TRIM69 Inhibits Vesicular Stomatitis Indiana Virus

Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

constraining viral pathogenesis and viral colonization is well established, because IFN responses involve so many ISGs, it is often unclear which individual gene products inhibit a given virus. Vesicular stomatitis Indiana virus (VSIV) is a virus that is mainly restricted to the Americas, where it causes vesicular stomatitis, a disease that primarily affects ungulates, and also rarely causes mild infections in humans (7)(8)(9). VSIV is transmitted by biting insects and causes characteristic vesicular lesions at bite sites around the hooves, mouth, nose, teats, and coronary bands (7). Although complications can occur, natural VSIV infection is typically mild and rapidly resolved. In contrast, experimental VSIV infection can be highly pathogenic and neurotropic in young mice (10).
In addition to being a notable veterinary pathogen, VSIV has been used extensively as a model virus and has been integral to our understanding of vesiculovirus and rhabdovirus biology. Notably, vesiculoviruses can be particularly sensitive to IFNs, leading to their inclusion in the IFN unit definition assay (11). Indeed, type I IFNs likely play a major role in limiting the severity of VSIV infections, and multiple ISGs have been ascribed anti-VSV activity (4,(12)(13)(14)(15).
Importantly, IFNs play a major role in constraining VSIV in vivo. Type I IFN receptor (IFNAR) knockout (KO) mice succumb to doses of VSIV that are several orders of magnitude lower than a lethal dose in wild-type (WT) mice (16). Moreover, while VSIV is largely restricted to the central nervous system (CNS) in lethally infected WT mice, VSIV colonizes multiple organs in IFNAR KO mice (16). Interestingly, it is likely that multiple ISGs are involved in limiting the tissue tropism of VSIV. Specifically, it appears that IFIT2 is crucial for preventing VSIV colonization of the brain, but it is not solely responsible for limiting VSIV replication in other organs (15). Thus, other ISGs must play key roles in limiting VSIV tissue tropism. Importantly, VSIV causes neurological disease in multiple species following intracranial inoculation (17,18), suggesting that the ability of ISGs to prevent VSIV from initially accessing the CNS is the cornerstone in limiting VSIV neuropathology across multiple species (19).
VSIV's low pathogenicity in humans, its rapid replication, and ease of genetic manipulation have made this virus the basis of multiple therapeutic strategies. For example, VSIV can be modified to express antigens from heterologous viruses that can be utilized as vaccine strategies (20). This approach has achieved recent notable success in conferring protection from Ebola virus infection (21). Similarly, VSIV has been used as the backbone of multiple oncolytic strategies (22). Just aa with natural VSIV infection, IFNs and ISGs appear to be critical for preventing oncolytic viruses from invading healthy tissues (23,24) and could be critical determinants governing whether oncolytic vesiculoviruses will be efficacious (25). Furthermore, ISGs likely play a key role in limiting the replication of VSIV-based vaccines and are an important safety feature of this immunization strategy.
The key roles that ISGs play in constraining VSIV pathogenesis and limiting VSIV replication (in natural infection, oncolytic therapies, and vaccine strategies) mean that there is a need to better understand both how and which ISGs inhibit VSIV. Using arrayed ISG expression screening, we identified the anti-VSIV activity of TRIM69, a relatively poorly characterized TRIM protein. Through exogenous expression and CRISPR/Cas9 knockout, we demonstrate that both exogenous and endogenous TRIM69 have potent anti-VSIV activity. Importantly, the inhibition is highly specific for VSIV as multiple other viruses were not inhibited by TRIM69. Notably, TRIM69 shows strong signatures of positive selection, and multiple common alleles circulate in human populations. Interestingly, murine orthologues of TRIM69 had no detectable anti-VSIV activity whereas rat TRIM69 possessed potent antiviral activity. We speculate that TRIM69 could be an important ISG for protecting healthy tissues from VSIV and might therefore limit VSIV pathogenesis and influence the specificity and efficacy of vesiculovirus-based therapeutic strategies.
(This article was submitted to an online preprint archive [26]).
isoform of macaque Mx1 included in the screen lacked 154 N-terminal residues relative to the sequence of the human counterpart (that exhibited anti-VSIV activity), potentially explaining the lack of inhibition conferred by the macaque variant. Importantly, TRIM69 was not identified in the previous ISG screen of VSIV as it was not present in the ISG library used (4). TRIM69 exhibits potent and highly specific antiviral activity. Because the anti-VSIV activities of type I IFN and Mx1 are well documented (13,16), we were immediately struck by how potent the TRIM69-mediated inhibition of VSIV was in our initial screen (Fig. 1D). At the time that these experiments were carried out, TRIM69 had not been ascribed any antiviral activity, so we examined the ability of doxycycline-inducible TRIM69 to inhibit a small panel of viruses. Notably, while VSIV-GFP infection was reduced by Ͼ100-fold by TRIM69, the other viruses in our panel [influenza A virus A/Puerto Rico/8/1934 (H1N1) (PR8), Rift Valley fever phlebovirus (RVFV 35/74), and HIV-1 (NHG)] were unaffected by TRIM69 expression (Fig. 1E). It is well documented that many TRIM proteins are involved in antiviral signaling, which can often be triggered by exogenous expression (5,(29)(30)(31). Moreover, exogenous or endogenous expression of many ISGs can promote cell death (32). However, the highly specific antiviral activity of TRIM69 against VSIV suggests that the antiviral mechanism does not involve global processes such as cellular toxicity or the induction of a polygenic antiviral state. Interestingly, a VSV-G (glycoprotein) pseudotyped variant of HIV-1 that does not express an HIV-1 envelope glycoprotein and was decorated with a VSV-G envelope derived from VSIV was also insensitive to TRIM69-mediated inhibition (Fig. 1E). This suggests that inhibition of VSIV by TRIM69 occurs after viral entry and that the VSIV glycoprotein is not directly targeted by TRIM69. Importantly, unmodified wild-type replication-competent VSIV was also potently inhibited by TRIM69 (Fig. 1F).
