RELATIVE REPLICATION COMPETENCE OF VIRUS ISOLATES RESISTANT TO ANTI-HIV COMPOUNDS

Abstract

The rapid selection of drug-resistant strains during antiviral therapy of HIV infected patients is the primary reason for treatment failure with both nucleoside and nonnucleoside reverse transcriptase inhibitors and protease inhibitors. Although resistance would appear to be detrimental to antiviral therapy, it may be possible to select for resistant strains with amino acid changes that result in reduced replication capacity of the virus. Mutations that engender drug resistance may actually handicap the viral enzymes, reducing the rate and/or extent of virus replication. This reduced rate of virus replication in a patient may prolong the interval between HIV infection and AIDS and allow the immune system to more effectively deal with the virus. We have developed an assay to evaluate and compare the replication kinetics of molecularly constructed viruses with specific amino acid changes engineered into the reverse transcriptase and compared the replication of these mutants to the replication of the wild-type NL4-3 virus. Transfection methodology, cell line, cell density, DNA concentration, and transfection efficiency were optimized to measure the extent of virus production. Mutations were chosen for evaluation based on their high frequency of selection in vitro and/or in vivo as reported in the scientific literature. We were able to broadly categorize the replication capacity of each virus possessing a single amino acid change as growing faster, slower, or at an equivalent rate as wild-type virus. Representative viruses from each category were then evaluated for their growth potential in the presence of wild-type virus using a dual transfection methodology. These assays measured relative growth potential of the mutant virus compared to the wild-type strain both in the absence of and presence of drug selective pressure. Monitoring virus replication using the dual infection method, rather than from parallel cultures, provided a more sensitive and relevent approach to differentiating the replication capacity of the mutant virus compared to wild-type virus. These results directly demonstrate that drug-induced mutations in the reverse transcriptase have both positive and negative effects on the ability of HIV to replicate in human cells. A complete understanding of the effect of each mutation, alone or in combination, may prove valuable in designing and evaluating therapeutic opportunities for HIV infected patients.