Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.     

Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle.

In this study, we have analyzed the effect of human alpha interferon (IFN-alpha) on a single replication cycle of human immunodeficiency virus type 1 (HIV-1) infection in the lymphocytic cell line CEM-174, which is highly sensitive to the antiviral effects of IFN. Pretreatment of cells with 50 to 500 U of recombinant human IFN-alpha per ml resulted in a marked reduction in viral RNA and protein synthesis. The effect of IFN-alpha was dose dependent and was amplified in multiple infection cycles. IFN-induced inhibition of viral protein synthesis could be detected only when cells were treated with IFN-alpha prior to infection or when IFN-alpha was added up to 10 h postinfection, but not if IFN-alpha was added at the later stages of HIV-1 replication cycle or after the HIV-1 infection was already established. Analysis of the integrated HIV-1 provirus showed a marked decrease in the levels of proviral DNA in IFN-treated cells. Thus, in contrast to the previous studies on established HIV-1 infection in T cells, in which the IFN block appeared to be at the posttranslational level, during de novo infection, IFN-alpha interferes with an early step of HIV-1 replication cycle that occurs prior to the integration of the proviral DNA. These results indicate that the early IFN block of HIV-1 replication, which has been previously observed only in primary marcophages, can also be detected in the IFN-sensitive T cells, indicating that the early IFN block is not limited to macrophages.     

Triterpene derivatives that inhibit human immunodeficiency virus type 1 replication

Triterpene derivatives were analyzed for anti-HIV-1 activity and for cellular toxicity. Betulinic aldehyde, betulinic nitrile, and morolic acid derivatives were identified to have anti-HIV-1 activity. These derivatives inhibit a late step in virus replication, likely virus maturation.     

Opposite effects of SDF-1 on human immunodeficiency virus type 1 replication

The alpha-chemokine SDF-1 binds CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1), and inhibits viral entry mediated by this receptor. Since chemokines are potent chemoattractants and activators of leukocytes, we examined whether the stimulation of HIV target cells by SDF-1 affects the replication of virus with different tropisms. We observed that SDF-1 inhibited the entry of X4 strains and increased the infectivity of particles bearing either a CCR5-tropic HIV-1 envelope or a vesicular stomatitis virus G envelope. In contrast to the inhibitory effect of SDF-1 on X4 strains, which is at the level of entry, the stimulatory effect does not involve envelope-receptor interactions or proviral DNA synthesis. Rather, we observed an increased ability of Tat to transactivate the HIV-1 long terminal repeat in the presence of the chemokine. Therefore, the effects of SDF-1 on the HIV-1 life cycle can be multiple and opposite, including both an inhibition of viral entry and a stimulation of proviral gene expression.     

Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection.

The matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein contains a highly basic region near its amino terminus. It has been proposed that this basic domain, in conjunction with the HIV-1 accessory protein Vpr, is responsible for the localization of the HIV-1 preintegration complex to the nucleus in nondividing cells. It has also been postulated that the matrix basic domain assists in the targeting of the HIV-1 Gag precursor Pr55Gag to the plasma membrane during virus assembly. To evaluate the role of this highly basic sequence during infection of primary human monocyte-derived macrophages, single- and double-amino-acid-substitution mutations were introduced, and the effects on virus particle production, Gag protein processing, envelope glycoprotein incorporation into virus particles, and virus infectivity in the CEM(12D-7) T-cell line, peripheral blood mononuclear cells, and primary human monocyte-derived macrophages were analyzed. Although modest effects on virus particle production were observed with some of the mutants, none abolished infectivity in primary human monocyte-derived macrophages. In contrast with previously reported studies involving some of the same matrix basic domain mutants, infectivity in monocyte-derived macrophages was retained even when combined with a vpr mutation.     

TAR-independent replication of human immunodeficiency virus type 1 in glial cells.

The molecular mechanisms involved in the replication of human immunodeficiency virus type 1 (HIV-1) may differ in various cell types and with various exogenous stimuli. Astrocytic glial cells, which can support HIV-1 replication in cell cultures and may be infected in vivo, are demonstrated to provide a cellular milieu in which TAR mutant HIV-1 viruses may replicate. Using transfections of various TAR mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated astrocytic cells. We further demonstrate, using viral constructs with mutations in the tat gene and in the nuclear factor kappa B (NF-kappa B)-binding sites (enhancer) of the HIV-1 long terminal repeat, that TAR-independent HIV-1 replication in astrocytic cells requires both intact NF-kappa B moiety-binding motifs in the HIV-1 long terminal repeat and Tat expression. We measured HIV-1 p24 antigen production, syncytium formation, and levels and patterns of viral RNA expression by Northern (RNA) blotting to characterize TAR-independent HIV-1 expression in astrocytic glial cells. This alternative regulatory pathway of TAR-independent, Tat-responsive viral production may be important in certain cell types for therapies which seek to perturb Tat-TAR binding as a strategy to interrupt the viral lytic cycle.     

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.     

In vitro suppression of human immunodeficiency virus type 1 replication by measles virus

During the acute phase of measles, human immunodeficiency virus type 1 (HIV-1)-infected children have a transient, but dramatic, decrease in plasma HIV-1 RNA levels (W. J. Moss, J. J. Ryon, M. Monze, F. Cutts, T. C. Quinn, and D. E. Griffin, J. Infect. Dis. 185:1035-1042, 2002). To determine the mechanism(s) by which coinfection with measles virus (MV) decreases HIV-1 replication, we established an in vitro culture system that reproduces this effect. The addition of MV to CCR5- or CXCR4-tropic HIV-1-infected human peripheral blood mononuclear cells (PBMCs) decreased HIV-1 p24 antigen production in a dose-dependent manner. This decrease occurred with the addition of MV before or after HIV-1. The inhibition of HIV-1 p24 antigen production was decreased when UV-inactivated MV or virus-free supernatant fluid from MV-infected PBMCs was used. Inhibition was not due to increased production of chemokines known to block coreceptor usage by HIV-1, a decrease in the percentage of CD4+ T cells, or a decrease in chemokine receptor expression by CD4+ T cells. Viability of PBMCs was decreased only 10 to 20% by MV coinfection; however, lymphocyte proliferation was decreased by 60 to 90% and correlated with decreased production of p24 antigen. These studies showed that an in vitro system of coinfected PBMCs could be used to dissect the mechanism(s) by which MV suppresses HIV-1 replication in coinfected children and suggest that inhibition of lymphocyte proliferation by MV may play a role in the suppression of HIV-1 p24 antigen production.     

Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 has been shown to affect the infectivity but not the production of virus particles. In this study, the effect of vif in the context of the HXB2 virus on virus replication in several CD4+ T-cell lines was investigated. vif was found to be required for replication in the CD4+ T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes. vif was not required for replication in the SupT1, C8166, and Jurkat T-cell lines. The infectivity of vif-defective viruses depended on the cell type in which the virus was produced. In CEM cells, vif was required for production of virus capable of initiating infection in all cell lines studied. vif-defective virus produced by SupT1, C8166, and Jurkat cells and the monkey cell line COS-1 could initiate infection in multiple cell lines, including CEM and H9. These results suggest that vif can compensate for cellular factors required for production of infectious virus particles that are present in some cell lines such as SupT1, C8166, and Jurkat but are absent in others such as CEM and H9 as well as peripheral blood T lymphocytes. The effect of vif was not altered by deletion of the carboxyl terminus of gp41, a proposed target for vif (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). These studies demonstrate that vif enhances viral infectivity during virus production and also suggest that vif is likely to be important for natural infections.     

Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-O-[3',3'-dimethylsuccinyl]-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.     

Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.