Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

BACKGROUND: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates. METHODS: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing. RESULTS: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells. CONCLUSIONS: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.

HIV type 1 integrase inhibitors: from basic research to clinical implications.

Similar to other retroviruses, productive infection with HIV-1 requires three key steps in the viral replication: (i) reverse transcription of viral genomic RNA into viral cDNA by the viral reverse transcriptase; (ii) integration of viral cDNA into host cell genome using the viral integrase; and (iii) cleavage of newly synthesized viral polypeptide by the viral protease into individual viral proteins during new virion assembly. Following their discovery, all three viral enzymes were considered as targets for antiretroviral drugs. However, while multiple reverse transcriptase and protease inhibitors have been used for more than 12 years to treat HIV-infected individuals, only recently has the viral integrase enzyme emerged as an alternative, clinically validated target to block HIV-1 replication. Here we review the biology of HIV-1 integration, the mechanisms of action and development of resistance to integrase inhibitors, and the latest data on the most recent clinical trials involving this promising, novel class of antiretroviral drugs.

Identification of low-molecular weight inhibitors of HIV-1 reverse transcriptase using a cell-based high-throughput screening system.

A cell-based drug screening system that utilizes a green fluorescent protein (GFP)-tagged recombinant lentiviral vector has been used to screen a chemical library of 34,000 small molecules for antiretroviral compounds. Thirty-three initial hits were analyzed and four compounds were selected based on their anti-human immunodeficiency virus type 1 (HIV-1) activity (EC(50) values ranging from 0.17 to 1.9 µM) and low cellular toxicity (CC(50) values >50 µM). The four compounds blocked reverse transcription and were able to inhibit the replication of a panel of different HIV-1 strains, including non-B subtype and viruses resistant to different drug classes. Serial in vitro passages of HIV-1(B-HXB2) in the presence of increasing drug concentrations selected for viruses with reduced susceptibility. Mutations previously associated with resistance to non-nucleoside reverse transcriptase (RT) inhibitors (L100I and Y181C for CBL-17 and CBL-21, respectively) or linked to nucleoside analogue resistance (A62V for CBL-4.0 and CBL-4.1) were identified. Viruses with reduced susceptibility to CBL-17 and CBL-21 but not the ones resistant to CBL-4.0 or CBL-4.1 showed a decrease in replicative fitness. Interestingly, two of the small molecules (CBL-4.0 and CBL-4.1) are indolopyridinones that were previously described as nucleotide-competing RT inhibitors.