Influence of Reverse Transcriptase Variants, Drugs, and Vpr on Human Immunodeficiency Virus Type 1 Mutant Frequencies

Abstract

The evolution of drug resistance is a major complication of human immunodeficiency virus type 1 (HIV-1) chemotherapy. HIV-1 reverse transcriptase (RT) is a major target of antiretroviral therapy and ultimately the target of drug resistance mutations. Previous studies have indicated that drug-resistant HIV-1 RTs can alter HIV-1 mutant frequencies. In this study, we have tested a panel of HIV-1 RT variants for their ability to influence virus mutant frequencies. The RT variants tested included drug-resistant RT variants as well as other variants analyzed in enzyme fidelity studies with the lacZ␣ gene as a mutation target and/or implicated as being important for enzyme fidelity by structural studies. Combinations of mutations that alone had a statistically significant influence on virus mutant frequencies resulted in different mutant frequency phenotypes. Furthermore, when virus replication occurred in the presence of drugs [e.g., 3-azido-3-deoxythymidine, (؊)2/,3-dideoxy-3-thiacytidine, hydroxyurea, thymidine, or thioguanine] with selected RT variants, virus mutant frequencies increased. Similarly, Vpr variants deficient for binding to the uracil DNA glycosylase repair enzyme were observed to influence HIV-1 virus mutant frequencies when tested alone or in combination with RT variants. In summary, these observations indicate that HIV-1 mutant frequencies can significantly change by single amino acid substitutions in RT and that these effects can be altered by additional mutations in RT, by drugs, and/or by expression of Vpr variants. Such altered virus mutant frequencies could impact HIV-1 dynamics and evolution in small population sizes.

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

“…We demonstrate that the Vpr-dependent incorporation of UNG into HIV-1 particles is directly responsible for the role of Vpr in the in vivo modulation of the virus mutation rate (11,12). Moreover, our results show that the incorporation of UNG into virions is critical for efficient replication of HIV-1 in primary non-dividing cells such as macrophages.…”

“…Although the viral integrase may also participate in this recruitment (22,23), the Vpr-dependent packaging of UNG2 into virions strikingly correlated with the ability of Vpr to influence the mutation rate of HIV-1 (24). This indicated that the interaction between Vpr and UNG2 may directly influence the reverse transcription accuracy and, thus, play a role in the modulation of the in vivo mutation rate of HIV-1 (11,12).…”

“…Infection of HeLa target cells was also done by cocultivation of virus-producing cells with target cells (29). The protocols for analysis of mutant frequencies have been previously described with minor modifications (12,28,30). Specifically, infected peripheral blood mononuclear cells (PBMCs) and monocytes-derived macrophages (MDMs) were not placed under drug selection, and after purification of proviral DNA with the lac repressor protein, the vector cassette containing the mutation target was PCR amplified before analysis of mutant frequencies in E. coli.…”

“…Analysis of HIV-1 Mutant Frequencies-The HIV-1 vectors used in these studies have been previously described (11,12,28). To produce vector virus, the HIV vectors were complemented in trans with pSVgagpol-rre-MPMV, the amphotropic murine leukemia virus env expression plasmid, pSV-A-MLV-env, and a Vpr expression plasmid derived from pAS1B (11).…”

“…First, expression of Vpr alters the cell cycle progression by arresting cells in the G 2 phase (7-10). Second, Vpr influences the reverse transcription process of the viral DNA, and this can modulate the in vivo mutation rate of HIV-1 (11,12). Finally, Vpr is required for the infection of nondividing cells, and this requirement is related, at least in part, to its role in the nuclear translocation of the preintegration complex (PIC) containing the viral DNA.…”