDNA3. Wild-type VV Flag-A51R and Flag-A51R^158-162Ala open reading frames were cloned into the SacII/PacI sites of p166 by SacII/PacI digest of Flag-A51R and Flag-A51R^158-162Ala pcDNA3 vectors [9]. The HA-hSpt16 and HA-hSpt16^∆NLS pEZ-M06 expression vectors have been described [9]. The Flag-GFP pcDNA3 and p166 vectors have been described [9,12]. All genes were sequence verified. Expression plasmids were transfected into U2OS cell lines as previously described using Lipofectamine 2000 in Opti-MEM I [12]. Plasmids transfected into 293T cells used a 1:6 ratio of DNA:PEI. Plasmids transfected into LD652 cells used a 1:3 ratio of DNA:Cellfectin II [49]. All cells were incubated with the transfection mixture for 6 h and then media was replaced with complete culture media Cells were incubated for the indicated times prior to further processing (e.g., protein extraction, see below).

General protein extraction and inhibitor treatments

Cells were washed with phosphate-buffered saline (PBS) prior to scraping and transfer into a 1.5 mL microcentrifuge tube for centrifugation at 4,000 x g at 4°C for 15 min. Cell pellets were resuspended in either 1x Reporter Lysis Buffer containing 1% Protease Inhibitor Cocktail (PIC) and 1 mM phenylmethylsulfonyl fluoride (PMSF) and freeze-thawed once, or were resuspended in Pierce RIPA buffer (containing 1% PIC and 1 mM PMSF) prior to addition of 5x SDS-PAGE loading buffer (100 mM tris HCl, pH 6.8, 4% SDS, 12% (v/v) glycerol, 4 mM DTT, 0.02% (w/v) Bromophenol Blue). Where indicated, cells were treated with 10 µM tannic acid for 4 h prior to protein harvest. Also, where indicated, TAK-243 (1 μM), bortezomib (25 nM), MG-132 (40 μM), or DMSO (vehicle control) was added to cells 6 h post-transfection and was kept in media until protein extraction.

Immunoblotting

WCE were boiled for 10 min prior to SDS-PAGE electrophoresis at 65–100 V for approximately 2–4 h. Separated proteins were transferred in Towbin Buffer onto nitrocellulose membranes at 150 mA at 4°C for 90 min and blocked with Li-COR Odyssey Blocking Buffer for 1 h at RT. Membranes were blotted with primary Ab overnight at 4°C, with actin serving as a loading control. After 3 x 5 min PBS-T (PBS, 0.1% Tween) washes, membranes were incubated in secondary Ab conjugated to an IRDye for 1 h, washed 3 x 5 min in PBS-T, then a final 5 min PBS wash. Membranes were then imaged with an Li-COR Odyssey Fc Imager. Densitometric analysis of immunoblots were performed using ImageJ (http://imagej.nih.gov/ij, Java 1.8.0_172[54-bit]). Digital images of immunoblots were analyzed by measuring the integrated density of each protein band following local background subtraction. The intensity of each target band (e.g., ETS-1, hSpt16^SUMO, hSpt16 or SSRP1) was normalized to the corresponding loading control band (e.g., actin) within the same lane from the same membrane. Normalized values were calculated as the ratio of target protein signal to actin signal and were subsequently expressed and plotted as “fold-change” relative to the indicated experimental control sample. The quantification shown represents the mean normalized values from at least three independent biological replicates with representative immunoblots shown in figures corresponding to one of these independent experiments.

Immunoprecipitation

Transient mammalian co-immunoprecipitations were performed by seeding 12e6 293T cells overnight, followed by PEI-mediated transfection for 6 h when bortezomib containing complete media was replaced. Infection-based co-immunoprecipitations were performed by seeding 15e6 A549 cells overnight, then infected for 24 h in the presence of bortezomib added after 6 hpi. Infection-based LD652 co-immunoprecipitation experiments were performed by seeding 1 x 10⁶ LD652 overnight, then infected with ∆A51R^FA51R (MOI = 25) for 24 h in the presence of AraC (0.2mg/ml) added with the replacement media. Regardless of co-immunoprecipitation condition, cells were washed with equal volumes of PBS twice prior to cell lysis in IP lysis buffer (1% PIC, 1 mM PMSF, 25 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1.0% NP-40) and subjected to shearing and sonication (two-15 second sonications with a 30 second interval on ice). Samples were benzonase treated (250 units/mL) for 1 h at RT. 10% of “input” was collected, and remaining lysate was end-over-end incubated with 10 µg of primary Ab overnight at 4°C (rabbit-anti-Flag, mouse-anti-VSV M or isotype control Ab). Then, lysates were incubated with PureProteome protein A/G magnetic beads for 1–2 h, extensively washed, and immunoprecipitants eluted in 60 μl 2x SDS-PAGE loading buffer. IPs of total SUMOylated protein fractions in mock-, VSV-eGFP, or VSV^M51R-eGFP-infected (MOI = 10) A549 cells for 12 h used either SUMO-1 or SUMO-2/3 Ab included with the Cytoskeleton Signal Seeker SUMOylation 1 or 2/3 Detection Kit and IPs were carried out according to the manufacturer instructions [9]. TUBE pulldowns were performed as per the manufacturer instructions. Briefly, cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, 50 µM PR-619, 1% PIC, 1 mM PMSF) and incubated with TUBE2 or control magnetic beads at 4 °C overnight, followed by extensive washing with TBS-T (20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% Tween-20).