Endogenous TRIM69 is IFN inducible and potently inhibits VSIV. We have previously used comparative transcriptomics to study the IFN response in a variety of species (1). Meta-analysis of these data indicated that TRIM69 expression was upregulated ϳ10-fold following IFN stimulation in primary human fibroblasts ( Fig. 2A). Similarly, TRIM69 has been previously identified as an ISG in multiple studies and is typically induced between ϳ2and ϳ10-fold by type I IFNs (2,33). To examine whether the endogenous protein exhibited antiviral activity, we knocked out TRIM69 using CRISPR/ Cas9 in diploid CADO-ES1 cells (34). We selected CADO-ES1 cells for these experiments as existing transcriptomics data suggested that TRIM69 was efficiently expressed in these cells (EMBL-EBI Expression Atlas [https://www.ebi.ac.uk/gxa/home]). TRIM69 knockout (KO) single-cell clones were derived, and the KO was confirmed by sequencing the genetic lesions. In accordance with the IFN-sensitive nature of vesiculoviruses, IFN treatment potently blocked VSIV infection in "no-guide" control clones (by Ͼ10,000fold) ( Fig. 2B and D). In striking contrast, when TRIM69 was knocked out, the protective effect of IFN was markedly reduced (by ϳ300-fold). This indicates that endogenous TRIM69 appears to play a major role in the anti-VSIV effects of IFN. Furthermore, the TRIM69 anti-VSIV activity was again specific as the magnitude of HIV-1 inhibition was largely similar in the presence or absence of TRIM69 ( Fig. 2C and E). Because CADO-ES1 cells do not support efficient HIV-1 infection, a pseudotyped HIV-1-based lentiviral vector (CSGW) was used for these experiments.
Not all TRIM69 isoforms confer antiviral activity. Because alternative splicing can produce divergent variants of antiviral factors, these spliced isoforms can exhibit differential antiviral activity. In the case of another TRIM protein, TRIM5, the spliced isoforms have been informative in understanding the mechanism of TRIM5's antiviral activity (35). We therefore considered whether all isoforms of TRIM69 conferred anti-VSIV activity. We cloned the five human TRIM69 isoforms listed on ENSEMBL, and we considered their activity in our doxycycline-inducible system. Using this strategy, we were able to detect expression of only isoforms A and B by Western blotting (Fig. 3A) even though the polyclonal antibody should recognize all isoforms. The only variant that conferred anti-VSIV activity was the longest isoform, isoform A ( Fig. 3B to D), which potently inhibited VSIV-GFP infection. Notably, isoform B was abundantly expressed (Fig. 3A), and we thus concluded that this isoform has no anti-VSIV activity. This suggests that the RING domain (or flanking sequence) is critically required for anti-VSIV activity or the correct folding or multimerization of TRIM69. Although we were unable to express or detect isoforms C, D, and E, because these isoforms also lack the N-terminal residues missing in isoform B, we find it unlikely that they possess anti-VSIV activity.
As well as lacking antiviral activity, shorter isoforms of the TRIM5 protein can form heteromultimers with TRIM5␣ and act as dominant negative inhibitors of endogenous and exogenous TRIM5␣-mediated restriction (35). We thus examined whether TRIM69 isoform B might similarly inhibit the anti-VSIV activity of isoform A. When TRIM69 (isoform A) was constitutively expressed, it conferred potent protection from VSIV-GFP infection (Fig. 3E), and this protection was further enhanced by the presence of inducible myc-tagged TRIM69 (Fig. 3E). In contrast, constitutively expressed TRIM69 isoform B had negligible effect on the ability of isoform A to inhibit VSIV (Fig. 3E), despite being abundantly expressed (Fig. 3F). Thus, although TRIM69 isoform B has no detectable antiviral activity, it does not appear to interfere with the ability of isoform A to block VSIV. It is therefore likely that heteromultimers between isoform A and isoform B either cannot form or are competent to restrict VSIV. Multiple TRIM69 alleles circulate in human populations, and TRIM69 exhibits strong signatures of positive selection. We next analyzed structural variation at the TRIM69 locus in human populations, using data from the 1000 Genomes Project collated by ENSEMBL (36). Interestingly, multiple TRIM69 alleles circulate at high frequencies in human populations. Surprisingly, the human TRIM69 variant NM_182985 (NCBI Reference Sequence) is only the third most common human TRIM69 allele, present at frequencies of ϳ4% (European) to ϳ17% (South Asian) in human populations. In light of this, we examined the ability of all the major TRIM69 alleles (with a frequency Ͼ5% in at least one population) to inhibit a small panel of vesiculoviruses (Fig. 4). We cloned the seven main alleles into our doxycycline-inducible expression system ( Fig. 4A to C) and challenged these cells with a small panel of vesiculoviruses ( Fig. 4D to G). In spite of the amino acid variation (Fig. 4A), all seven major alleles conferred potent protection from VSIV infection (Fig. 4E). Although some variation in the magnitude of protection was observed, we attributed this to slight variations in TRIM69 expression levels, as opposed to variation in the anti-VSIV activities of the different alleles (Fig. 4C). Upon further viral challenges, TRIM69 again exhibited exquisite antiviral specificity as none of the TRIM69 human alleles inhibited either VSV New Jersey (VSNJV) (Fig. 4F) or Chandipura virus (37) (CHNV-GFP) (Fig. 4G), both of which are closely related to VSIV (Fig. 4D).
