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Title: The Interferon Response Inhibits HIV Particle Production by Induction of TRIM22}
Article:
Abstract:
Treatment of human cells with Type 1 interferons restricts HIV replication. Here we report that the tripartite motif protein TRIM22 is a key mediator. We used transcriptional profiling to identify cellular genes that were induced by interferon treatment and identified TRIM22 as one of the most strongly up-regulated genes. We confirmed, as in previous studies, that TRIM22 over-expression inhibited HIV replication. To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector. Further studies showed that TRIM22 inhibited budding of virus-like particles containing Gag only, indicating that Gag was the target of TRIM22. TRIM22 did not block the release of MLV or EIAV Gag particles. Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking. Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication.
Introduction:
The interferon system is a well studied branch of the innate immune system active against viruses. Infection of vertebrate cells by many viruses provokes synthesis and secretion of interferons( IFNs), which mediate induction of a cellular antiviral state that obstructs further viral spread. Type I IFNs( α and β) are produced by many cell types, while Type II IFN( gamma) is produced by immune cells. IFN-induced signaling pathways begin with IFN binding to IFN receptors at the cell surface. This results in signal transduction via the Jak/Stat pathway, which leads to activation of interferon-responsive genes and synthesis of effector proteins, including PKR, RNAseL, Mx, and many others( reviewed in[1],[2],[3],[4]). For HIV, previous studies have implicated IFNs in blocking both early and late stages of the HIV-1 lifecycle[5],[6],[7],[8],[9],[10]. In many cell types, the inhibition of later steps in the life cycle following integration seems to be most potent( reviewed in[2]). Although the effector mechanisms acting late have not been fully clarified, one report suggested that the IFN-inducible protein ISG15 interfered with the endosomal trafficking pathway used by HIV-1 to exit 293T cells by blocking the interaction of TSG101 with HIV-1 Gag[11]. Many tripartite motif( TRIM) proteins may function in innate immunity to restrict viral replication. TRIM5α from rhesus macaque( RhTRIM5α) blocks early replication steps of HIV-1 and other retroviruses( reviewed in[12]); in addition, one group has proposed that RhTRIM5α acts late during HIV replication as well[13]. The early target of RhTRIM5α is the capsid( CA) protein, which forms the protein shell of the viral core. Anti-HIV-1 activity has also been reported for other TRIM family members. TRIM1 has been shown to target the CA protein at an early stage pre-reverse transcription, TRIM22 has been suggested to affect HIV transcription and TRIM19 and TRIM32 have been suggested to affect trafficking of viral proteins( reviewed in[14]). Recently, TRIM28 was shown to repress transcription from a retroviral promoter by binding to proviral DNA[15]. TRIM proteins contain a highly conserved RBCC motif comprised of a RING domain, one or two B-box domains, and a predicted coiled-coil region( reviewed in[14]). The RING domain contains a specialized zinc finger[16] and has been shown to possess E3 ubiquitin ligase activity[17],[18],[19],[20],[21],[22]. Little is known about the B-box domain, which is unique to TRIM proteins. The coiled-coil domain is believed to promote protein oligomerization[23]. Several TRIM proteins also contain a C-terminal SPRY domain that is proposed to be involved in protein-protein interactions and RNA binding[24],[25]. Many TRIM family members are inducible by IFNs, providing candidate mediators of IFN inhibition. In an effort to identify TRIM proteins mediating anti-retroviral activity, we carried out transcriptional profiling of IFN-treated human osteosarcoma( HOS) cells and found that TRIM22 was the most up-regulated TRIM queried on the microarray used. HOS-CD4/CXCR4 cells were studied because they support robust HIV replication and show a strong antiviral response to IFNβ. Human TRIM22( also known as Staf-50) was previously identified from a search for IFN-regulated genes in Daudi cells. Over-expression of TRIM22 was reported to repress transcription from the HIV-1 LTR in a plasmid reporter system in COS-1 cells[26]. In a later study by another group, over-expression of TRIM22 was reported to inhibit HIV-1 replication in human macrophages and 293T cells and postulated to act by repressing transcription from the HIV-1 LTR[27]. TRIM22 has also been implicated in normal haematopoietic differentiation[28],[29] and in diseases such as systemic lupus erythematosus[30] and Wilms tumor[31]. The expression of TRIM22 is altered in response to a variety of stimuli in addition to IFN including T-cell co-stimulation by CD2 and CD28[32], viral antigens or infection[27],[33],[34],[35] and inflammatory cytokines[28]. Here we investigate the mechanism by which TRIM22 inhibits HIV infection. We mapped the primary block to late stages of assembly and release in the viral life cycle using over-expression studies. Short hairpin RNA against TRIM22 substantially reversed the inhibition of late steps induced by IFN treatment, implicating TRIM22 as a functional effector when expressed at biological levels. TRIM22 blocked the release of HIV-1 Gag-only particles, and the release of HIV particles was dependent on the RING domain of TRIM22. These findings clarify an IFN-inducible effector arm that disrupts the late stages of HIV infection.