Protein purification

GST-His only, GST-VSV M-His, and GST-VSV M^M51R-His plasmids were transformed in BL21-CodonPlus(DE3)-RIPL cells in the presence of 100 μg/ml ampicillin. Once overnight cultures reached an OD₆₀₀ of 0.5, protein expression was induced with 0.5mM isopropyl-D-1-thiogalactopyranoside (IPTG) at 18˚C for 20 h. Cells were lysed using an Emulsiflex C5 in GST Buffer A (50 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, 1% PIC, 1 mM PMSF, 1 μM Pepstatin, 2 μM Aprotinin, 10 μM Leupeptin, 0.2 mM AEBSF, and 10% glycerol, pH 7.5). Proteins were affinity purified using Glutathione Superflow Agarose resin and washed in GST Buffer A prior to elution using GST Buffer B (50 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, 1% PIC, 1 mM PMSF, 10% glycerol, and 10 mM glutathione, pH 7.5). The protein was then passed through a HisTrap HP column, subsequently washed with 10 volumes of His Buffer A (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 50 mM imidazole, 10% glycerol, pH 7.5) then eluted in His Buffer B (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 500 mM imidazole, 10% glycerol, pH7.5). Proteins were concentrated in a 10 kDa MWCO ultra centrifugal filter prior to storage. Flag-hSpt16 proteins were purified as previously described [9].

In vitro pulldown

10 μg of purified GST-His, GST-VSV M-His or GST-VSV M^M51R-His proteins were co-incubated with 10 μg of purified Flag-Spt16 proteins in Pull Down Buffer (0.1% NP40, 100 mM NaCl, 50 mM Tris-HCl pH 7.4 with 1% PIC and 1 mM PMSF) overnight. The next day, 10% input aliquots were taken and the rest of the sample was incubated with glutathione agarose resin for 1 h, washed 5 times in Pull Down Buffer, then eluted in 2 x SDS-PAGE loading dye for SDS-PAGE analysis.

RNAi in cell culture

All siRNAs were obtained from Sigma’s pre-designed siRNA library (see S1 Table). The hSpt16 and ETS-1-targeted siRNAs have been previously validated [9]. Transient siRNA-mediated knockdown was achieved by reverse transfecting mammalian cells with Lipofectamine 2000 according to manufacturer’s protocol for 48–72 h prior to viral infection [9]. RNAi knockdowns in LD652 cells were performed as previously described using in vitro-transcribed dsRNAs transfected with Cellfectin II in Sf-900 II serum free medium [12]. Transfection medium was replaced with complete culture medium after overnight culture. Sequences of primers used to generate dsRNA targeting GFP (control) or LdSpt16 gene sequences are in S1 Table.

Luciferase assays

Luciferase assays in LD652 WCE were performed as previously described with minor modifications [12,49]. Briefly, at the indicated times post-infection, cells were washed in PBS, collected by centrifugation (2000 rpm, 10 min, 4°C) and lysed in reporter lysis buffer (Promega). Lysates were spotted to 96-well dishes, mixed with Luciferase Assay Reagent (Promega) and arbitrary light units (LU) were measured using an FLUOstar Omega Plate reader (BMG Labtech).

Antibodies

Information regarding antibodies used in this study, including their source, is available in S1 Table.

Fluorescence microscopy

At indicated times post-infection, cells were stained with serum free media containing NucBlue (Thermo Fisher) for 30 min followed by replacement with equal volumes of PBS. Cells were imaged using a 4X objective on an EVOS-FL fluorescence microscope (Thermo Fisher) using DAPI and GFP cubes. Each condition had three replicate wells, and at least four images/well were collected for analysis. Image analysis was conducted using Fiji (NIH) to quantify the normalized GFP signal/field by dividing total GFP signal by total DAPI (NucBlue) signals. Positive signal was determined by using uninfected wells lacking NucBlue stain to set a minimum threshold in both DAPI and GFP channels that was applied to the remaining dataset. Fold change in GFP signals were calculated by dividing normalized GFP values of experimental treatments with normalized GFP values in control treatments (indicated in each Figure).

Immunofluorescence microscopy

For staining U2OS cells transiently transfected with HA-hSpt16 or HA-hSpt16^∆NLS, cells were seeded at a density of 30,000 cells per coverslip, cultured overnight, then transiently transfected with 500 ng of HA-hSpt16 or HA-hSpt16^∆NLS expression plasmids with Lipofectamine2000 for 6 h before replacing with complete media. Cells were fixed with 4% paraformaldehyde at 24 h post-transfection, incubated with blocking buffer (PBS with 1% BSA and 0.1% Triton-X) for 1 h, stained with rabbit-anti-HA Ab for 2 h, washed extensively with blocking buffer, incubated with Alexa Fluor-conjugated secondary Ab for 1h, and then again washed extensively. Coverslips were then mounted onto glass slides using ProLong™ Diamond anti-fade with DAPI and imaged with an Olympus FV10i confocal laser scanning microscope (version 2.1) equipped with Olympus Fluoview software (version 4.2a). Images were deconvoluted with CellSens Imaging software (version 1.18).

Protein sequence alignment and structure prediction

VSV M (NCBI accession: NP_041714.1) structure was predicted using AlphaFoldv3 [43] and analyzed using PyMOL (The PyMOL Molecular Graphics System version 1.2r3pre, Schrödinger, LLC.). To determine structural similarity between VSV M 4OWR and our Alphafold3-generated VSV M structure, pairwise structural alignments were conducted using RSCB PDB [44] to assign RMSD and TM scores. RMSD values of <3 Å and TM scores >0.5 were considered to have similar overall fold, while pairwise comparisons producing RMSD values >3 Å and TM scores <0.5 were considered to be structurally unrelated [45].

Statistical analyses

All statistical analyses were conducted using GraphPad Prism (version 8.0) software and P values <0.05 were considered statistically significant. Sample sizes, statistical tests used, and P value information are indicated in the respective Figure or Figure legend for each quantitative experiment.