The cellular protein lyric interacts with HIV-1 Gag

Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interaction requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.     

Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative

The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.     

A heterologous, high-affinity RNA ligand for human immunodeficiency virus Gag protein has RNA packaging activity

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.     

The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells

Background

Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before.

Methodology/Principal Findings

Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA.

Conclusion

Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.     

A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55(Gag). Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.     

The host protein Staufen1 participates in human immunodeficiency virus type 1 assembly in live cells by influencing pr55Gag multimerization

Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.     

Human immunodeficiency virus type 2 Gag interacts specifically with PRP4, a serine-threonine kinase, and inhibits phosphorylation of splicing factor SF2

Using a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that bind to the human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified PRP4, a serine-threonine protein kinase. Specific interaction of PRP4 and HIV-2 Gag was confirmed in in vitro and in vivo assays. The interacting region of HIV-2 Gag is located in the conserved matrix and capsid domains, while both the RS (arginine-serine-rich) domain and the KS (kinase) domain of PRP4 are able to bind to HIV-2 Gag. PRP4 is not incorporated into virus particles. HIV-2 Gag is able to inhibit PRP4-mediated phosphorylation of the splicing factor SF2. This is also observed with Gag from simian immunodeficiency virus, a closely related virus, but not with Gag from human T-cell lymphotropic virus type 1. Our results provide evidence for a novel interaction between Gag and a cellular protein kinase involved in the control of constitutive splicing in two closely related retroviruses. We hypothesize that as Gag accumulates in the cell, down regulation of splicing occurs through reduced phosphorylation of SF2. At late stages of infection, this interaction may replace the function of the early viral regulatory protein Rev.     

trans-Complementation rescue of cyclophilin A-deficient viruses reveals that the requirement for cyclophilin A in human immunodeficiency virus type 1 replication is independent of its isomerase activity

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.     

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.     

Chimeric human immunodeficiency virus type 1 (HIV-1) virions containing HIV-2 or simian immunodeficiency virus Nef are resistant to cyclosporine treatment

The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and SIV Nef are able to compensate for the need for cyclophilin A for full infectivity and that amino acids present at the C termini of these proteins are important for this function.     

Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.     

Small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between Vif and ElonginC

The HIV-1 viral infectivity factor (Vif) protein is essential for viral replication. Vif recruits cellular ElonginB/C-Cullin5 E3 ubiquitin ligase to target the host antiviral protein APOBEC3G (A3G) for proteasomal degradation. In the absence of Vif, A3G is packaged into budding HIV-1 virions and introduces multiple mutations in the newly synthesized minus-strand viral DNA to restrict virus replication. Thus, the A3G-Vif-E3 complex represents an attractive target for development of novel anti-HIV drugs. In this study, we identified a potent small molecular compound (VEC-5) by virtual screening and validated its anti-Vif activity through biochemical analysis. We show that VEC-5 inhibits virus replication only in A3G-positive cells. Treatment with VEC-5 increased cellular A3G levels when Vif was coexpressed and enhanced A3G incorporation into HIV-1 virions to reduce viral infectivity. Coimmunoprecipitation and computational analysis further attributed the anti-Vif activity of VEC-5 to the inhibition of Vif from direct binding to the ElonginC protein. These findings support the notion that suppressing Vif function can liberate A3G to carry out its antiviral activity and demonstrate that regulation of the Vif-ElonginC interaction is a novel target for small-molecule inhibitors of HIV-1.     

Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.     

Impaired infectivity of ritonavir-resistant HIV is rescued by heat shock protein 90AB1

Certain ritonavir resistance mutations impair HIV infectivity through incomplete Gag processing by the mutant viral protease. Analysis of the mutant virus phenotype indicates that accumulation of capsid-spacer peptide 1 precursor protein in virus particles impairs HIV infectivity and that the protease mutant virus is arrested during the early postentry stage of HIV infection before proviral DNA synthesis. However, activation of the target cell can rescue this defect, implying that specific host factors expressed in activated cells can compensate for the defect in ritonavir-resistant HIV. This ability to rescue impaired HIV replication presented a unique opportunity to identify host factors involved in postentry HIV replication, and we designed a functional genetic screen so that expression of a given host factor extracted from activated T cells would lead directly to its discovery by rescuing mutant virus replication in nonactivated T cells. We identified the cellular heat shock protein 90 kDa α (cytosolic), class B member 1 (HSP90AB1) as a host factor that can rescue impaired replication of ritonavir-resistant HIV. Moreover, we show that pharmacologic inhibition of HSP90AB1 with 17-(allylamino)-17-demethoxygeldanamycin (tanespimycin) has potent in vitro anti-HIV activity and that ritonavir-resistant HIV is hypersensitive to the drug. These results suggest a possible role for HSP90AB1 in postentry HIV replication and may provide an attractive target for therapeutic intervention.