Reverse transcriptases (RTs) frequently switch templates, leading to an increase in genetic variation and retroviral recombination. Based on several studies using high-frequency deletion of homologous repeats as a model system, we proposed a dynamic copy-choice model for retroviral recombination, which postulated that there is a steady state between the rates of DNA synthesis and RNA degradation and that conditions affecting this steady state influence the frequency of RT template switching. We have determined the influence of antiviral drug resistance mutations on the frequency of RT template switching and retroviral recombination. These studies suggest that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution. Based on the dynamic copy choice model for retroviral recombination and the effect of AZT on RT template switching, we proposed that an equilibrium exists between 1) NRTI incorporation and degradation of the RNA template by RNase H activity, and 2) NRTI excision and resumption of DNA synthesis. Degradation of template RNA leads to dissociation of the template and primer unless the NRTI is excised and DNA synthesis is resumed; the dissociation of the template and primer leads to abrogation of HIV-1 replication. Studies of RNase H mutants provided strong evidence in support of this hypothesis and novel insights into the mechanism of NRTI drug resistance. We are extending these studies to determine whether mutations in the RNase H domain that confer drug resistance are selected in response to therapy in adult and pediatric patients infected with subtype B or subtype C HIV-1. A prerequisite to retroviral recombination is infection of virus producing cells with two different virions followed by copackaging of the two viral genomic RNAs. In collaboration with Dr. Wei-Shau Hu's laboratory, we have shown that double infection of cells with HIV-1 is nonrandom and occurs at a higher than expected rate. In addition, we have shown that viral entry is an important but not the sole determinant of nonrandom double infection. To elucidate the mechanisms of retroviral reverse transcription in cells, we are developing a novel strand-specific amplification assay (SSA) that allows quantitative strand-specific determination of viral DNA products in infected cells. We are using the SSA method to measure the rate of viral DNA synthesis in infected primary CD4+ T cells. In other studies, we have investigated whether MLV- and HIV-1-based vectors containing two primer-binding site (PBS) regions can initiate reverse transcription more than once and have used this system as a sensitive ex vivo assay to analyze the effects of cis-acting mutations in the PBS regions on the efficiency of DNA synthesis initiation. These studies have elucidated the relative influence of various RNA-RNA interactions between viral RNA and tRNALys3 on the efficiency of DNA synthesis initiation.
Showing the most recent 10 out of 14 publications
