Proteasomal Degradation of TRIM5α during Retrovirus Restriction

The host protein TRIM5α inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5α. Here, we show that TRIM5α is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5α-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5α protein but not the nonrestrictive human TRIM5α protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5α was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV) but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5α and TRIMCyp proteins. We also detected degradation of endogenous TRIM5α in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5α degradation by a proteasome-dependent mechanism.


Introduction
Retroviruses exhibit a restricted host range due to the requirement for specific interactions between viral and host proteins to complete the viral life cycle. Also limiting retroviral tropism are several recently identified intracellular antiviral factors ( [1][2][3][4][5]); reviewed in [6][7][8][9][10]). The prototypical restriction activity, Fv1, was first detected in the 1970s as differential susceptibility of inbred mice strains to the Friend leukemia virus [11][12][13]. Fv1 blocks infection of murine leukemia viruses (MLV) at a stage following fusion but prior to integration [14,15]. The block to infection can be overcome at high multiplicities of infection (m.o.i.) or by pretreatment of target cells with non-infectious virus like particles (VLPs) [11,16]. Susceptibility to Fv1 restriction is determined by the sequence of the viral capsid protein (CA) [17][18][19]. The gene encoding Fv1 was identified in 1996 by positional cloning [1]; yet the molecular mechanism by which Fv1 inhibits MLV infection remains poorly defined.
Shortly after the identification of TRIM5a, a second HIV-1 restriction factor was identified in owl monkeys [4,5]. This protein, TRIMCyp, is the apparent result of a LINE1-mediated retrotransposition event in which the cyclophilin A (CypA) mRNA was inserted into the TRIM5 locus resulting in a functional fusion protein [4]. TRIMCyp potently inhibits HIV-1 infection by interacting with an exposed loop on the surface of the CA via the CypA domain. The discovery of TRIMCyp provided a simple explanation for the ability of cyclosporin A (CsA), which inhibits CypA binding to CA, to render owl monkey cells permissive to HIV-1 infection [38]. Mutations in the CypA binding loop that result in a failure to bind CypA also result in a loss of restriction by TRIMCyp [4,5]. More recently, novel TRIM5-CypA proteins have also been identified in other primate species [39][40][41][42].
TRIM5a and TRIMCyp are members of the tripartite motif family of proteins, which encode RING, B-Box, and coiled-coil (RBCC) domains [43]. TRIM5a is the longest of the three isoforms (a, c, and d) generated from the TRIM5 locus by alternative splicing of the primary transcript. While all three TRIM5 isoforms contain identical RBCC domains, the atranscript also encodes the B30.2/SPRY domain required for recognition of the incoming viral capsid and restriction specificity [29,30,33,34,36,[44][45][46]. The coiled-coil domain promotes the multimerization of TRIM5a molecules that is required for efficient restriction [44,47,48]. While the precise function of the B-Box domain is unclear, deletion of this region results in total loss of restriction potential thus indicating its importance [44,49]. The RING domain of TRIM5a is also required for full restriction activity, as mutants that lack this domain or in which proper folding is impaired are severely impaired for restriction and have altered cellular localization [3,44,49]. Substitution of RING domains from other human TRIM proteins results in changes in both the timing of restriction (i.e. pre-vs. post-reverse transcription) and the intracellular localization of the restriction factor [37,[50][51][52].
RING domains are commonly associated with ubiquitin ligase (E3) activity facilitating specific transfer of ubiquitin from various ubiquitin-conjugating (E2) proteins to substrates (reviewed in [53,54]). Polyubiquitylation of proteins commonly targets them for intracellular degradation by proteasomes. TRIM5a can be ubiquitylated in cells [55], but a role for this modification in TRIM5a stability or restriction has not been established. The d isoform of TRIM5, which encodes an identical RING domain to TRIM5a, exhibits E3 activity in vitro and mutation of the RING domain abolishes this activity [56]. The presence of a RING domain on TRIM5a suggested that the restriction factor might function by transferring ubiquitin to a core-associated viral protein, thus targeting it for proteasomal degradation. However, such a modification has not been detected, and the magnitude of restriction imposed by TRIM5a was not altered in cells in which the ubiquitination pathway was disrupted [57]. Nonetheless, recent studies have shown that proteasome inhibitors relieve the TRIM5a-dependent inhibition of reverse transcription, yet a block to HIV-1 nuclear entry remains [58,59].
Based on these findings implicating the proteasome in TRIM5a-dependent retroviral restriction, we hypothesized that restriction by TRIM5a leads to proteasomal degradation of a TRIM5a-viral protein complex. Here we show that inoculation of TRIM5a-expressing cells with a restricted retrovirus results in accelerated degradation of TRIM5a itself. Destabilization of TRIM5a was tightly correlated with the ability of the restriction factor to block infection by the incoming virus. Proteasome inhibitors prevented HIV-1-induced degradation of TRIM5a rh when added to cells prior to virus inoculation. These data suggest a functional link between proteasomal degradation of TRIM5a and the ability of TRIM5a to restrict an incoming retrovirus.