Discussion:
Here we present data that TRIM22 is an interferon inducible effector that is responsible at least in part for the late IFNβ block to HIV-1 replication. We observed, as have others, that over-expression of TRIM22 in cells obstructed HIV replication. We show here that knockdown of TRIM22 in HOS-CD4/CXCR4 cells in the context of a natural IFN response abrogated the late block to HIV replication, implicating TRIM22 as an antiviral effector. TRIM22 expression was able to block release of HIV-1 Gag-only virus-like particles, but not MLV or EIAV Gag particles, indicating that TRIM22 acts specifically on the HIV Gag protein. In binding studies, TRIM22 associated with HIV-1 Gag but not MLV or EIAV Gag. TRIM22 expression altered Gag localization–cells containing Gag-GFP fusions showed punctate structures at the plasma membrane, potential sites of virus assembly and budding[41],[54],[55]. In contrast, Gag-GFP expressed in the presence of TRIM22 alone or IFNβ showed only diffuse staining. TRIM22 with substitutions in the E3 catalytic residues were unable to restrict HIV-1 release, indicating that inhibition was dependent on the active site. Taken together, these data implicate TRIM22 as an effector arm of the IFN-mediated antiviral state that selectively disrupts Gag sorting and release. Complications in studying mechanisms of the IFN response arise from i) the pronounced cell type specificity of IFN action, ii) differential effects of different doses, and iii) differences among the effects of interferon types. Previous studies have reported that Type I IFNs can block HIV gene expression, Gag sorting, and Gag release[9],[11],[26],[56],[57],[58],[59],[60],[61],[62]. Here we show, using HOS-CD4/CXCR4 cells, that TRIM22 acts on HIV Gag, allowing efficient Gag expression but blocking assembly and/or release. Similar results were seen in HeLa cells. However, in U2OS and 143B cells, TRIM22 expression resulted in reduced accumulation of Gag in cells, indicating that TRIM22 inhibited either Gag synthesis or increased Gag turnover. A previous study, analyzing TRIM22 in COS cells, indicated that TRIM22 inhibited LTR-driven transcription. A similar mechanism may operate in U2OS cells and 143B cells. Recently, rhesus TRIM5α( but not its human ortholog) was shown to cause the rapid degradation of Gag in 293T cells[63]. We found no evidence that TRIM22 drastically altered the stability of Gag protein levels in HOS-CD4/CXCR4 or HeLa cells, however it is possible that altered Gag sorting results in faster degradation in U2OS and 143B cells than seen in HOS-CD4/CXCR4 and HeLa cells. Against this background of cell type specific effects, comparisons of our results to previous reports are quite tentative. In one study, addition of 500 units/ml of IFNα to 293T cells was reported to inhibit HIV particle release about two-fold, and an siRNA against the interferon-induced ubiquitin-like molecule ISG15 blocked IFN inhibition[64],[65],[66]. In HOS-CD4/CXCR4 cells treated with IFNβ at 1000 units/ml, the inhibition of particle release was stronger( 3. 0-fold to 7. 4-fold), but the shRNA against TRIM22 did not completely reverse IFN inhibition. One possibility is that TRIM22 and ISG15 both act in the same pathway. For example, TRIM22 might act as an E3 ligase for ISG15. Further experiments will be needed to assess the relationship of inhibition by TRIM22 to that of ISG15. Another recent report suggested that IFNα treatment caused HIV-1 virions to become tethered to cell surfaces, resulting in eventual re-uptake by endocytosis[67]. The viral Vpu protein was reported to overcome this block in some cell types. It is possible that the mis-sorting of Gag observed here in HOS-CD4/CXCR4 cells results from virions trafficking to the membrane followed by re-uptake, though in HOS-CD4/CXCR4 cells there was no evidence for Gag accumulating at membranes at early times as might be expected from the re-uptake model. Also, in the HOS-CD4/CXCR4 cell model, expression of Gag in the presence or absence of Vpu did not noticeably affect particle accumulation in the supernatant. It will be useful to identify the cell type or experimental differences responsible for these divergent observations. How human TRIM22 alters intracellular Gag trafficking in HOS-CD4/CXCR4 cells is unknown but a fascinating problem for further study. One candidate model would be that TRIM22 acts by interfering with Gag binding to membranes via the linked myristate residue. Membrane binding of Gag has been reported to be regulated by a multimerization-dependent myristoyl switch[68],[69],[70],[71]. We found that Gag was myristoylated normally in the presence of TRIM22( Figure 5C) but TRIM22 might affect Gag multimerization and resulting exposure of the myristate residue. We also found that that the E3 ligase activity of TRIM22 is important for restricting viral particle release, which implies that TRIM22 probably disrupts Gag trafficking by transferring ubiquitin( or an ubiquitin-related moiety) to one or more targeted proteins. Although TRIM22 interacts with Gag, we have yet to detect a convincing effect of TRIM22 on the levels of Gag ubiquitylation or a size-altering modification of Gag( data not shown), raising the possibility that a cellular protein involved in Gag trafficking is the target. Thus studies of TRIM22 link E3 ligases, IFN, and HIV late replication steps, providing a new experimental route to investigating these issues.
Interferons are produced by cells in response to challenge by foreign pathogens such as viruses . The molecular mechanisms by which Type I interferons ( e . g . , IFNβ ) inhibit the replication of HIV-1 are not fully clarified . We identified a gene called TRIM22 that belongs to the tripartite motif ( TRIM ) family that was strongly induced by IFNβ . Using RNA interference to reduce the expression of TRIM22 , we showed that TRIM22 is a key mediator of the IFNβ response when expressed at natural levels . We demonstrate that TRIM22 blocks the intracellular trafficking of the viral structural protein Gag to the surface of the cell , and that the antiviral activity of TRIM22 is dependent on two cysteine residues ( Cys15 and Cys18 ) that are critical for the E3 ligase activity of RING-containing proteins . This report describes a mechanism by which Type I interferons block HIV-1 replication .