Sebla B. Kutluay

MX2 is an interferon-induced inhibitor of HIV-1 infection

HIV-1 replication can be inhibited by type I interferon (IFN), and the expression of a number of gene products with anti-HIV-1 activity is induced by type I IFN. However, none of the known antiretroviral proteins can account for the ability of type I IFN to inhibit early, preintegration phases of the HIV-1 replication cycle in human cells. Here, by comparing gene expression profiles in cell lines that differ in their ability to support the inhibitory action of IFN-α at early steps of the HIV-1 replication cycle, we identify myxovirus resistance 2 (MX2) as an interferon-induced inhibitor of HIV-1 infection. Expression of MX2 reduces permissiveness to a variety of lentiviruses, whereas depletion of MX2 using RNA interference reduces the anti-HIV-1 potency of IFN-α. HIV-1 reverse transcription proceeds normally in MX2-expressing cells, but 2-long terminal repeat circular forms of HIV-1 DNA are less abundant, suggesting that MX2 inhibits HIV-1 nuclear import, or destabilizes nuclear HIV-1 DNA. Consistent with this notion, mutations in the HIV-1 capsid protein that are known, or suspected, to alter the nuclear import pathways used by HIV-1 confer resistance to MX2, whereas preventing cell division increases MX2 potency. Overall, these findings indicate that MX2 is an effector of the anti-HIV-1 activity of type-I IFN, and suggest that MX2 inhibits HIV-1 infection by inhibiting capsid-dependent nuclear import of subviral complexes.

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.

Global Changes in the RNA Binding Specificity of HIV-1 Gag Regulate Virion Genesis

The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.

Curcumin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity.

Curcumin, a phenolic compound from the curry spice turmeric, exhibits a wide range of activities in eukaryotic cells, including antiviral effects that are at present incompletely characterized. Curcumin is known to inhibit the histone acetyltransferase activity of the transcriptional coactivator proteins p300 and CBP, which are recruited to the immediate early (IE) gene promoters of herpes simplex virus type 1 (HSV-1) by the viral transactivator protein VP16. We tested the hypothesis that curcumin, by inhibiting these coactivators, would block viral infection and gene expression. In cell culture assays, curcumin significantly decreased HSV-1 infectivity and IE gene expression. Entry of viral DNA to the host cell nucleus and binding of VP16 to IE gene promoters was not affected by curcumin, but recruitment of RNA polymerase II to those promoters was significantly diminished. However, these effects were observed using lower curcumin concentrations than those required to substantially inhibit global H3 acetylation. No changes were observed in histone H3 occupancy or acetylation at viral IE gene promoters. Furthermore, p300 and CBP recruitment to IE gene promoters was not affected by the presence of curcumin. Finally, disruption of p300 expression using a short hairpin RNA did not affect viral IE gene expression. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity.

Fates of retroviral core components during unrestricted and TRIM5-restricted infection.

TRIM5 proteins can restrict retroviral infection soon after delivery of the viral core into the cytoplasm. However, the molecular mechanisms by which TRIM5α inhibits infection have been elusive, in part due to the difficulty of developing and executing biochemical assays that examine this stage of the retroviral life cycle. Prevailing models suggest that TRIM5α causes premature disassembly of retroviral capsids and/or degradation of capsids by proteasomes, but whether one of these events leads to the other is unclear. Furthermore, how TRIM5α affects the essential components of the viral core, other than capsid, is unknown. To address these questions, we devised a biochemical assay in which the fate of multiple components of retroviral cores during infection can be determined. We utilized cells that can be efficiently infected by VSV-G-pseudotyped retroviruses, and fractionated the cytosolic proteins on linear gradients following synchronized infection. The fates of capsid and integrase proteins, as well as viral genomic RNA and reverse transcription products were then monitored. We found that components of MLV and HIV-1 cores formed a large complex under non-restrictive conditions. In contrast, when MLV infection was restricted by human TRIM5α, the integrase protein and reverse transcription products were lost from infected cells, while capsid and viral RNA were both solubilized. Similarly, when HIV-1 infection was restricted by rhesus TRIM5α or owl monkey TRIMCyp, the integrase protein and reverse transcription products were lost. However, viral RNA was also lost, and high levels of preexisting soluble CA prevented the determination of whether CA was solubilized. Notably, proteasome inhibition blocked all of the aforementioned biochemical consequences of TRIM5α-mediated restriction but had no effect on its antiviral potency. Together, our results show how TRIM5α affects various retroviral core components and indicate that proteasomes are required for TRIM5α-induced core disruption but not for TRIM5α-induced restriction.