Since multiple TRIM69 alleles currently circulate in human populations and because antiviral TRIM proteins can possess strong signatures of positive selection (38), we conducted positive-selection analysis of TRIM69 sequences from primates. We retrieved and aligned the TRIM69 coding region from 18 primate species representing Ͼ40 million years of divergent evolutionary pressures (39). Using the maximum likelihood approach in PAML (40), we tested whether models that permit positive selection on individual codons (ratio of nonsynonymous to synonymous changes [dN/dS] of Ͼ1) were a better fit to these data than models that do not allow positive selection. In each case, permitting sites to evolve under positive selection gave a better fit ( Fig. 5A and B), with a high proportion of codons exhibiting dN/dS values greater than 1 (32.8% with an average dN/dS of 1.9). These analyses identified six residues exhibiting relatively strong signatures of positive selection ( Fig. 5A and B). One of the sites identified using this approach (S404) is within the SPRY domain (the domain that forms the hostpathogen interface that defines the antiretroviral specificity of TRIM5 [38]). These analyses suggest that while the majority of sites (67.2%) in TRIM69 have evolved under purifying selection (in order to maintain the overall structure and function of TRIM69), positive selection has likely occurred at specific sites, perhaps influencing the antiviral activity of TRIM69 (38).
Based on the signatures of positive selection at the TRIM69 locus in primates, we hypothesized that divergent TRIM69 proteins might exhibit divergent antiviral specificities. We therefore cloned a variety of TRIM69 orthologs from a selection of species (including primates and a variety of other mammals) into our doxycycline-inducible expression system. Similar to human TRIM69, orthologs from rhesus macaques, rats, cows, alpacas, dogs, ferrets, and horses all potently inhibited VSIV infection (Fig. 5C). Although the magnitude of inhibition was variable, we attributed the majority of this variability to different TRIM69 expression levels (Fig. 5C). Strikingly, murine TRIM69 did not inhibit VSIV, despite being expressed at higher levels than multiple inhibitory orthologs (Fig. 5C). Furthermore, the anti-VSIV activity of TRIM69 appears to have been lost in the Mus genus, as TRIM69 orthologs from Mus caroli and Mus pahari were also noninhibitory, whereas rat TRIM69 potently inhibited VSIV (Fig. 5D). This is not simply due to the murine orthologs lacking specific cofactors/interactions within human cells, as stable expression of murine TRIM69 (Mus musculus) in mouse cells indicated that murine TRIM69 still possessed no apparent anti-VSIV activity (Fig. 5E). Crucially, murine TRIM69 was abundantly expressed in these cells, and human TRIM69 conferred potent protection from VSIV infection when expressed at similar levels in the identical murine background ( Fig. 5E and F). Thus, while murine cells support TRIM69-mediated anti-VSIV activity, all tested murine orthologs of TRIM69 have lost the ability to inhibit VSIV.
We also challenged the species variants of TRIM69 with related vesiculoviruses (VSNJV and CHNV), but none of the orthologs considered possessed substantial antiviral activity against these viruses ( Fig. 5G and H).
In light of the positive-selection analysis and species-dependent activity of TRIM69, we compared the murine TRIM69 amino acid sequence to sequences of the restrictive human and rat TRIM69 variants (Fig. 6). While human and mouse TRIM69 sequences were relatively well conserved (79% identity), the rat and mouse variants (which were the closest orthologs we tested that displayed differential activity against VSIV), appeared highly similar, differing at only 38 amino acid positions (92% identity). Although site-specific signatures of positive selection were detected in TRIM69, all of the signature sites were conserved between rats and mice (Fig. 6). Thus, the genetic basis of the differential anti-VSIV activity observed in rodent TRIM69 variants is currently unknown.