Exposure of Cells to HIV-1 Destabilizes TRIM5a
We hypothesized that TRIM5a itself might be degraded as a consequence of the post-entry restriction process. To test this, TRIM5a rh -expressing 293T cells were cultured in the presence of cycloheximide to arrest protein synthesis and then challenged with VSV-G-pseudotyped HIV-1 particles. At various times postinfection, cells were harvested for analysis of TRIM5a levels by quantitative immunoblotting. In control cells not exposed to virus, the TRIM5a level declined at a slow rate, eventually leveling off to 55% of the original level after 4 hours ( Figure 1A). By contrast, inoculation with HIV-1 induced a more rapid decrease in the TRIM5a level resulting in 85% loss after 4 hours. Analysis of data from 4 experiments indicated that the decay of TRIM5a was significantly faster in the HIV-1-inoculated cultures relative to the control ( Figure 1B). The stability of TRIM5a in our cells differs in terms of time as compared to previously published reports using Hela cells [55]. In additional studies we observed a similar destabilizing effect of HIV-1 exposure on TRIM5a rh in HeLa cells (data not shown).
Exposure of target cells to saturating levels of virus or VLPs can overcome restriction by TRIM5a. To determine whether the decay of TRIM5a rh was related to saturation of restriction, we inoculated TRIM5a rh -expressing cells with various doses of a GFP-encoding virus in the presence of cycloheximide for a fixed period of time and harvested the cells to quantify TRIM5a levels. To probe the relationship between saturation of restriction and TRIM5a degradation, a portion of the harvested cells were replated and cultured for 48 hours, and the extent of infection determined by flow cytometric analysis of GFP expression. The results showed that the ability to detect degradation of TRIM5a rh was strongly dependent on the dose of virus used ( Figure 1C). Furthermore, the TRIM5a level following inoculation was inversely related to the overall extent of infection ( Figure 1D). These results indicate that HIV-1-induced degradation of TRIM5a is correlated with saturation of restriction, likely due to a requirement to engage most of the restriction factor to detect the loss of the protein.

Human TRIM5a Stability is Not Affected by HIV-1
Human TRIM5a does not efficiently restrict HIV-1 infection. To further probe the link between restriction and TRIM5a destabilization, we analyzed the stability of the human TRIM5a protein following challenge of cells with HIV-1. As previously shown in Figure 1, HIV-1 challenge of TRIM5a rh -expressing 293T cells resulted in a more rapid loss of the protein vs. mockinfected cells (Figure 2A and B). TRIM5a hu was intrinsically less stable than TRIM5a rh , as indicated by its more rapid decay in the mock-infected cultures ( Figure 2B and C). However, inoculation with HIV-1 did not result in further destabilization of TRIM5a hu , indicating that the HIV-1-induced degradation of TRIM5a rh is not a nonspecific cellular response to the viral challenge. These results suggest that the loss of TRIM5a rh depends on its ability to recognize the HIV-1 core.

Author Summary
Recent studies have identified several cellular proteins that restrict infection by a variety of retroviruses. One of these restriction factors, TRIM5a, is partially responsible for the differences in susceptibility of monkeys and humans to SIV and HIV-1, respectively. TRIM5a inhibits retrovirus infection soon after penetration into the target cell by associating with the viral protein CA, which forms the polymeric capsid shell of the viral core. Although the detailed mechanism of restriction is unknown, TRIM5a is postulated to alter the stability of the viral core, resulting in a failure to complete reverse transcription. The activity of cellular proteasomes, which are responsible for intracellular protein degradation, has also been implicated in TRIM5adependent attenuation of retroviral reverse transcription. In this study, we show that cellular TRIM5a is rapidly degraded in cells exposed to a restriction-sensitive retrovirus but not in cells infected with an unrestricted virus. Virus-induced degradation of TRIM5a was dependent on cellular proteasome activity, as inhibition with drugs blocking proteasome function also inhibited degradation of TRIM5a. These results provide additional support for a role of proteasomal degradation in TRIM5a-dependent retrovirus restriction and suggest a novel mechanism by which binding of TRIM5a to the viral capsid prevents infection.

