| |
Measuring the Replicative Fitness of Recombinant HIV-1 Vectors Expressing Protease and Reverse Transcriptase Derived from Patient Viruses T. WRIN*, A. GAMARNIK, E. PAXINOS, R. ZIERMANN, W. HUANG, K. LIMOLI, G. HEILEK-SNYDER, M. SARTORIS, J. WHITCOMB, N. PARKIN, N. HELLMANN, and C. PETROPOULOS.
ViroLogic, S. San Francisco, CA Background: Suboptimal antiretroviral therapy frequently promotes the emergence of HIV-1 variants containing reverse transcriptase (RT) and/or protease (PR) mutations that enable the virus to replicate in the presence of drug. Some investigators have ascribed a potential clinical benefit to the reduced replication efficiency of drug resistant viruses in the face of incomplete viral suppression during antiretroviral therapy.
Methods: A replicative fitness assay was developed which uses recombinant HIV-1 vectors containing patient derived PR and RT coding regions and a luciferase gene. Vectors are used to generate virus stocks and the ability of these viruses to complete a single round of replication (in the absence of antiretroviral drug) is assessed by measuring luciferase activity in infected cells. Relative fitness measures are derived by comparing the luciferase activity generated by recombinant viruses containing patient derived PR/RT to a reference vector derived from NL4-3. A relative fitness value less than 1 reflects impaired replication.
Results: Replicative fitness of recombinant viruses containing site directed mutations in PR or RT was evaluated. Generally, mutations that reduce drug susceptibility (D30N/N88D; G48V/L90M) impair the replication of recombinant test vectors in the absence of drug. Certain mutations associated with drug resistance (L63P, V77I; gag cleavage sites) partially restored vector replication. A survey of patient viruses demonstrated a broad range of replicative fitness. Many patient viruses demonstrating severely impaired replicative fitness contained PR and RT mutations associated with drug resistance.
Conclusions: A single replication cycle assay has been developed that enables rapid quantitative assessments of the replicative fitness of recombinant HIV-1 vectors expressing PR and RT derived from patient viruses. Measurements of the replicative fitness of HIV-1 may be useful in the management of patients receiving antiretroviral drug treatment.
Key Words: assay, fitness, resistance
|
