A Novel Recombinant Marker Virus Assay for Comparing the Relative Fitness of HIV-1 Reverse Transcriptase Variants

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Abstract
Summary:A novel recombinant marker virus assay (RMVA) has been developed to perform growth competition assays for assessing fitness of HIV-1. This assay allowed the generation of replication-competent viruses by homologous recombination of polymerase chain reaction (PCR)-derived reverse transcriptase (RT) coding sequences in RT-deleted proviral clones of HIV-1 in which the nef gene was replaced by the Salmonella typhimurium histidinol dehydrogenase (hisD) or human heat-stable placental alkaline phosphatase (PLAP) gene (pHIVδRTBalIδnefhisD and pHIVδRTBalIδnefPLAP, respectively). The proportion of a given RT species in a mixed culture was determined by quantifying the linked hisD or PLAP marker gene using real-time PCR. The RMVA was tested by comparing the relative fitness of wild-type and lamivudine-resistant recombinant viruses. The RMVA reproducibly detected differences in the fitness of these two viruses in growth competition assays. With appropriate modification of the recombination vectors, the RMVA should be useful for analyzing the fitness of viruses resistant to protease, integrase, or fusion inhibitors and should be applicable in clinical research. Address correspondence to Daniel R. Kuritzkes, University of Colorado Health Sciences Center, 4200 East 9th Avenue, B-168, Denver, CO 80262, U.S.A.; e-mail: [email protected] A portion of this work was presented at the Fourth International Workshop on HIV Drug Resistance and Treatment Strategies, Sitges, Spain, June 12-16, 2000 [abstract 140]. Manuscript received December 18, 2000; accepted March 26, 2001. © 2001 Lippincott Williams & Wilkins, Inc.

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