1st International Workshop on HIV Drug Resistance & Treatment Strategies 25-28 June 1997, St. Petersburg, Florida, USA

ANTIVIRAL AND RESISTANCE STUDIES OF RPR103611, AN INHIBITOR OF HIV REPLICATION

Antiviral Therapy 1997;2 (Suppl 5):# (abstract no. 33)

B Labrosse¹, O Pleskoff¹, N Sol¹, C Jones¹, Y Henin² and M Alizon¹

The antiviral activity of RPR103611 seems to be due to the blocking of membrane fusion mediated by the HIV-1 envelope proteins (Env). RPR103611 neutralized HIV-1LAI but did not neutralize infection by HIV-2 or by HIV-1_NDK. HIV-1 entry was blocked when RPR103611 was present during virus-cell contact.

OBJECTIVE: Identification of the HIV-1 Env domain responsible for the sensitivity or resistance to neutralization by a triterpene derived from betulinic acid (RPR103611).

METHODS: A CD4+ LTR LacZ cell line (HeLa-P4), in which HIV infectious titres can be determined after a single cycle of reverse transcription, was used to study the antiviral and strain-specific activities of RPR103611. HeLa-P4 cells were infected with three viral strains of HIV (LAI, NDK, ROD) or cocultured with HeLa cell lines transiently transfected with recombinant env cDNA. Selection of drug-resistant HIV-1 (Lai-R) was performed by growing infected CEM cells (CD4+ T lymphoid cell line) in the presence of a single dose of RPR103611. Virus production was monitored every 3 days by p24 production in the supernatant. After 32 days, virus production was delayed and susceptibility of viral supernatants were tested on fresh CEM cells in the presence or absence of the drug. To obtain a drug-resistant env gene, PCR was performed on DNA from HeLa-P4 cells infected with Lai-R in the presence of 10 µM RPR103611. Then the Lai-R env gene was completely sequenced.

RESULTS: The HIV-1_NDK strain was used to define domains in Env responsible for the sensitivity. The substitution of the NDK V3 or NDK gp120 into LAI Env did not confer drug resistance. RPR103611 blocked fusion mediated by all chimeric Env bearing the LAI gp41 but had a limited effect on fusion mediated by chimeras with NDK gp41. To complete this study, we compared the env gene of a selected drug-resistant HIV-1_LAI and the parental LAI strain. The gp120 of LAI and LAI-R were identical, except for two amino acid substitutions in gp41, Arg to Ala at position 22 and Ile to Ser at position 84. These mutations in gp41, responsible for the drug-resistant phenotype of LAI-R, were consistent with previous results with the LAI/NDK env chimeras.

CONCLUSIONS: RPR103611 is to our knowledge the only chemical reagent blocking HIV-1 entry by affecting gp41, except for peptides such as DP-107 and DP-178, derived from the alpha-helix domain. Drugs acting on a gp41 target could be interesting candidates for the chemotherapy of HIV infection since gp41 is far more conserved than gp120 across HIV-1 strains. A critical parameter in determining the clinical efficacies of antiviral drugs is the rate of resistance development. We have isolated resistant variants of the HIV-1_LAI after long-term passaging in the presence of RPR103611. The drug resistance was due to a non-conservative mutation at a highly conserved residue of gp41, suggesting that the genetic constraints on gp41 are much lower than expected.

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1997-06-25
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