Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.

We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an “encyclopedia” of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3 end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metaldependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg 2؉ . The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.A n essential step in HIV replication is the integration of the reverse-transcribed viral genome into host chromosomal DNA by the virally encoded integrase (IN) protein (1-3). Integration is required for efficient long terminal repeat-driven transcription of the provirus for the production of viral proteins and RNA progeny. IN represents an important chemotherapeutic target, as its inactivation, either by mutagenesis or inhibition, blocks productive infection by HIV-1 (4-7).Integration is carried out in the cell in a series of distinct steps (8-10). First, IN cleaves the two terminal nucleotides from each 3Ј end of the viral DNA. The 3Ј processing reaction is carried out concurrently with or soon after reverse transcription in the cytoplasm. In the second step, strand transfer, IN catalyzes staggered nicking of the target chromosomal DNA and joining of each 3Ј end of the viral DNA to the 5Ј ends of the host DNA. Strand transfer is temporally and spatially separated from 3Ј processing and occurs after transport of the preintegration complex from the cytoplasm into the nucleus.Divalent metals such as Mg 2ϩ or Mn 2ϩ are required for both 3Ј processing and strand transfer and for the assembly of IN onto specific viral donor DNA to form a complex competent to carry out either function (11)(12)(13) (18), L-731,988 and related DKAs inhibit integration and viral replication in cell culture (7). Mutations that confer resistance to DKAs have been identified and map to IN residues adjacent to D64 and E152. The in...

Histone deacetylases (HDACs) act on histones within the nucleosome-bound promoter of human immunodeficiency virus type 1 (HIV-1) to maintain proviral latency. HDAC inhibition leads to promoter expression and the escape of HIV from latency. We evaluated the ability of the potent inhibitor recently licensed for use in oncology, suberoylanilide hydroxamic acid (SAHA; Vorinostat), selective for Class I HDACs, to induce HIV promoter expression in cell lines and virus production from the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients. In J89, a Jurkat T cell line infected with a single HIV genome encoding the enhanced green fluorescence protein (EGFP) within the HIV genome, SAHA induced changes at nucleosome 1 of the HIV promoter in chromatin immunoprecipitation (ChIP) assays in concert with EGFP expression. In the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients clinically achievable exposures to SAHA induced virus outgrowth ex vivo. These results suggest that potent, selective HDAC inhibitors may allow improved targeting of persistent proviral HIV infection, and define parameters for in vivo studies using SAHA.