The VSIV phosphoprotein confers sensitivity/resistance to TRIM69. The short generation times inherent to the lifecycles of most viruses mean that resistance to inhibition can be rapidly selected in vitro (5,27,41,42). Approaches using in vitro evolution can rapidly identify antiviral sensitivity/resistance determinants in viruses targeted by antiviral factors (41,43). We used a diverse swarm of replication-competent, full-length (FL) VSIV-GFP (44) that had been propagated in mammalian cells. This parental virus stock was potently inhibited by TRIM69 following overnight infection (Fig. 7A). We used this virus to inoculate a culture expressing TRIM69, using a dose in which Ͻ1% of cells were GFP positive following overnight incubation. Four days later, VSIV had begun to overwhelm this culture, and the supernatant was filtered and transferred to a new culture (also induced to express TRIM69). In this second passage, VSIV replicated far more efficiently in the presence of TRIM69, suggesting that resistance had emerged. Such rapid acquisition of resistance is a documented property of VSIV populations, and VSIV fitness has been previously shown to increase Ͼ1,000-fold in a single passage (within a new cellular environment) (45). We titrated the filtered supernatant containing the TRIM69-passaged swarm, in the presence and absence of TRIM69, and observed that VSIV had been selected to resist TRIM69 inhibition (Fig. 7A). We sequenced the viral population and observed four substitutions that had been selected to near uniformity in the viral swarm ( Fig. 7B and C). These substitutions included two synonymous changes and two nonsynonymous substitutions. Because we had previously observed that the VSV-G protein was not targeted by TRIM69 when used to pseudotype HIV-1 (Fig. 1E), we reasoned that one substitution, E92K in VSIV-G, was unlikely to confer resistance to TRIM69. This left only one nonsynonymous substitution, D70Y, located in the VSIV phosphoprotein (P protein) that might confer TRIM69 resistance. We mutated this residue in isolation in the FL VSIV-GFP plasmid background and rescued the parental and D70Y mutant viruses. While the rescued parental virus was potently inhibited by TRIM69, the P protein D70Y mutant was completely insensitive to inhibition by TRIM69 (Fig. 7D). Thus, the VSIV P protein is the genetic target of TRIM69 and can determine sensitivity or resistance to TRIM69. Moreover, just a single amino acid within the P protein can determine TRIM69 sensitivity. We next considered the variation at position 70 in VSIV sequences deposited in GenBank. Interestingly, VSIV was uniformly aspartic acid at position 70 ( Fig. 7E), suggesting that the D70Y escape mutant either cannot thrive in vivo or that TRIM69 does not exert enough pressure to provide a selective advantage to this variant (within these sequences). Importantly,  antiviral factors that target highly conserved viral peptides are more likely to form an effective antiviral defense. TRIM69 inhibits VSIV but not DENV-2. During the preparation of the manuscript, it was reported that TRIM69 is an ISG that targets dengue virus type 2 (DENV-2) through an interaction with NS3 that targets NS3 for degradation (33). This TRIM69-mediated degradation of NS3 interrupts the life cycle of DENV-2 (33). This observation was of immediate interest to us as it is unusual for an antiviral factor to specifically target viruses as divergent as DENV-2 (a positive-sense ssRNA virus) and VSIV (a negativesense ssRNA virus). Moreover, murine TRIM69, which is inactive against VSIV, was reported to inhibit DENV-2 (33), suggesting that species variants of TRIM69 might have divergent antiviral specificities. We thus compared the ability of TRIM69 to inhibit

TRIM69 Inhibits VSIV
Journal of Virology DENV-2 and VSIV in the same experiment. Because Vero cells are susceptible and permissive to both DENV-2 and VSIV, we selected these cells as the background for our experiments and generated Vero cells that stably expressed human, rat, or mouse TRIM69 and infected these cells with a titrated challenge of DENV-2 or VSIV-GFP. We used the New Guinea C strain of DENV-2 as this strain was previously reported to be inhibited by TRIM69 (33). Unexpectedly, DENV-2 produced in either mammalian or insect cells was not inhibited by TRIM69 under these conditions (Fig. 8A to D). In contrast, VSIV was potently inhibited by human and rat, but not mouse, TRIM69 in parallel experiments (Fig. 8E and F). Because the previously published work had not specifically investigated exogenous TRIM69 expression in Vero cells, we examined whether TRIM69 expression in HEK 293T cells would induce the degradation of transfected NS3 but not NS3 K104R (in order to recapitulate the published observations) (33). Surprisingly, TRIM69 had no effect on NS3 expression levels (Fig. 8G). In contrast, VSIV was potently blocked in parallel experiments using the equivalent cells (Fig. 8H). We conclude that DENV-2 is not always inhibited by TRIM69, even under conditions where TRIM69 exhibits substantial antiviral activity against VSIV.
The anti-VSIV activity of TRIM69 is not dependent upon IFN signaling or E3 Ub ligase activity. TRIM69 has a RING domain that is predicted to have E3 ubiquitin (Ub) ligase activity. Moreover, proteasomal degradation has previously been reported to be involved in the TRIM69-mediated inhibition of DENV-2 (33). In addition, TRIM69 isoform B (which lacks a RING domain) possesses no anti-VSIV activity (Fig. 3C). We therefore considered whether inhibiting proteasomal degradation might affect TRIM69 antiviral activity. As a less toxic alternative to the proteasome inhibitor MG132, we used bortezomib (Bort), an inhibitor of the 26S proteasome, which is also licensed for clinical use (46,47). In order to validate the efficacy of inhibition, we transduced cells with a lentiviral vector encoding ubiquitin fused to GFP. This fusion protein is rapidly degraded, and GFP expression is not visible under normal culture conditions (Fig. 9A and  B). However, when the 26S proteasome was inhibited by bortezomib treatment, abundant GFP expression was visible, suggesting that proteasomal inhibition was efficient in our culture system. In parallel experiments, we considered the ability of proteasome inhibition to influence TRIM69-mediated restriction of VSIV. When normal- ized to infection in the absence of TRIM69, proteasomal inhibition appeared to partially rescue the restriction of VSIV (Fig. 9C). However, bortezomib treatment caused noticeable toxicity in these experiments, consistent with bortezomib's proapoptotic and anti-tumor cell growth properties (47). Visual inspection of the titration curves indicated that the majority of the rescue was due to reduced VSIV infection in control cells (in the presence of bortezomib), as opposed to proteasomal inhibition actually enhancing infection in the presence of TRIM69 (Fig. 9D). Importantly, TRIM69 potently restricted VSIV (Ͼ50-fold) in the presence of efficient proteasome inhibition, indicating that proteasomal degradation is not necessary for effective TRIM69-mediated restriction.