Regulation of Histone Deposition on the Herpes Simplex Virus Type 1 Genome during Lytic Infection

ABSTRACT During lytic infection by herpes simplex virus type 1 (HSV-1), histones are present at relatively low levels on the viral genome. However, the mechanisms that account for such low levels—how histone deposition on the viral genome is blocked or how histones are removed from the genome—are not yet defined. In this study, we show that histone occupancy on the viral genome gradually increased with time when transcription of the viral immediate-early (IE) genes was inhibited either by deletion of the VP16 activation domain or by chemical inhibition of RNA polymerase II (RNAP II). Inhibition of IE protein synthesis by cycloheximide did not affect histone occupancy on most IE promoters and coding regions but did cause an increase at delayed-early and late gene promoters. IE gene transcription from HSV-1 genomes associated with high levels of histones was stimulated by superinfection with HSV-2 without altering histone occupancy or covalent histone modifications at IE gene promoters. Moreover, RNAP II and histones cooccupied the viral genome in this context, indicating that RNAP II does not preferentially associate with viral genomes that are devoid of histones. These results suggest that during lytic infection, VP16, RNAP II, and IE proteins may all contribute to the low levels of histones on the viral genome, and yet the dearth of histones is neither a prerequisite for nor a necessary result of VP16-dependent transcription of nucleosomal viral genomes.

Transcriptional Coactivators Are Not Required for Herpes Simplex Virus Type 1 Immediate-Early Gene Expression In Vitro

ABSTRACT Virion protein 16 (VP16) of herpes simplex virus type 1 (HSV-1) is a potent transcriptional activator of viral immediate-early (IE) genes. The VP16 activation domain can recruit various transcriptional coactivators to target gene promoters. However, the role of transcriptional coactivators in HSV-1 IE gene expression during lytic infection had not been fully defined. We showed previously that transcriptional coactivators such as the p300 and CBP histone acetyltransferases and the BRM and Brg-1 chromatin remodeling complexes are recruited to viral IE gene promoters in a manner dependent mostly on the presence of the activation domain of VP16. In this study, we tested the hypothesis that these transcriptional coactivators are required for viral IE gene expression during infection of cultured cells. The disrupted expression of the histone acetyltransferases p300, CBP, PCAF, and GCN5 or the BRM and Brg-1 chromatin remodeling complexes did not diminish IE gene expression. Furthermore, IE gene expression was not impaired in cell lines that lack functional p300, or BRM and Brg-1. We also tested whether these coactivators are required for the VP16-dependent induction of IE gene expression from transcriptionally inactive viral genomes associated with high levels of histones in cultured cells. We found that the disruption of coactivators also did not affect IE gene expression in this context. Thus, we conclude that the transcriptional coactivators that can be recruited by VP16 do not contribute significantly to IE gene expression during lytic infection or the induction of IE gene expression from nucleosomal templates in vitro.

Role of chromatin during herpesvirus infections.

DNA viruses have long served as model systems to elucidate various aspects of eukaryotic gene regulation, due to their ease of manipulation and relatively low complexity of their genomes. In some cases, these viruses have revealed mechanisms that are subsequently recognized to apply also to cellular genes. In other cases, viruses adopt mechanisms that prove to be exceptions to the more general rules. The double-stranded DNA viruses that replicate in the eukaryotic nucleus typically utilize the host cell RNA polymerase II (RNAP II) for viral gene expression. As a consequence, these viruses must reckon with the impact of chromatin on active transcription and replication. Unlike the small DNA tumor viruses, such as polyomaviruses and papillomaviruses, the relatively large genomes of herpesviruses are not assembled into nucleosomes in the virion and stay predominantly free of histones during lytic infection. In contrast, during latency, the herpesvirus genomes associate with histones and become nucleosomal, suggesting that regulation of chromatin per se may play a role in the switch between the two stages of infection, a long-standing puzzle in the biology of herpesviruses. In this review we will focus on how chromatin formation on the herpes simplex type-1 (HSV-1) genome is regulated, citing evidence supporting the hypothesis that the switch between the lytic and latent stages of HSV-1 infection might be determined by the chromatin state of the HSV-1.

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis

While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

Assisted Evolution Enables HIV-1 to Overcome a High TRIM5α-Imposed Genetic Barrier to Rhesus Macaque Tropism

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an ‘assisted evolution’ approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection.