Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains

Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4(+) cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection.     

Expression of the c-myc proto-oncogene is essential for HIV-1 infection in activated T cells

We previously found that activation of primary CD4+ T cells via both the T cell antigen receptor (TCR) and CD28 is required for HIV-1 DNA to be translocated from the cytoplasm to the nucleus. Here we report that expression of c-Myc protein in CD4+ T cells is induced only after such costimulation. In addition, cyclosporin A not only inhibits nuclear import of HIV-1 DNA but also inhibits expression of c-Myc protein. Because of these correlations, we tested whether c-Myc is necessary for nuclear import of HIV-1 DNA. Specific c-myc antisense, but not sense or non-sense, phosphorothioate oligodeoxynucleotides selectively induced the accumulation of two NH2-terminally truncated c-Myc proteins and abolished HIV-1 genome entry into host nuclei. Consequently, both virus replication and HIV-1-induced apoptotic cell death were inhibited. Synthesis of viral full-length DNA was not affected. Specific c-myc antisense oligonucleotide inhibited HIV-1 infection under conditions that did not affect cell cycle entry or proliferation. Thus, c-Myc appears to regulate HIV-1 DNA nuclear import via a mechanism distinct from those controlling entry into the cell cycle.     

Statins inhibit HIV-1 infection by down-regulating Rho activity

Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft-associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We show that statins decreased viral load and increased CD4+ cell counts in acute infection models and in chronically HIV-1-infected patients. Viral entry and exit was reduced in statin-treated cells, and inhibition was blocked by the addition of l-mevalonate or of geranylgeranylpyrophosphate, but not by cholesterol. Cell treatment with a geranylgeranyl transferase inhibitor, but not a farnesyl transferase inhibitor, specifically inhibited entry of HIV-1-pseudotyped viruses. Statins blocked Rho-A activation induced by HIV-1 binding to target cells, and expression of the dominant negative mutant RhoN19 inhibited HIV-1 envelope fusion with target cell membranes, reducing cell infection rates. We suggest that statins have direct anti-HIV-1 effects by targeting Rho.     

HIV-1 replication cycle

The HIV-1 is a formidable pathogen with establishment of a persistent infection based on the ability to integrate the proviral genome into chronically infected cells, and by the rapid evolution made possible by a high mutation rate and frequent recombination during the viral replication. HIV-1 has a variety of novel genes that facilitate viral persistence and regulation of HIV replication, but this virus also usurps cellular machinery for HIV replication, particularly during gene expression and virion assembly and budding. Recent success with antiretroviral therapy may be limited by the emergence HIV drug resistance and by toxicities and other requirements for successful long-term therapy. Further investigation of HIV-1 replication may allow identification of novel targets of antiretroviral therapy that may allow continued virus suppression in patients of failing current regiments, particularly drugs that target HIV-1 entry and HIV-1 integration.     

HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse

Direct cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4(+)/CXCR4(+) target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.     

Blockade of HIV-1 infection of New World monkey cells occurs primarily at the stage of virus entry

HIV-1 naturally infects chimpanzees and humans, but does not infect Old World monkeys because of replication blocks that occur after virus entry into the cell. To understand the species-specific restrictions operating on HIV-1 infection, the ability of HIV-1 to infect the cells of New World monkeys was examined. Primary cells derived from common marmosets and squirrel monkeys support every phase of HIV-1 replication with the exception of virus entry. Efficient HIV-1 entry typically requires binding of the viral envelope glycoproteins and host cell receptors, CD4 and either CCR5 or CXCR4 chemokine receptors. HIV-1 did not detectably bind or utilize squirrel monkey CD4 for entry, and marmoset CD4 was also very inefficient compared with human CD4. A marmoset CD4 variant, in which residues 48 and 59 were altered to the amino acids found in human CD4, supported HIV-1 entry efficiently. The CXCR4 molecules of both marmosets and squirrel monkeys supported HIV-1 infection, but the CCR5 proteins of both species were only marginally functional. These results demonstrate that the CD4 and CCR5 proteins of New World monkeys represent the major restriction against HIV-1 replication in these primates. Directed adaptation of the HIV-1 envelope glycoproteins to common marmoset receptors might allow the development of New World monkey models of HIV-1 infection.