Many TRIM proteins are involved in antiviral signaling and appear to have antiviral activity because their exogenous expression promotes or potentiates a polygenic antiviral state (30,31). Although the observation that most viruses escape inhibition by TRIM69 suggests that a polygenic response is not involved, we investigated whether TRIM69 could inhibit VSIV in the absence of JAK-STAT signaling. We therefore used the Janus kinase (JAK) inhibitor ruxolitinib to block interferon signaling in TRIM69expressing cells (48). We then examined the ability of TRIM69 to block VSIV infection in the absence of IFN signaling. As with bortezomib, some toxicity was observed, and ruxolitinib slightly reduced the level of infection in the absence of TRIM69 ( Fig. 9E and F). Thus, the majority of the apparent ϳ7-fold rescue (Fig. 9F) was caused by reduced infection in the absence of TRIM69 (as opposed to ruxolitinib solely enhancing infection in the presence of TRIM69). However, in contrast to bortezomib, ruxolitinib did modestly increase the amount of VSIV infection in the presence of TRIM69 (increasing the titer ϳ2.5-fold) (Fig. 9E). Importantly, the ruxolitinib treatment effectively blocked IFN signaling, and no phosphorylated STAT1 was detectable following IFN treatment in the presence of ruxolitinib (Fig. 9G). Notably, TRIM69 expression alone was insufficient to trigger the phosphorylation of STAT1 (Fig. 9G), and TRIM69 potently blocked VSIV (Ͼ20-fold) in the presence of ruxolitinib. Thus, IFN signaling is not necessary for TRIM69 to inhibit VSIV (although JAK-STAT signaling might modestly enhance this inhibition).

DISCUSSION
Although vesiculoviruses can be particularly sensitive to IFN-induced inhibition, the specific ISGs that mediate the anti-VSIV activity of IFNs (and how this inhibition is achieved) have not yet been fully defined. Here, we show that one ISG, TRIM69, can potently and specifically inhibit VSIV. Moreover, the high degree of specificity of this inhibition overwhelmingly suggests that this inhibition is direct. TRIM69 potently blocked VSIV, whereas other vesiculoviruses (and more divergent RNA viruses) were entirely resistant. Furthermore, the inhibition of VSIV appeared to be remarkably specific as a single substitution conferred complete resistance to TRIM69. Thus, although we cannot exclude the possibility that TRIM69 expression elicits some kind of antiviral signaling, such a signaling event would have to be independent of proteasomal degradation and JAK-STAT signaling and also result in an outcome that specifically targeted VSIV. Therefore, the more parsimonious explanation is that TRIM69, or a complex containing TRIM69, directly targets VSIV (and interferes with the virus life cycle). This potentially adds TRIM69 to an expanding list of directly antiviral TRIM proteins that includes the classical restriction factors TRIM5 (35) and PML (49). Nonetheless, inhibition of IFN signaling modestly rescued VSIV infection, and TRIM69 KO slightly reduced the IFN-induced inhibition of HIV-1. Thus, it is possible that TRIM69 might also play a signaling role analogous to the signaling capacity of established directly antiviral factors such as TRIM5, TRIM21, and tetherin (50)(51)(52).
Perhaps the most surprising aspect of the highly specific anti-VSIV activity of TRIM69 was that DENV-2, previously reported to be inhibited by TRIM69 (33), was not inhibited in our experiments. Importantly, this lack of TRIM69 sensitivity was observed while parallel cultures (of the equivalent cells) fiercely resisted VSIV infection. There are many possible explanations for these apparently contradictory observations. The most likely explanation is that due to the highly specific nature of TRIM69-mediated inhibition, some aspect of the virus strain, cellular background, or method used led to DENV-2 appearing insensitive to inhibition (in our experiments). We were careful to use the same strain as used previously (specifically, the New Guinea C strain, which we obtained from Public Health England). We confirmed the presence of the TRIM69-sensitive lysine (at position 104 of NS3) in our virus stocks, and more work will be required to understand why we did not observe DENV-2 inhibition in the face of antiviral TRIM69. Importantly, with both VSIV and DENV-2, a single amino acid substitution conferred resistance to TRIM69. Moreover, we considered only a single human allele (allele 3), which differs at two residues from the variant used previously (GenBank accession no. AY305385.1) (33,53). Thus, seemingly minor differences in viral strains could easily reconcile these apparently contradictory observations.