Exposure to Restriction-Sensitive HIV-1 Destabilizes TRIMCyp
The owl monkey restriction factor TRIMCyp restricts HIV-1 by binding to an exposed loop on the surface of CA. Restriction can be prevented by addition of CsA or amino acid substitutions in CA that reduce CypA binding. We therefore asked whether TRIMCyp would also be destabilized following encounter of HIV-1. 293T cells expressing TRIMCyp were treated with cycloheximide and then challenged with VSV-G pseudotyped HIV-1 particles. As a control, parallel cultures were inoculated in the presence of a CsA concentration known to abolish TRIMCyp restriction of HIV-1. In the control mock-inoculated cells, TRIMCyp was stable in the cells during the six-hour time course ( Figure 3A). Challenge with HIV-1 resulted in accelerated loss of TRIMCyp. In the cultures containing CsA, the HIV-1-induced loss of TRIMCyp was markedly reduced ( Figure 3B).  Next we asked whether the HIV-1-induced degradation of TRIMCyp is correlated with the specificity of restriction. HIV-1 containing the G89V mutation in the CypA binding loop of CA is incapable of binding CypA and is also not restricted by TRIMCyp. However, this viral mutant is susceptible to TRIM5a rh restriction. Parallel cultures of 293T cells expressing either TRIMCyp or TRIM5a rh were treated with cycloheximide and then challenged with equivalent quantities of VSV-G pseudotyped HIV-GFP particles or the G89V CA mutant virus. As seen in Figure 3C, exposure to wild type HIV-1 induced accelerated loss of both TRIMCyp and TRIM5a rh . By contrast, exposure to the G89V mutant particles resulted in loss of TRIM5a rh but not TRIMCyp. These results indicate that exposure of cells to HIV-1 results in destabilization of TRIMCyp by a mechanism requiring recognition of the incoming HIV-1 core by the restriction factor.

Human TRIM5a is Destabilized Upon Encounter of Ntropic MLV
TRIM5a hu cannot restrict HIV-1 or B-tropic MLV but potently restricts N-MLV. To further test the link between TRIM5a destabilization and retrovirus restriction, we challenged 293T cells stably expressing TRIM5a hu with N-and B-tropic MLV viruses and measured TRIM5a levels following infection. The GFPtransducing N-and B-tropic MLV stocks were first titrated on nonrestrictive CrFK cells ( Figure S2, detailed in Text S1) then normalized to ensure equivalent dosing. Mock-treated cells lost TRIM5a hu at a slow rate (t 1/2 ,2.5 h; Figure 4A). Challenge with B-MLV did not significantly affect the rate of TRIM5a hu decay ( Figure 4A). By contrast, cells challenged with an equivalent quantity of N-MLV showed accelerated loss of TRIM5a hu (t 1/2 ,1 h) ( Figure 4A and 4B). The relative band intensities of the TRIM5a levels for this experiment were calculated and are represented in the graph in Figure 4B. These results, together with the TRIM5a and TRIMCyp data, establish a strong correlation between virus-induced TRIM5a destabilization and the specificity of restriction.

Virus-induced TRIM5a Destabilization is Correlated with Lentiviral Restriction in Old and New World Monkeys
TRIM5a proteins from different primates differ in their ability to restrict specific lentiviruses. For example, tamarin monkey TRIM5a (TRIM5a tam ) restricts SIV mac but not HIV-1, while spider monkey TRIM5a (TRIM5a sp ) restricts both viruses. To further test the correlation between virus-induced loss of TRIM5a and antiviral specificity, we stably expressed the TRIM5a tam and TRIM5a sp proteins in 293T cells and challenged them with equivalent titers of VSV-pseudotyped HIV-1 and SIV mac239 GFP reporter viruses (as determined by titration on permissive CrFK cells). The cell lines were found to restrict the respective viruses by at least ten-fold (data not shown). Immunoblot analysis of post-nuclear lysates revealed that TRIM5a rh was specifically destabilized when challenged with HIV-1 but not upon SIV mac challenge ( Figure 5A). By contrast, the SIV-restrictive TRIM5a tam was destabilized only in response to SIV mac challenge ( Figure 5A). TRIM5a sp , which restricts both viruses, was degraded in response to challenge with either virus ( Figure 5A and B). These results further strengthen the correlation between the specificity of retrovirus restriction and virus-induced destabilization of TRIM5a.

