Development of resistance to VIR-353 with cross-resistance to the natural HIV-1 entry virus inhibitory peptide (VIRIP)

Objective: Virus-inhibitory peptide (VIRIP) has been identified as a component of human hemofiltrate that blocks HIV-1 gp41-dependent fusion by interacting with the fusion peptide. A VIRIP analogue (VIR-576) has been shown to be effective in a phase I/II clinical trial. We have evaluated the activity and mechanism of HIV-1 resistance to VIRIP and its analogue, VIR-353. Methods: Anti-HIV activity and passage of HIV-1 strains in cell culture were used to generate and identify mutations that confer resistance to VIRIP and VIR-353. Recombinant viruses harboring the most relevant mutations were generated and characterized. Results: VIRIP and VIR-353 showed anti-HIV-1 activity with EC50 of 28 and 0.3 mu mol/l, respectively, and were active against virus resistant to BMS-155, AMD3100, T20, TAK-779 or nevirapine. Time of addition experiments showed that VIR-353 targets a time/site of action corresponding to gp41-dependent fusion. VIR-353-resistant virus was generated after 450 days in cell culture, suggesting a high genetic barrier for resistance. The VIR-353-resistant virus was cross-resistant to VIRIP but remained sensitive to T20, AMD3100 or zidovudine. Recombination of gp41 into a wild-type backbone partially recovered the resistant phenotype, but both gp120 and gp41 from the resistant virus were necessary to restore resistance to VIRIP or VIR-353. Site-directed mutagenesis confirmed the role of specific mutations and identified a combination of three mutations (A433T/V489I/V570I) as the most relevant to VIRIP resistance. Conclusion: VIRIP may interact with a region of gp41 that is essential for fusion but not the fusion peptide. Our results highlight interactions between gp41 and gp120 that may be required during the fusion process. (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins