The objectives of this research proposal are to investigate the interaction of Vpr with the DNA repair enzyme uracil DNA glycosylase (UNG) and to test whether this interaction is important in human immunodeficiency virus type 1 (HIV-1) replication. Two lines of investigation led to the unexpected observation that the Vpr-UNG interaction can modulate the HIV mutation rate. First, it was observed that lack of Vpr expression led to a fourfold increase in the HIV mutation rate in one round of replication. Second, the yeast two-hybrid assay was used to identify that UNG was a binding partner of Vpr. The Vpr-UNG interaction was not associated with the ability of Vpr to cause cell cycle arrest. When the interaction of Vpr and UNG was prevented by site-directed mutations in Vpr, there was an increase in the virus mutation rate equivalent to that observed when Vpr was not expressed. The interaction of Vpr with UNG was also found to recruit UNG into HIV particles. In particular, the nuclear form of UNG was preferentially packaged into HIV particles. The lack of UNG recruitment into HIV-1 particles was found to correlate to the increase in the HIV mutation rate. Most mutations that occur during HIV-1 replication are likely due to errors by reverse transcriptase. However, the interaction of Vpr and UNG suggests that cellular DNA repair pathways could contribute to the accuracy of the reverse transcription process. A model system developed by the P.I. to study the in vivo mutation rate of HIV-1 will be used in this project to achieve a portion of the goals of the proposal. First, UNG variants which prevent the interaction of Vpr and UNG will be tested for their ability to influence the HIV-1 mutation rate. The testing of such mutants will verify the correlation between the interaction of Vpr and UNG, and the subsequent virion incorporation of UNG, on the modulation of the virus mutation rate. Second, site-directed mutations in UNG will be used to test for a correlation between the known properties of UNG (i.e., catalytic activity, uracil binding activity, and DNA binding activity) and the ability of the Vpr-UNG interaction to influence the HIV-1 mutation rate. Third, the mechanisms for how UNG modulates HIV-1 variation will be investigated. Finally, cell culture experiments will be done to test mutations in either Vpr or reverse transcriptase that can alter the in vivo fidelity of reverse transcription.
| Tabah, Azah A; Tardif, Keith; Mansky, Louis M (2014) Anti-HIV-1 activity of Trim 37. J Gen Virol 95:960-7 |
| Dapp, Michael J; Bonnac, Laurent; Patterson, Steven E et al. (2014) Discovery of novel ribonucleoside analogs with activity against human immunodeficiency virus type 1. J Virol 88:354-63 |
| Clouser, Christine L; Bonnac, Laurent; Mansky, Louis M et al. (2014) Characterization of permeability, stability and anti-HIV-1 activity of decitabine and gemcitabine divalerate prodrugs. Antivir Chem Chemother 23:223-30 |
| Holtz, Colleen M; Mansky, Louis M (2013) Variation of HIV-1 mutation spectra among cell types. J Virol 87:5296-9 |
| Dapp, Michael J; Patterson, Steven E; Mansky, Louis M (2013) Back to the future: revisiting HIV-1 lethal mutagenesis. Trends Microbiol 21:56-62 |
| Holtz, Colleen M; Sadler, Holly A; Mansky, Louis M (2013) APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure. Nucleic Acids Res 41:6139-48 |
| Dapp, Michael J; Heineman, Richard H; Mansky, Louis M (2013) Interrelationship between HIV-1 fitness and mutation rate. J Mol Biol 425:41-53 |
| Rawson, Jonathan M; Heineman, Richard H; Beach, Lauren B et al. (2013) 5,6-Dihydro-5-aza-2'-deoxycytidine potentiates the anti-HIV-1 activity of ribonucleotide reductase inhibitors. Bioorg Med Chem 21:7222-8 |
| Greggs 3rd, Willie M; Clouser, Christine L; Patterson, Steven E et al. (2012) Discovery of drugs that possess activity against feline leukemia virus. J Gen Virol 93:900-5 |
| Dapp, Michael J; Holtz, Colleen M; Mansky, Louis M (2012) Concomitant lethal mutagenesis of human immunodeficiency virus type 1. J Mol Biol 419:158-70 |
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