HIV-1-Induced Destabilization of TRIM5a Requires Proteasome Activity
A major mechanism for cellular protein degradation is via the 26S proteasome. Previous studies have shown that the turnover of TRIM5a is dependent on cellular proteasome activity. Furthermore, inhibition of proteasome activity overcomes the early block to reverse transcription imposed by TRIM5a. We asked whether HIV-1-induced destabilization of TRIM5a rh is dependent on proteasome activity. As previously reported [55], treatment of cells with the proteasome inhibitor MG132 resulted in an accumulation of TRIM5a protein (Figure 1, 0 H.p.i.). MG132 also prevented the HIV-1-induced destabilization of TRIM5a rh ( Figure 6A and B). Additional studies revealed that epoxomicin, a more specific proteasome inhibitor, also blocked the HIV-1-induced degradation of TRIM5a rh (data not shown). By contrast, infection by HIV-1 in the presence of the S-cathepsin inhibitor E64 did not prevent HIV-1induced TRIM5a rh degradation (data not shown), suggesting that endosomal proteases are not responsible for TRIM5a rh destabilization. We conclude that the virus-induced degradation of TRIM5a is dependent on cellular proteasome activity.
To determine whether HIV-1-induced destabilization of TRIMCyp depends on proteasome activity, we challenged TRIMCyp-expressing 293T cells with either restricted HIV-GFP or unrestricted HIV.G89V-GFP in the presence or absence of MG132. As shown in Figure 6C, MG132 prevented the HIV-1induced loss of TRIMCyp. Infection with the unrestricted G89V virus did not alter TRIMCyp stability, while addition of MG132 stabilized the restriction factor.

HIV-1-Induced Destabilization of Endogenous TRIM5a in Primate Cells
All of the previous experiments studying TRIM5a stability were conducted in transduced 293T cell lines in which TRIM5a was detected by virtue of a hemagglutinin epitope tag. In this setting, it PLoS Pathogens | www.plospathogens.org was necessary to treat the cells with cycloheximide to detect virusinduced degradation of the restriction factor, potentially leading to artifacts due to general inhibition of protein synthesis. Virus titration experiments demonstrated markedly greater restriction in the transduced cells vs. rhesus macaque FRhK-4 cell line, indicating that the 293T cells overexpress TRIM5a rh (our unpublished observations). Furthermore, while cycloheximide treatment had only a minor effect on restriction in FRhK-4 cells, the drug markedly reduced restriction in 293T cells ( Figure  S4). To probe the physiological relevance of our observations made in 293T cells, we sought a means of detecting endogenous TRIM5a protein in rhesus macaque cells. Using a monoclonal antibody against native TRIM5a for immunoblotting, we detected a band that was consistent in terms of molecular weight with TRIM5a rh that was also absent in cells lacking TRIM5a rh (data not shown). To confirm that the band is TRIM5a, we transfected FRhK-4 cells with either a TRIM5a rhspecific siRNA duplex or a non-targeting control siRNA duplex and quantified the intensity of this band by immunoblotting. As shown in Figure 7A and B, transfection with TRIM5a rh -specific siRNA resulted in a 72% decrease in intensity of the relevant band vs. FRhK-4 cells treated with the non-targeting control. Cells treated with the TRIM5a rh -specific also exhibited a tenfold increase in permissiveness to infection with HIV-1 (data not shown). HIV-1 infection of FRhK-4 cells was not altered by treatment with the non-targeting siRNA control. As expected, treatment with either siRNA duplex did not affect permissiveness to SIV infection (data not shown). These results indicated that the monoclonal antibody is capable of detecting endogenous TRIM5a rh in FRhK-4 cells. They further demonstrated that the transduced 293T cells express a 3.3 fold higher level of TRIM5a than FRhK-4 cells ( Figure 7B).
We next sought to determine if endogenous TRIM5a rh was destabilized by HIV-1 in rhesus macaque cells. FRhK-4 cultures were inoculated with HIV-1 in the presence or absence of cycloheximide and the stability of TRIM5a rh in response to infection was analyzed by immunoblotting. Initial experiments showed no effect of cycloheximide treatment on TRIM5a rh levels in HIV-1-exposed cells (data not shown); therefore the drug was removed in all subsequent experiments. We observed that TRIM5a rh levels were stable in FRhK-4 cells over the 4 hour period ( Figure 7C and D). Infection with HIV-1 resulted in accelerated decay of endogenous TRIM5a rh in rhesus macaque cells without any requirement of inhibition of protein synthesis.
We next sought to determine if the loss of TRIM5a rh was specifically due to restriction or was a non-specific effect resulting from viral infection. In the absence of cycloheximide we infected FRhK-4 cells with equivalent titers of HIV-1 or SIVmac239 GFP reporter viruses. As seen in Figure 8A and B, infection with HIV-1 resulted in a potent loss of TRIM5a rh while infection with SIV resulted in only a slight loss of TRIM5a rh as compared to the control cells. We conclude that infection by HIV-1 results in a rapid loss of TRIM5a rh in target cells and that this loss is directly related to the ability of TRIM5a rh to restrict infection by the incoming virus.

