Hiv 1 Gag And Env Proteins In Virus Assembly And Infecti

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Abstract

The viral Gag proteins control many aspects of the HIV-1 replication cycle. The Gag precursor drives the assembly of virus particles in the infected cell, and, through putative interactions with the transmembrane envelope (Env) glycoprotein gp41, directs Env incorporation into virus particles. Following infection, the Gag proteins play a central role in uncoating and assist in the reverse transcription process. The assembly of type C retroviruses and lentiviruses, including HIV-1, takes place at the plasma membrane (PM). It is well established that this process is promoted by Gag proteins; however, the mechanisms by which assembly is specifically targeted to the PM remain to be determined. Recent studies have suggested that the PM contains microdomains with distinct protein and lipid compositions. One type of microdomain, the lipid raft, is enriched in sphingolipids and cholesterol and can be isolated as detergent-resistant membrane (DRM). Rafts have been shown to play essential roles in a variety of biological processes, often by acting as a target site for proteins involved in a common pathway. Last year, we reported that a large portion of membrane-bound Gag was recovered in DRM (Ono and Freed, PNAS, 2001). To address the specificity of Gag-raft association, we examined the kinetics of this association. We found that DRM association of Gag occurred more slowly than binding of Gag to membranes, suggesting that HIV-1 Gag is specifically targeted to PM rafts after it binds membrane. We observed that the determinant for raft association maps to the N-terminus of Gag, but that Gag-Gag interactions facilitate or stabilize raft association. To address the physiological relevance of Gag-raft association, we analyzed the impact of cholesterol depletion, which disrupts raft structure, in HIV-1-producing cells. We found that cholesterol depletion markedly and specifically reduces virus release. Moreover, virus released from cholesterol-depleted cells displays reduced infectivity. Interestingly, cells expressing a mutant Gag with substitutions in the p6 late domain motif show no reduction in virus release. These results identify the association of Gag with PM rafts as an important step in HIV-1 production, and suggest possible links between rafts and late steps in virus assembly. Recent work has been aimed at defining the mechanism by which raft disruption impairs virus production. These results also suggest that cholesterol depletion prevents the efficient binding of Gag to membrane. The p6 domain of HIV-1 is located at the C-terminus of the Gag precursor protein Pr55Gag. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. We performed a detailed mutational analysis of the N-terminus of p6 to define sequences required for efficient virus release. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we determined the role of p6 in particle release in a panel of cell lines as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). The results (Demirov et al., J. Virol. 2002) indicate that: i) the highly conserved P-T/S-A-P motif located near the N-terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence induce profound virus-release defects in HeLa cells. ii) Single and double amino acid substitutions outside the P-T/S-A-P motif have no significant effect on particle release. iii) The introduction of stop codons one or two residues beyond the P-T-/S-A-P motif blocks virion release, whereas truncation four residues beyond P-T/S-A-P has no effect on particle production in HeLa cells. iv) Though the effects of p6 mutation on virus replication in T-cell lines are cell-type dependent, even in T-cell lines in which p6 mutations block virus replication, these changes have little or no effect on particle release. v) p6-mutant particles produced in T-cell lines exhibit a defect in virion-virion detachment, resulting in the production of tethered chains of virions. vi) Transient heterokaryons produced between HeLa cells and a T-cell line display a T cell-like phenotype with respect to the requirement for p6 in particle release, indicating that the activity that suppresses the release defect phenotype is dominant in this cell system. vii) Finally, in primary MDM, mutation of p6 results in marked defects in both particle release and virus replication. These results reveal a strong cell-type dependent requirement for p6 in virus particle release. A variety of lines of evidence suggest a connection between retroviral L domains function and the host ubiquitination machinery. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N-terminus a domain highly related to ubiquitin conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle production. Intriguingly, we observe that this domain (referred to as TSG-5') potently inhibits virus production (Demirov et al., PNAS 2002). Examination of cells coexpressing HIV-1 Gag and TSG-5' by electron microscopy reveals a defect in virus budding very reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5' is dependent upon an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5'. Furthermore, assembly and release of murine leukemia virus and Mason-Pfizer monkey virus are insensitive to TSG-5'. TSG-5' is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5' incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and raise the possibility that TSG101 derivatives could serve as potent and specific inhibitors of HIV-1 replication by blocking virus budding. To define the mechanism by which TSG-5' inhibits virus release, we investigated the importance of TSG-5'/Gag interaction in the virus release inhibition. We observed that a mutation in TSG-5' that prevents Gag from interacting with this truncated protein eliminates its ability to disrupt particle release. These results suggest that direct binding to Gag, rather than disruption of cellular sorting machinery, is responsible for the ability of TSG-5' to block budding. We also investigated the effect of overexpressing longer forms of TSG101, including the full-length protein, on virus budding. We observe that overexpressing other truncated mutants as well as the full-length protein, disrupt budding. Interestingly, the ability of full-legth TSG101 to inhibit virus release when overexpressed does not require binding to Gag. Rather, as indicated by confocal microscopy experiments, overexpressing the full-length protein intereferes with the cellular endosomal sorting machinery. To complement our studies on HIV-1, we are investigating the role that TSG101 and other host factors play in the budding of a diverse array of retroviruses, including murine leukemia virus, human T cell leukemia virus, bovine leukosis virus, Mason-Pfizer monkey virus, and equine infectious anemia virus.