HIV-1 viral protein R (Vpr) has been shown to induce cell cycle arrest, and this has been suggested to play an important to role in viral replication and pathogenesis. Our recent studies have revealed that Vpr activates ATR, suggesting that it may tigger cell cycle arrest as a consequence of the induction of the DNA damage pathway. In yeast, the ATR homolog Mec1 has been shown to not only regulate cell cycle progression, but also to control the biosynthesis of cellular dNTP in response to DNA damage, by modulating the activity of ribonucleotide reductase. In preliminary experiments, we have found that cellular dNTP levels also increase upon DNA damage in several human cell lines. This presumably allows repair DNA polymerases, which are also induced by DNA damage, to execute efficient DNA gap repair. Numerous studies have demonstrated that non-dividing cells contain lower dNTP levels than actively dividing cells. However, the dNTP level of human macrophages has not been determined, due to the lack of a highly sensitive dNTP assay. Recently, we established an enzymatic dNTP assay that has allowed the determination of dNTP levels in human macrophages. This analysis revealed that human macrophages contain much a lower intracellular concentration of dNTPs (20 approximately 40nM) than either activated or resting T cells (approximately 2 mu M). In fact, the dNTP concentration in human macrophages is considerably lower than the known dNTP binding affinity of the host DNA polymerases that execute the repair of the 5-nucleotide DNA gaps generated during HIV-1 proviral DNA integration. This suggests that dNTP availability may represent a rate-limiting step in HIV- 1 infection of macrophages. In this proposal, we hypothesize that, like DNA damage, HIV-1 Vpr elevates cellular dNTP levels, and that this has a stimulatory effect on HIV- 1 replication in macrophages, which normally contain very low levels of dNTPs. To test this hypothesis, we will examine the effect of HIV-1 Vpr on cellular dNTP levels and HIV-1 gap repair activity in macrophages; we will also directly test whether the elevation of cellular dNTP levels by deoxynucleosides (dNs) results in an enhancement of the proviral DNA integration and proviral DNA synthesis kinetics of HIV-1 in macrophages. We will also identify host repair proteins essential for Vpr to alter dNTP biosynthesis and gap repair. This study will 1) elucidate a mechanism that HIV-1 employs to achieve its unique ability to infect non-dividing cells and 2) identify host repair proteins that may be potential antiviral targets.
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