HIV-1-Induced Destabilization of Endogenous TRIM5a Requires Active Proteasomes
We sought to determine if inhibition of proteasome function would restore TRIM5a rh stability in rhesus macaque cells. FRhK-4 cells were exposed to HIV-1 in the presence or absence of MG132 for a period of four hours, and the levels of TRIM5a rh were measured by immunoblotting. As can be seen in Figure 8C  and D, MG132 stabilized TRIM5a rh in HIV-1-exposed cells. Flow cytometry analysis of GFP signal in a small subset of the infected cells showed no difference in infection levels resulting from inhibition of proteasome function, which is consistent with previously published results. These results indicate that HIV-1induced destabilization of TRIM5a rh in rhesus macaque cells requires proteasome activity. They further suggest that the results we observed with TRIM5a-transduced 293T cells are unlikely to be an artifact of cycloheximide treatment.

Discussion
While it is well established that TRIM5a limits the host range of many retroviruses, the precise mechanism of restriction remains undefined. TRIM5a can specifically associate with assemblies of HIV-1 CA-NC protein in vitro, and genetic evidence indicates that TRIM5a and TRIMCyp require an intact or semiintact viral capsid for binding [60,61]. However, the detailed molecular consequences of the binding interaction to the viral core remain poorly defined. Two lines of evidence have implicated the ubiquitin-proteasome system in restriction. First, the d isoform of TRIM5, which has a RING domain identical to that of TRIM5a, exhibits E3 activity in vitro [56]. Deletion or mutation of the RING domain in TRIM5a results in significant loss of restriction efficacy [44,49]. TRIM5a is ubiquitinated in cells, although a role of this modification in retrovirus restriction has not been established [55]. Second, inhibition of proteasome activity alters the stage at which TRIM5a-mediated restriction occurs [58,59]. The latter observation led us to hypothesize that the proteasome may participate in restriction by degrading a complex of TRIM5a with one or more incoming viral proteins. To test this, we asked whether exposure of cells to HIV-1 alters the stability of TRIM5a rh . We observed that inoculation with HIV-1 results in an accelerated turnover of the restriction factor. Similar effects were observed in both 293T and HeLa cells (data not shown), suggesting that TRIM5a destabilization is not specific to a unique cell type. HIV-1 challenge resulted in destabilization of TRIM5a rh but not TRIM5a hu . Likewise, TRIM5a hu was destabilized by inoculation of cells with restriction-sensitive N-MLV particles but not by unrestricted B-MLV. Similar results were seen in cells expressing the HIV-1specific restriction factor TRIMCyp. Treatment of target cells with CsA, which blocks TRIMCyp restriction of HIV-1, or infection with virus containing mutations that prevent CypA binding [4,5,38], did not affect TRIMCyp stability. Specific loss of TRIM5a from cells expressing different primate alleles of the protein also correlated very well with the ability of those alleles to restrict HIV or SIV. The HIV-1-induced destabilization of TRIM5a rh and TRIMCyp was prevented by inhibition of cellular proteasome activity. Destabilization of TRIM5a rh by HIV-1 was also observed in a primate derived cell line without the need of cycloheximide to inhibit protein synthesis. This destabilization was specific for the restricted HIV-1 and was not observed in cells infected with an unrestricted virus. Inhibition of proteasome function restored TRIM5a rh stability in response to infection by HIV-1 in the rhesus macaque cells. We conclude that TRIM5related restriction factors are targeted for degradation by a proteasome-dependent mechanism following encounter of a restriction-sensitive retroviral core.
TRIM5a forms heterogenous structures in cells referred to as cytoplasmic bodies (CBs). While the role of CBs in restriction is unclear, TRIM5a protein in these structures rapidly exchanges with soluble TRIM5a, indicating that the protein is highly dynamic within cells [62]. We observed that most of the cellular TRIM5a can be degraded in response to exposure to a restriction-sensitive retrovirus, which implies that a majority of cellular TRIM5a molecules can engage incoming viral cores. If the CB-associated TRIM5a is inaccessible to incoming virus, our observation that a restricted virus can induce degradation of the majority of the TRIM5a molecules suggests that this protein rapidly redistributes to a compartment accessible to incoming virus.
TRIM5a and TRIMCyp are subject to proteasome-dependent turnover under steady-state conditions, yet its rapid turnover is not a prerequisite for restriction activity [55,63]. Accordingly, proteasome inhibitors do not overcome restriction ( [57]; Figure   S5). Nonetheless, the effect of virus exposure on TRIM5a stability had heretofore not been reported. While alterations of specific individual portions of TRIM5a may alter its intrinsic stability, our results indicate that TRIM5a encounter with a restricted core results in degradation of the restriction factor by a proteasomedependent mechanism.
