HIV-host interactome revealed directly from infected cells.

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection.

Mass spectrometry-based proteomic approaches for discovery of HIV-host interactions.

A molecular understanding of viral infection requires a multi-disciplinary approach. Mass spectrometry has emerged as an indispensable tool to investigate the complex and dynamic interactions between HIV-1 and its host. It has been employed to study protein associations, changes in protein abundance and post-translational modifications occurring after viral infection.

Suppression of Foxo1 activity and down-modulation of CD62L (L-selectin) in HIV-1 infected resting CD4 T cells.

HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection in vitro, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state.

HIVToolbox, an integrated web application for investigating HIV.

Many bioinformatic databases and applications focus on a limited domain of knowledge federating links to information in other databases. This segregated data structure likely limits our ability to investigate and understand complex biological systems. To facilitate research, therefore, we have built HIVToolbox, which integrates much of the knowledge about HIV proteins and allows virologists and structural biologists to access sequence, structure, and functional relationships in an intuitive web application.

Sequential deletion of the integrase (Gag-Pol) carboxyl terminus reveals distinct phenotypic classes of defective HIV-1.

A requisite step in the life cycle of human immunodeficiency virus type 1 (HIV-1) is the insertion of the viral genome into that of the host cell, a process catalyzed by the 288-amino-acid (32-kDa) viral integrase (IN). IN recognizes and cleaves the ends of reverse-transcribed viral DNA and directs its insertion into the chromosomal DNA of the target cell. IN function, however, is not limited to integration, as the protein is required for other aspects of viral replication, including assembly, virion maturation, and reverse transcription.

Prospective strategies for targeting HIV-1 integrase function.

Integration is a key step in the HIV-1 life cycle in which the ends of linear viral DNA are covalently joined with host chromosomal DNA. Integrase is the highly conserved and essential viral protein that performs two catalytically related reactions that ultimately lead to the insertion of the viral genome into that of the host cell. The only chemotherapeutic agents against integrase currently available for HIV-1 infected individuals are those that interrupt strand transfer, the second step of catalysis.

Natural polymorphisms of human immunodeficiency virus type 1 integrase and inherent susceptibilities to a panel of integrase inhibitors.

We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drug-resistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance.

The role of lysine 186 in HIV-1 integrase multimerization.

HIV-1 integrase (IN) catalyzes biochemical reactions required for viral cDNA insertion into host cell chromosomal DNA, an essential step in the HIV-1 replication cycle. In one of these reactions, the two ends of the linear viral cDNA are believed to be simultaneously ligated to chromosomal DNA by a tetrameric form of IN. The structure of the full-length IN tetramer is not known but a model consisting of the N-terminal domain and the catalytic core revealed basic residues 186 to 188 at the interface between the two IN dimers.

Posttranslational acetylation of the human immunodeficiency virus type 1 integrase carboxyl-terminal domain is dispensable for viral replication.

A recent report sought to demonstrate that acetylation of specific lysines within integrase (IN) by the histone acetyltransferase (HAT) p300 regulates human immunodeficiency virus type 1 (HIV-1) integration and is essential for viral replication (A. Cereseto, L. Manganaro, M.

Characterization of HIV-1 integrase N-terminal mutant viruses.

During infection, human immunodeficiency virus type 1 integrase engages a number of molecules and mechanisms, both of viral and cellular origin. In one of such instances, integrase is thought to be degraded by the N-end rule proteasome pathway a process that targets the N-terminal residue of its substrates. Here we describe the properties of HIV-1 viruses in which the first amino acid residue of integrase has been substituted to render it resistant to the N-end rule pathway.

Effect of DNA repair protein Rad18 on viral infection.

Host factors belonging to the DNA repair machineries are assumed to aid retroviruses in the obligatory step of integration. Here we describe the effect of DNA repair molecule Rad18, a component of the post-replication repair pathway, on viral infection. Contrary to our expectations, cells lacking Rad18 were consistently more permissive to viral transduction as compared to Rad18(+/+) controls.

A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons.

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins.

Shifts in cell-associated HIV-1 RNA but not in episomal HIV-1 DNA correlate with new cycles of HIV-1 infection in vivo.

The significance of distinct classes of HIV-1 nucleic acids as correlates of recent HIV-1 replication was assessed in peripheral blood mononuclear cells (PBMC) obtained from 14 patients during 2 weeks of structured interruption of antiretroviral therapy (STI) and 2 weeks of resuming therapy. Levels of HIV RNA in plasma (HIV-RNAplasma) and of unspliced cell-associated HIV-1 RNA (HIV-UsRNAPBMC) were significantly increased as a result of STI, whereas no significant shifts in the levels of 2-LTR episomal HIV-1 DNA (2-LTR circles) and total late HIV-1 reverse transcripts (late-DNA) were observed. Thus, limited viral replication had occurred, which had no effect on the pool size of infected cells in the periphery.

Cellular inhibitors with Fv1-like activity restrict human and simian immunodeficiency virus tropism.

Many nonhuman primate cells are unable to support the replication of HIV-1, whereas others are nonpermissive for infection by simian immunodeficiency virus from macaques (SIVmac). Here, we show that restricted HIV-1 and SIVmac infection of primate cell lines shares some salient features with Fv1 and Ref1-mediated restriction of murine retrovirus infection. In particular, the nonpermissive phenotype is most evident at low multiplicities of infection, results in reduced accumulation of reverse transcription products, and is dominant in heterokaryons generated by fusion of permissive and nonpermissive target cells.

Interaction of HIV-1 integrase with DNA repair protein hRad18.

We have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase.