A novel recombinant marker virus assay for comparing the relative fitness of hiv-1 reverse transcriptase variants.
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 (pHIVDeltaRTBalIDeltanefhisD and pHIVDeltaRTBalIDeltanefPLAP, 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.
Lu
J
J
Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, Colorado, USA.
Kuritzkes
D R
DR
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015754:Genes, nef; D005819:Genetic Markers; D015658:HIV Infections / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D010920:Placenta / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D014779:Virus Replication
D005819:Genetic Markers; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D054303:HIV Reverse Transcriptase; D000469:Alkaline Phosphatase