Retrovirus uncoating is a poorly characterized process, but can be defined as the disassembly of the viral capsid following penetration of the viral core into the target cell cytoplasm. Studies of HIV-1 CA mutants indicate that the stability of the viral capsid is properly balanced for productive uncoating in target cells: mutants with unstable capsids are impaired for viral DNA synthesis, suggesting that premature uncoating is detrimental to reverse transcription [64]. Thus a plausible mechanism for restriction is that binding of TRIM5a to the viral capsid inhibits infection directly by physically triggering premature uncoating in target cells [65,66]. In this model, TRIM5a, perhaps with one or more cofactors, promotes the physical decapsidation of the virus core independently of proteolysis. Consistent with this view are studies demonstrating that TRIM5a restriction is associated with decreased recovery of sedimentable CA protein in lysates of acutely-infected cells [65,66]. However, these studies fell short of demonstrating that the sedimentable CA protein was associated with intact viral cores. Furthermore, a recent study reported that treatment of cells with proteasome inhibitors prevented TRIM5a-dependent loss of particulate CA protein [67], indicating the potential involvement of proteasome activity in TRIM5ainduced virus uncoating.
Other studies further implicate the activity of the proteasome in TRIM5a-dependent restriction. Inhibition of proteasome activity rescues HIV-1 reverse transcription in TRIM5a-expressing cells, revealing a downstream block to nuclear import mediated by the restriction factor [58,59]. Engagement of the viral capsid by TRIM5a may lead to proteasomal degradation of a TRIM5a-CA complex, resulting in functional decapsidation of the viral core and a premature uncoating phenotype. Consistent with this model, TRIM5a restriction has been associated with decreased intracellular accumulation of HIV-1 CA [68]. In addition, a recent study of MLV particle-mediated RNA cellular transfer reported reduced accumulation of viral CA protein in cells in a manner that was correlated with restriction by TRIM5a, and this effect was reversed by proteasome inhibition [69]. Unfortunately, our own efforts to detect an effect of TRIM5a on the stability of the incoming HIV-1 CA have thus far yielded negative results; thus we are reluctant to conclude at this stage that a specific component of the viral core is degraded as a complex with TRIM5a. Another potential mechanism is that proteasomal engagement of TRIM5a bound to the virus core results in physical dissociation of CA from the core followed by its release from TRIM5a, thus leading to destruction of the restriction factor and decapsidation of the core but not necessarily degradation of CA [70]. Genetic evidence from abrogation-of-restriction studies indicates that TRIM5a binding requires an intact or semiintact viral capsid [60], suggesting that TRIM5a binding to CA is highly dependent on avidity resulting from multivalent interactions with the polymeric viral capsid. It is thus plausible that CA is released from TRIM5a following forced uncoating. This model is attractive in its ability to reconcile most, if not all, of the reported data regarding the mechanism of restriction by TRIM5a.
HIV-1 infection in many primate cell lines exhibits biphasic titration curves, and restriction can be abrogated in trans by high concentrations of VLPs, indicating that virus restriction is saturable. While it is generally assumed that the saturation occurs via sequestration of the restriction factor by the incoming virus, our results reveal another potential mechanism. Degradation of TRIM5a rh by HIV-1 was tightly correlated with cellular susceptibility to infection by incoming virus, suggesting that loss of restriction at high virus input may occur via degradation of the restriction factor itself. Consistent with this view, treatment with MG132 resulted in a three-fold decrease in HIV-1 infection of FRhK-4 as well as OMK cells, while infection by unrestricted SIV was inhibited only marginally ( Figure S5). This result, coupled with our observations of proteasome-dependent degradation of TRIM5a proteins in restrictive cells, suggests that depletion of TRIM5a via the proteasome contributes to the saturability of restriction.
The potential involvement of ubiquitylation in virus-induced degradation of TRIM5a degradation warrants further study. The autoubiquitylation of TRIM5d observed in vitro suggests that TRIM5a may be ubiquitylated in trans upon polymerization of the restriction factor on a retroviral capsid. However, we have been unable to detect accumulation of cellular ubiquitylated TRIM5a species following HIV-1 inoculation either in the presence or absence of proteasome inhibitors (our unpublished observations). While many cellular proteins are regulated by ubiquitin-dependent proteolysis, ubiquitin-independent proteasomal degradation is also well documented (reviewed in [71]). Most E3 ligases are not degraded following ubiquitylation of a substrate, yet notable exceptions exist. The E3 enzyme Mdm2 is degraded following its ubiquitylation of its target, p53 [72], and the stability of several E3 ligases is related to their ubiquitylation status resulting from autoubiquitylation [73][74][75]. It will therefore be of interest to determine whether HIV-1-induced degradation of TRIM5a is dependent on host cell ubiquitylation and the TRIM5a RING domain.
The early post-entry stage of infection remains a fundamentally obscure part of the retrovirus life cycle. Our results provide novel evidence for a role for proteasome activity in TRIM5a restriction. Further mechanistic studies of TRIM5a may reveal novel approaches to antiviral therapy and fundamental insights into the molecular details of HIV-1 uncoating.