Although we do not know how TRIM69 impedes VSIV, the block must occur relatively early in the viral life cycle. TRIM69 is able to block VSIV prior to the expression of GFP, which in the case of the rVSVΔG-GFP system is encoded in place of the VSV-G protein. Thus, the TRIM69-mediated block must occur at a step prior to translation of VSV-G subgenomic RNA (sgRNA). In the absence of direct evidence, the D70Y mutation in the VSIV phosphoprotein does provide some potential mechanistic clues. Importantly, D70 is present in a region of VSV-P that is heavily phosphorylated (phosphorylation of residues 60, 62, and 64 has been described previously) (54). Phosphorylation of these sites has been proposed to be important in P protein dimerization and interaction with the L protein (large polymerase protein) (55)(56)(57)(58)(59), and phosphorylation of this region is essential for functional RNA-dependent RNA polymerase activity (60). Because tyrosine residues are known to be phosphorylated in the VSV-P protein (61), it is therefore tempting to speculate that a tyrosine at position 70 could be phosphorylated and that this phosphorylation somehow overcomes the block mediated by TRIM69. A speculative mechanism that involves TRIM69-mediated inhibition of VSIV RNA-dependent RNA polymerase activity (by inhibiting the association of P and L proteins), preventing subgenomic RNA transcription, would be entirely consistent with the early block described herein.
Although TRIM69 isoform B (which lacks the RING domain) does not possess anti-VSIV activity, proteasomal inhibition was unable to rescue VSIV infectivity. Thus, it is unclear whether functional E3 ubiquitin ligase activity is required to inhibit VSIV. Importantly, isoform B is ϳ30% shorter than isoform A, and it is possible that such a large deletion perturbs the tertiary or quaternary structure of TRIM69. Such an inability to form multimers could simultaneously explain why isoform B is both inactive and lacks dominant negative activity. More work will be required to understand the role that E3 ubiquitin ligase activity plays in TRIM69's immune role.
The N-terminal regions of vesiculoviral P proteins are relatively divergent, providing a possible explanation for the observed insensitivity of VSNJV and CHNV to TRIM69. Thus, examining the more closely related Morreton virus (MORV), vesicular stomatitis Alagoas virus (VSAV), Cocal virus (COCV), or Maraba virus (MARV) might identify more viruses which are sensitive to TRIM69 and potentially identify further evidence of species specificity. Notably, after analyzing 50 VSIV sequences (deposited in GenBank), we did not observe a tyrosine at position 70 of VSIV-P in any VSIV sequences. This suggests that if TRIM69 substantially inhibits VSIV in vivo, the D70Y mutation must be deleterious for some other reason and is therefore negatively selected (despite conferring resistance to TRIM69). Potential explanations include a reduction in replicative fitness or sensitization to other immune processes. Although we have not directly compared the replicative fitness of the D70Y mutant to that of the parental virus, virus preps of this mutant have slightly lower titers (less than a 5-fold reduction [our unpublished observations]), providing a possible explanation as to why this mutant was not observed in reported VSIV sequences.
Our failure to identify any viruses other than VSIV that were inhibited by TRIM69 limited our ability to identify whether species variants of TRIM69 possess divergent antiviral specificities. Despite this, differential activity was observed as anti-VSIV activity has apparently been lost in the Mus genus. Whether murine TRIM69s have been selected to target other viruses or whether murine orthologs have no antiviral activity at all remains to be determined.
The lack of anti-VSIV activity conferred by murine TRIM69 could have important implications for our understanding of how the IFN response constrains VSIV. Many key VSIV experiments have been conducted in mice (15,16), and it will be important to establish in the future whether nonmurine variants of TRIM69 possess anti-VSIV activity in vivo, potentially limiting the ability of VSIV to invade and colonize certain tissues. It is possible that the potent inhibition of VSIV observed in vitro will be recapitulated in vivo. In this way, IFN-induced TRIM69 might inhibit natural VSIV infections and similarly influence the safety and/or efficacy of therapeutic interventions (based upon VSIV). Thus, an improved understanding of how TRIM69 inhibits VSIV may help us to better understand VSIV pathogenesis and eventually lead to tangible benefits in the clinical use of VSIV derivatives.
The VSIV-GFP virus (rVSVΔG-GFP) competent to undergo a single round of infection but not encoding the VSV-G envelope (rVSV-ΔG-GFP decorated with VSV-G expressed in trans) system was used (28). Virus stocks were generated as described previously (42). Briefly, HEK 293T cells were transfected with a VSV-G expression plasmid. The next day, the cells were infected with rVSV-ΔG-GFP at a multiplicity of infection (MOI) of 1. Progeny virus-like particle (VLP) stocks were harvested at 24 h postinfection and clarified using a 0.45-m-pore-size filter. The replication-competent FL-VSIV-GFP, VSIV, and VSNJV viruses were a generous gift from Megan Stanifer (Heidelberg University). Virus stocks were generated through infection of BHK-21 cells using a low MOI. Once cytopathic effect (CPE) was readily apparent, supernatants were harvested and clarified using a 0.45-m-pore-size filter.
