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.

Degradation of HIV-1 integrase by the N-end rule pathway.

Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA. We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins. Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells.

Enhancement of human immunodeficiency virus type 1 infection by the CC-chemokine RANTES is independent of the mechanism of virus-cell fusion.

We have studied the effects of CC-chemokines on human immunodeficiency virus type 1 (HIV-1) infection, focusing on the infectivity enhancement caused by RANTES. High RANTES concentrations increase the infectivity of HIV-1 isolates that use CXC-chemokine receptor 4 for entry. However, RANTES can have a similar enhancing effect on macrophagetropic viruses that enter via CC-chemokine receptor 5 (CCR5), despite binding to the same receptor as the virus.

Inhibition of influenza virus hemagglutinin-mediated membrane fusion by a compound related to podocarpic acid.

Entry of influenza virus into the host cell is dependent on the fusion of the viral envelope with the endosomal membrane and is mediated by a low-pH-induced change of the viral hemagglutinin (HA) to a conformation that is fusogenic. A compound related to podocarpic acid (180299) was identified that inhibits multicycle replication of influenza A/Kawasaki/86 (H1N1) virus in culture. Treatment of Madin-Darby canine kidney (MDCK) cells with 180299 at 1 h before infection resulted in the inhibition of viral protein synthesis.

Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease.

Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively.

Complementation of integrase function in HIV-1 virions.

Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses.

Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs.

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent.

Potent human immunodeficiency virus type 1 protease inhibitors that utilize noncoded D-amino acids as P2/P3 ligands.

Noncoded D-amino acids have been designed to replace the quinaldic amide-asparaginyl moiety (P2/P3 ligand) found in several potent human immunodeficiency virus (HIV) protease inhibitors such as LY289612. The substituted nitrogen, optimally an N-methanesulfonyl moiety, served as a CH2CONH2 (asparagine side chain mimic), while the amino acid side chain became the backbone and P3 ligand of these novel inhibitors. Compounds derived from S-aryl-D-cysteine proved to be potent HIV protease inhibitors which also exhibited potent whole cell antiviral activity.

Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene.

We have created a temperature-sensitive (ts) mutant of human immunodeficiency virus type 1, using the technique of charge-cluster-to-alanine scanning mutagenesis to introduce specific changes into the integrase coding region. In the ts mutant virus, the lysine at amino acid 136 and the glutamic acid at amino acid 138 of integrase have been replaced with alanines (K136A/E138A). When K136A/E138A is synthesized at 35 degrees C, it replicates to a similar degree as wild-type virus during infection of CEM cells at 35 degrees C on the basis of syncytium formation, levels of core antigen, and reverse transcriptase activity.

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells.

Integrase is the only viral protein necessary for integration of retroviral DNA into chromosomal DNA of the host cell. Biochemical analysis of human immunodeficiency virus type 1 (HIV-1) integrase with purified protein and synthetic DNA substrates has revealed extensive information regarding the mechanism of action of the enzyme, as well as identification of critical residues and functional domains. Since in vitro reactions are carried out in the absence of other viral proteins and they analyze strand transfer of only one end of the donor substrate, they do not define completely the process of integration as it occurs during the course of viral infection.

The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1.

The recently described human immunodeficiency virus type 2 (HIV2) is significantly divergent in sequence from the more frequently isolated human immunodeficiency virus type 1 (HIV1). Both HIV1 and HIV2 encode a transactivator that is capable of strongly stimulating expression directed by the viral long terminal repeat (LTR). Here, we define the region of the HIV2 genome encoding the transactivator and show that the specificity of the transactivator differs from that of HIV1.