Chemicals
MG132 and cycloheximide were purchased from Sigma-Aldrich and used at final concentrations of 25 mM and 50 mM, respectively. Cyclosporin A was purchased from CalBiochem used at 2.5 mM final concentration. Epoxomicin was purchased from Boston Biochem and used at 10 mM. The cathepsin inhibitor E64 was purchased from Sigma-Aldrich and was used at 40 mM.

Cells and Viruses
FRhK-4 cells were purchased from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/ streptomycin. VSV-G-pseudotyped HIV-1 NL4.3 , HIV-GFP, and SIV-GFP viruses were produced by calcium phosphate transfection of 293T cells with proviral plasmid DNA (23 mg) and pHCMV-G (7 mg). N-and B-tropic MLV virus stocks were prepared by cotransfection of 23 mg pCIG-N or pCIG-B plasmids with pHCMV-G (7 mg) onto the cell line 293TeGFP. This cell line is a clone generated from 293T cells previously transduced with the retroviral vector pBABE-eGFP and isolated by limiting dilution and selected for high levels of GFP expression. Transfected cells were washed after 24 hours and replenished with fresh media. Supernatants were harvested 48-72 hours after transfection, clarified by passing through 0.45 mm filters, and stored in aliquots at 280uC. Retrovirus stocks for transduction of TRIM5a alleles were harvested from 293T cells transfected with the plasmids pCL-ampho (10 mg), the appropriate TRIM5a vector (15 mg), and pHCMV-G (5 mg). Viruses were collected 48 hours after transfection and used to transduce 293T cells. All 293T cell lines expressing TRIM5a proteins were polyclonal cell populations obtained by selection of transduced cells with puromycin. TRIMCyp-expressing cells were obtained by isolation of a single cell clone via limiting dilution. HIV-1 was strongly restricted in these cells, and restriction was prevented by the addition of 5 mg/ml cyclosporin A (CsA).

