Virions of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses contain conical cores consisting of a protein shell composed of the viral capsid protein (CA) surrounding an internal viral ribonucleoprotein complex. Although genetic studies have implicated CA in both early and late stages of the virus replication cycle, the mechanism of core disassembly following penetration of target cells remains undefined. Using quantitative assays for analyzing HIV-1 core stability in vitro, we identified point mutations in CA that either reduce or increase the stability of the HIV-1 core without impairing conical core formation in virions. Alterations in core stability resulted in severely attenuated HIV-1 replication and impaired reverse transcription in target cells with only minimal effects on viral DNA synthesis in permeabilized virions in vitro. We conclude that formation of a viral core of optimal stability is a prerequisite for efficient HIV-1 infection and suggest that disassembly of the HIV-1 core is a regulated step in infection that may be an attractive target for pharmacologic intervention

The human immunodeficiency virus type 1 (HIV-1) vif gene encodes a 23-kDa protein of unknown function, also produced by most other known lentiviruses. Vif was found to be essential for the spread of HIV-1 in peripheral blood lymphocytes and in primary macrophages, as well as in some but not all established T-cell lines. Vifwas required at the stage of viral particle formation, for cell-to-cell as well as for cell-free transmission of HIV-1. Accordingly, vif-defective viruses could be complemented by the expression of vifin the producer but not in the target cell. vif-defective virions contained wild-type amounts of Gag and Env proteins, reverse transcriptase, integrase, genomic RNA, and partial reverse transcripts. Most importantly, they could enter cells normally, and the vif defect could not be rescued through the use of HIV(MLV [murine leukemia virus]) pseudotypes. Instead, vif-mutant viruses were severely impaired in their ability to complete the synthesis of proviral DNA, once internalized in the target cell. These results suggest that Vif plays a role which is novel for a retroviral protein, in allowing the processing and/or the transport of the internalized HIV core.

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6Gag bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101-and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.The structural proteins of many enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1) Gag, contain short cis-acting sequence motifs, termed "late domains," that recruit host factors to facilitate virus budding (5,29,35,59). To date, three well-characterized late-domain sequences and their cellular binding partners have been described: PT(S)AP late domains bind TSG101 (6, 12, 31, 57), YPXL late domains bind ALIX/AIP1 (51) (as well as the AP-2 adaptor complex [2]), and PPXY late domains bind various members of the large family of mammalian NEDD4 ubiquitin E3 ligases (hereafter called NEDD4-like proteins; reviewed in references 22, 29, and 46). Each of the late domain binding partners can be linked to the cellular ESCRT (endosomal sorting complex required for transport) pathway, indicating that this pathway functions in virus release.Within the cell, the ESCRT pathway also plays important roles in helping to sort membrane proteins into vesicles that bud into late endosomes/multivesicular bodies (MVB) (21, 38, 61). These vesicles carry membrane proteins from the limiting endosomal membrane into the endosomal lumen, where their contents can ultimately be degraded when MVBs fuse with lysosomes. The ESCRT pathway can also be recruited to function in the abscission stage of cytokinesis (1), a process in which the thin membranous midbody that connects two dividing cells is severed to complete the process of cell division. In addition to their shared dependence on the ESCRT pathway, the processes of MVB vesicle formation, abscission, and virus budding are similar in that the membrane fission events required for ve...

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual among retroviruses in their ability to infect nondividing cells. The matrix proteins of several lentiviruses contain a short stretch of amino acids reminiscent of known nuclear localization signals. In HIV-1, this motif has been shown to function as a nuclear targeting sequence when conjugated to a heterologous protein, and to permit the active nuclear import of the HIV-1 preintegration complex in growth-arrested cells. In the present work, mutations were introduced in the matrix nuclear localizaion region of T-cell-and macrophage-tropic HIV-1 clones. The resulting viral mutants replicated with normal or even accelerated kinetics in dividing cells, including activated peripheral blood lymphocytes. However, in sharp contrast with wild-type virus, the mutants could not grow efficiently in terminally differentiated macrophages or establish a stable and inducible infection intermediate in unstimulated peripheral blood lymphocytes.Because macrophages represent a major viral reservoir in vivo, and because at any given time most T cells in the body are quiescent, these results strongly suggest that the karyophilic properties ofthe matrix protein are critical for the spread ofthe virus in HIV-infected individuals, and consequently for AIDS pathogenesis.

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.