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VIRIP has been identified as a highly specific natural inhibitor of HIV-1 that blocks HIV-1 gp41-dependent fusion by interacting with the gp41 fusion peptide. Two analogues of VIRIP (VIR-353 and VIR-576) with a few amino acid changes increase its antiretroviral potency by two orders of magnitude in cell culture. VIR-576 has been shown effective in a phase I/II clinical trial. Resistance to VIRIP and its analogue VIR-353 were generated after long-term passage in cell culture suggesting a high genetic barrier to resistance. Mutations conferring resistance to VIRIP and VIR-353 significantly reduced virus fitness. However, accumulation of additional mutations rescued the replication capacity of the virus while retaining resistance to VIR-353 and full sensitivity to T20. Combinations of VIR-353 and T20 had an additive effect on the inhibition of wild type HIV-1 replication, but only a single agent was active when combinations were tested against T20-resistant HIV-1, suggesting that both gp41 peptides do not interfere with each other in their binding to gp41. Our results provide additional support to the development of a new class of antiretroviral agents targeting gp41-dependent fusion., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A transmembrane glycoprotein of HIV-1, gp41, plays a central role in membrane fusion of HIV-1 and host cells. Peptides derived from the amino- and carboxyl-terminal heptad repeat (N-HR and C-HR, respectively) of gp41 inhibit this fusion. The mechanism of resistance to enfuvirtide, a C-HR-derived peptide, is well defined; however the mechanism of resistance to N-HR-derived peptides remains unclear. We characterized an HIV-1 isolate resistant to the N-HR-derived peptide, N36. This HIV-1 acquired a total of four amino acid substitutions, D36G, N126K and E137Q in gp41, and P183Q in gp120. Among these substitutions, N126K and/or E137Q conferred resistance to not only N36, but also C34, which is the corresponding C-HR-derived peptide fusion inhibitor. We performed crystallographic and biochemical analysis of the 6-helix bundle formed by synthetic gp41-derived peptides containing the N126K/E137Q substitutions. The structure of the 6-helix bundle with N126K/E137Q was identical to that in wild-type HIV-1 except for the presence of a new hydrogen bond. Denaturing experiments revealed that the stability of the 6-helix bundle of N126K/E137Q is greater than in the wild-type. These results suggest that the stabilizing effect of N126K/E137Q provides resistance to N36 and C34., (Copyright 2010 Elsevier B.V. All rights reserved.)

Mutations that are selected at low frequency and/or reside outside the enfuvirtide target region, amino acid 36-45 of gp41, might still be important determinants for drug resistance. This study aimed to investigate the phenotypic impact against enfuvirtide and sifuvirtide of uncharacterized gp41 mutations 42G, 43T and 50V, selected in patients failing enfuvirtide-containing regimens. As single mutations, neither 42G, 43T nor 50V conferred resistance to enfuvirtide. However, 50V increased slightly resistance levels for 36D, 38M, 43D or 43T as did 43T for 38M. All mutants displayed a reduced replication capacity, except 42S, 50V and 36D+/-50V. None of the mutants displayed resistance to the next-generation fusion inhibitor sifuvirtide. This study highlights the necessity to confirm the in vitro effect of infrequently selected mutations as 42G was not associated with enfuvirtide resistance whereas 43T and 50V should be considered as secondary enfuvirtide resistance mutations., (Copyright 2010 Elsevier B.V. All rights reserved.)

Infection of target cells by HIV is a complex, multi-stage process involving attachment to host cells and CD4 binding, coreceptor binding, and membrane fusion. Drugs that block HIV entry are collectively known as entry inhibitors, but comprise a complex group of drugs with multiple mechanisms of action depending on the stage of the entry process at which they act. Two entry inhibitors, maraviroc and enfuvirtide, have been approved for the treatment of HIV-1 infection, and a number of agents are in development. This review covers the entry inhibitors and their use in the management of HIV-1 infection. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010., (Copyright 2009 Elsevier B.V. All rights reserved.)

Primary mutations in HIV-1 that are directly involved in the resistance to enfuvirtide have been well documented. However, secondary mutations that are associated with primary mutations and contribute little to the resistance still remain to be elucidated. This study reveals that synonymous mutations at gp41 Q41 (CAG to CAA) or L44 (UUG to CUG) act as secondary mutations. Complementary mutations in the nucleotide level are located in the Rev responsive element (RRE) of the HIV-1 RNA-genome and maintain the replication kinetics of HIV-1 through increasing the structural stability of stem-loop III in the RRE. Therefore, synonymous mutations in the gp41/RRE sequence improve the viral replication impaired by the primary mutations and play a key role as secondary (complementary) mutations.

Several polyanionic compounds with potential for use as topically applied microbicides to prevent HIV-1 sexual transmission, such as PRO 2000, are currently in phase III clinical efficacy trials. Microbicidal formulations may well comprise combinations of inhibitors to increase potency, reduce dose and minimize problems of HIV-1 resistance. We have therefore evaluated in vitro, the anti-HIV-1 activity of two leading polyanionic microbicides combined with other antiretroviral agents with microbicidal potential. Dextran sulfate (DS) and PRO 2000 were combined with the neutralizing antibody IgG1b12, the peptide-based fusion inhibitor T20, the CCR5 antagonist TAK779 and the cyanobacterial protein cyanovirin-N. Anti-HIV-1 activity was assessed in a single cycle replication assay using pseudoviruses carrying a luciferase reporter gene and the envelope glycoproteins from HIV-1 isolates JR-FL (R5) and HxB2 (X4), against both immortalized and primary CD4+ cell targets. The data were analyzed for synergy using Calcusyn software. Results indicate that PRO 2000 and DS can act synergistically with most inhibitors tested, although the degree of synergy depends on inhibitor concentration and combination. These data provide a rational basis for testing of microbicide combinations in vivo.

The gp41 subunit of HIV-1 has been recently recognized as a target for antiviral therapy. C-34 is a peptide that mimics the heptad repeat 2 in the ectodomain of gp41. Here, we describe two HIV-1 strains selected after 5 and 17 passages in culture with increasing concentrations of C-34 (breakthrough concentration of 10 microg/ml). The HXB2-derived strain was more than 1000-fold resistant and contained a V38E mutation in the gp41 coding DNA sequence. The NL4-3-derived strain was more than 500-fold resistant and contained a L33S mutation in gp41. No cross-resistance to the RT inhibitor AZT, the HIV binding inhibitor dextran sulfate (DS), or the chemokine receptor antagonist ALX-40-4C was detected. These data indicate that HIV-1 can overcome C-34 inhibition through mutations at residues not involved in the formation of the hydrophobic cavity of gp41.