Infection Protocol
Cells were seeded in 6-well plates at a density of 1 to 1.25610 6 cells/well and incubated overnight. Prior to infection, cultures were treated for 1 hour in 50 mM cycloheximide to block protein synthesis. In experiments involving proteasome inhibitors, cells were incubated with both cycloheximide and the appropriate inhibitor for 1 hour prior to infection. Viral stocks containing cycloheximide, polybrene (5 mg/mL), CsA (2.5 mM), and proteasome inhibitors were prewarmed to 37uC prior to addition to cells. After culturing for 1 hr, media from zero hour timepoints was removed and 1 ml of PBS was added. Cells were then detached from the plate by flushing, pelleted, washed in PBS, repelleted, and the pellets frozen at 280uC. Cells that were challenged with virus had media removed and replaced with viral stock and were returned to 37uC. Individual cultures were harvested hourly using same procedure as previously described for the zero hour timepoints. All cell pellets were frozen at 280uC prior to analysis. For experiments utilizing FRhK-4 cells the cells were seeded in 6 well plates at a density of 3610 5 cells/well and incubated overnight. Prewarmed viral stocks containing polybrene (5 mg/ mL) were added the following day with a well harvested at the time of viral addition serving as the zero hour timepoint. Cells were incubated with the viral stock for the indicated time period then trypsinized, placed in fresh D10 media at a 1:1 volume, pelleted, washed in 1 mL complete D10 media to inactivate trypsin, repelleted, washed 2 times in 1 mL PBS, then frozen at 280uC. In experiments with FRhK-4 cells involving MG132, the cells were incubated with inhibitor for one hour prior to viral addition with the zero hour timepoint being an uninfected well harvested after 1 hour pretreatment.
siRNA Knockdown of TRIM5a rh 293T and FRhK-4 cells were seeded at a density of 2610 5 cells per well in 6-well plates and incubated overnight. 24 hours later, TRIM5a rh -specific siRNA [3], or a non-targeting control siRNA (Dharmacon), were diluted to a concentration of 3 mM in 16siRNA buffer then transfected into cells using Dharmafect 1 transfection reagent and OptiMEM I (Gibco) according to manufacturers protocol (Dharmacon). Cells were then incubated overnight and retransfected with siRNAs again the following day utilizing the identical protocol. 48 hours after the first siRNA transfection the cells were removed from the 6-well plates and plated onto a 10 cm dish in complete D10 media at a ratio of 1 well to 1 10 cm dish and incubated for either 24 or 48 hours. 24 hours later, one 10 cm dish of either TRIM5a rh -specific siRNA treated cells or non-targeting control treated cells were trypsinized and replated in 24 well plates at a density of 2610 5 cells/well then incubated overnight. The following day the remaining two 10 cm dishes of siRNA treated cells were trypsinized, diluted 1:1 in D10 media, pelleted, washed 16 in D10 media to inactivate trypsin, repelleted, washed 26 in 1 mL PBS per wash, repelleted, then frozen at 280uC. Cells that had been seeded the prior day in the 24 well plates were then infected with dilutions of HIV and SIV-GFP, incubated for 48 hours, then analyzed for GFP expression by flow cytometry.

Protein Analyses
Cell pellets were thawed and lysed in a solution containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 0.5% NP-40. Nuclei were pelleted via centrifugation at 16,0006g for 10 minutes and post-nuclear supernatants were removed. Protein levels were quantified via BCA assay (Pierce). Samples, normalized for total protein, were denatured in SDS and subjected to electrophoresis on 4-20% acrylamide gradient gels (BioRad). Proteins were transferred to nitrocellulose and probed with HA-epitope tagspecific rat monoclonal antibody (3F10, Roche) and Alexa Fluor 680 conjugated goat anti-rat IgG (Molecular Probes). Cells expressing TRIMCyp were probed with the myc epitope-specific mouse monoclonal antibody (9E10, Invitrogen) and Alexa Fluor 680-conjugated goat anti-mouse IgG (Molecular Probes). Proteins extracted from FRhK-4 cells were probed the TRIM5a-specific mouse polyclonal antibody (IMG-5354, Imgenex) and Alexa Fluor 680 conjugated goat anti-mouse IgG (Molecular Probes). All immunoblots were probed with b-actin-specific rabbit monoclonal antibody (A2228, Sigma) and IRDye800-conjugated goat antirabbit IgG (Rockland). Dilutions of antibodies were 1:1000 and 1:5000 for primary and secondary respectively with the exception of IMG-5354 which was used at a dilution of 1:2000. Bands were detected by scanning blots with the LI-COR Odyssey Imaging System using both 700 and 800 channels, and integrated intensities were determined using the LI-COR Odyssey band quantitation software with the median top-bottom background subtraction method. The TRIM5a band intensities were then normalized to the signals from the corresponding b-actin bands. All signals were then expressed as a percentage of the initial TRIM5a/actin band intensity ratio.