The D70Y mutant (VSV-P) was made using an Agilent QuikChange Lightning site-directed mutagenesis kit, in accordance with the manufacturer's instructions, using the parental FL-rVSIV-GFP plasmid as the template [pVSV1(ϩ)-GFP] (44) and the following oligonucleotides, 5=-GCT TCC GGA TCT GGT ACA  TAC AAG CCT TGA TTG TAT TCA ATT TCT GGT TCA GAT TCT GT-3= and 5=-TGA TTC TGA CAC AGA ATC  TGA ACC AGA AAT TGA ATA CAA TCA AGG CTT GTA TGT ACC AG-3=. The entire coding region was subsequently sequence verified to confirm the presence of the sole D70Y (VSV-P) mutation. To rescue the parental and mutant virus, BSR-T7 cells were seeded and infected with fowlpox T7 virus (equivalent to an MOI of ϳ2 determined using DF-1 cells) (63). After 1 h of incubation, 1.66 g of the FL-VSIV-GFP rescue plasmid [pVSV1(ϩ)-GFP] or mutant D70Y [pVSV1(ϩ)-GFP P-D70Y] was cotransfected with pBS-N (0.83 g), pBS-P (0.5 g), and pBS-L (0.33 g) (KeraFAST) using FuGENE6 (Promega). After 48 h, GFP-positive cells and CPE were observed in the transfected BSR-T7 cells. Supernatant containing VSIV was harvested and clarified using a 0.45-m-pore-size filter and stored at Ϫ80°C. VSIV was propagated in BHK-21 cells in T-25 flasks. Cells were infected at a low MOI of VSV harvested from BSR-T7 cells. At 48 h postinfection (or when the majority of cells were GFP positive), supernatant was harvested and clarified using a 0.45-m-pore-size filter.
DENV-2 was obtained from Public Health England (catalogue number 0006041v). The NS3 of the DENV-2 stocks (propagated in Vero cells) used herein was directly sequenced. Briefly, RNA was extracted from infected cells using the same approaches described below for passaged FL-VSIV-GFP, and the NS3 region was amplified using the primers 5=-CGA AGA GGA AGA ACA AAT ACT GAC C-3= and 5=-GAT TGT ACG CCC TTC CAC CTG CTT C-3=; PCR products were sequenced using these primers and an additional primer (5=-GTG GAG CAT ATG TGA GTG CTA TAG C-3=). The NS3 sequence differed by one amino acid, a threonine at position 442, from GenBank accession number AAC59275 (strain New Guinea C).
The replication-competent HIV-1 proviral clone NHG (GenBank accession no. JQ585717) and Δenv derivatives have been described previously (5,64). Virus stocks were generated through transient transfection of HEK 293T cells in isolation (NHG) or in conjunction with a VSV-G expression plasmid (NHG Δenv). The single-cycle HIV-1 lentiviral vector CSGW is a self-inactivating, VSV-G-pseudotyped lentiviral vector that encodes GFP expressed from an internal spleen focus-forming virus (SFFV) long terminal repeat (LTR) (65), packaged using GagPol derived from HIV-1 NL4-3 (pNLGP). The single-round RVFV system has also been described previously (66). Briefly, BHK-Rep cells were transiently transfected with pCAGGs-M. The cells were washed the following day, and supernatant was harvested at 48 h posttransfection and clarified using a 0.45-m-pore-size filter.
An NS1-enhanced green fluorescent protein (EGFP) construct expressing influenza A/Puerto Rico/8/ 1934 (H1N1) (PR8) virus was designed based on the previously described Color-Flu system (67). A DNA sequence was synthesized (Genewiz) with flanking BsmBI sites corresponding to the NS segment of PR8 (GenBank accession no. EF467817.1) in which the NS1 open reading frame (ORF) had been altered to code for a C-terminally EGFP-tagged NS1 protein with a CSGG linker. This was immediately followed in frame by a CSG linker, the 2A protease of porcine teschovirus (PTV), and the nuclear export protein (NEP) ORF. A splice acceptor site in the NS1 ORF was removed by introducing A527C and A530G (numbering is in accordance with GenBank accession no. EF467817.1). The sequence was subcloned into the pHW2000 reverse genetics plasmid. The PR8-NS1-EGFP virus was rescued using a well-established reverse genetics system previously described (68) TCC AGG CGA CTA TGT TGA AAT GAA TG-3=, and 3=-TCT CTC GGC CAG AGA GGC CTT ACT  GTG GAT GTA AGA TGT GCA ATG G-5=; allele 7, 5=-CTC TCT GGC CGA GAG GGC CAT GGA GGT ATC CAC  CAA CCC CTC CTC-3=, 3=-GAG ACA TTG CTC CTG AAG CTG GCT CAG TTT CAA CTC CAT CTC ATT CAA GGC  TTT C-5=, 5=-GCC TTG AAT GAG ATG GAG TTG AAA CTG AGC CAG CTT CAG GAG CAA TGT CTC TTA GC-3=, and 3=-TCT CTC GGC CAG AGA GGC CTT ACT GTG GAT GTA AGA TGT GCA ATG G-5=. Allele 4 was used as a template for allele 5 PCRs, and allele 5 was used as a template for allele 7. The N-terminal fusion of a mutated uncleavable ubiquitin moiety fused to GFP (Ub-G76V GFP) has been described previously (69). Plasmid DNA containing Ub-G76V GFP[pcDNA3.1(ϩ).Ub-G76V GFP], a gift from A. Fletcher, was digested with BamHI and NotI and inserted into the similarly digested lentiviral vector pCSGWΔNotI (65) (a gift from G. Towers and A. Thrasher).
Gene editing was achieved using a lentiGuide-Puro system (70) in accordance with the protocols of the Zhang lab. The following oligonucleotides were used to make TRIM69 guides: 5=-CAC CGC AAC CCT GTA CTG GAC AAG T-3= and 5=-AAA CAC TTG TCC AGT ACA GGG TTG C-3= (guide 1); 5=-CAC CGA AGA AGT TAC CCT TAC TCA A-3= and 5=-AAA CTT GAG TAA GGG TAA CTT C-3= (guide 2). Diploid CADO-ES1 cells were either transduced with vectors encoding Cas9 and the relevant TRIM69-targeting sgRNAs or transduced in parallel with vectors encoding Cas9 and no sgRNA (no guide). Single-cell clones were generated using limiting dilution, and the KO was confirmed by extracting genomic DNA (DNeasy; Qiagen), followed by PCR amplification of the guide target regions in exon 2 using the following oligonucleotides 5=-CAC TTT CAA AGG AGA GAT TAT GTG C-3= and 5=-GAG CAG TCT GGG CTT TCT AAT CAT C-3=. The PCR products were cloned into pGEM-T-Easy (Promega), and multiple clones were Sanger sequenced.
Viral vectors were produced using transient transfection of HEK 293T cells (5 g of vector/genome plasmid, 5 g of the relevant GagPol expression vector, and 1 g of a VSV-G expression plasmid). Vector-containing supernatants were filtered (using a 0.45-m-pore-size filter) and used to transduce the relevant cell types.
Virus infections and titrations. For assays using GFP-encoding viruses, cells were seeded in 96-well plates. Adherent and semiadherent cells were seeded 24 h before challenge or treatment whereas suspension cells were seeded immediately prior to infection or treatment. In experiments using doxycycline-inducible TRIM69 expression, cells were treated with 125 ng/ml of doxycycline hyclate (Sigma) 24 h before infection. Where stated in the figures, figure legends, or text, cells were treated with 2 M ruxolitinib (INCB018424) (catalog no. S1378-SEL; Stratech), 50 nM bortezomib (2204; Cell Signaling Technology), or 1,000 units of IFN-␤ (11420-1; PBL Assay Science) immediately before infection (or in the case of IFN-␤, 24 h before infection, except for the experiments in Fig. 8, where a 4-h treatment was used). Cells were then infected with titrated challenges of the indicated virus and incubated overnight (ϳ16 h) or for 48 h (HIV-1 and RVFV) prior to fixation using 4% formaldehyde and enumeration of infected GFP-positive cells using flow cytometry.
For 50% tissue culture infective dose (TCID 50 ) assays, MT4 cells were seeded in 96-well plates and treated with 125 ng/ml of doxycycline hyclate (Sigma) 24 h prior to infection. The cells were infected with eight replicates of 3-fold serially diluted full-length VSNJV or VSIV. At 72 h postinfection (hpi), CPE was analyzed, and the TCID 50 was calculated using the Spearman and Kärber algorithm and a modified TCID 50 calculator from Marco Binder.
VSIV in vitro evolution, RNA extraction, PCR, and sequencing. MT4 cells expressing TRIM69 were seeded and induced with doxycycline hyclate (200 ng/ml) 24 h prior to challenge with FL-VSIV-GFP at a low MOI (Ͻ1% infection following 16 h of incubation). The level of infection based on the percentage of GFP-positive cells was monitored daily. Once the culture was overwhelmed, supernatant was filtered (0.45-m pore size) and used to infect a second culture. Following passage 2, CPE was observed at 24 h postinfection (in the presence of TRIM69). The supernatant of the passaged virus was filtered (0.45-m pore size) and stored. Cell pellets were resuspended in TRIzol (Invitrogen), and viral RNA was isolated from infected cells using a hybrid TRIzol and RNeasy extraction (Qiagen) protocol. Viral cDNA was reverse transcribed (SuperScript III) using random hexamer primers. All VSIV coding regions were PCR amplified, and the PCR products were directly sequenced using Sanger sequencing (Eurofins Genomics).
Sequence collection, alignment, and expression analysis. TRIM69 sequences were obtained from publicly available databases such as ENSEMBL and GenBank using TBLASTN (72). The protein sequences were aligned using MAFFT (73), and a codon alignment was generated based on the protein alignment using PAL2NAL (74). The alignment was screened for recombination in HyPhy (75) using singlebreakpoint recombination (SBP) and GARD, a genetic algorithm for recombination detection (76). TRIM69 expression in primary human fibroblasts was meta-analyzed from published data (1), and counts per million (CPM) of TRIM69 expression were extracted as described previously (1). TRIM69 expression levels in human cells were assessed by meta-analyzing existing gene expression data through the EMBL-EBI Expression atlas (https://www.ebi.ac.uk/gxa/home), leading to the selection of CADO-ES1 cells for TRIM69 knockout.
To explore the amino acid variation surrounding amino acid D70, 50 sequences from NCBI were aligned, and identical sequences were removed from the alignment to produce a nonredundant set of protein sequences. A WebLogo sequence logo (77) for the protein region was produced using a web server (https://weblogo.berkeley.edu/). Phylogenetics and positive selection analyses. The best substitution model was selected using Bayesian information criteria (BIC) in jModeltest (78), and the maximum likelihood phylogeny was reconstructed with this model in PhyML with 1,000 bootstrap replicates (79). The primate lineage of the gene tree, which is more extensively sampled for species, was used in CODEML (40) to detect sites under positive selection.