SAMHD1-mediated dNTP pool regulation in macrophage : effect on HIV-1 reverse transcription kinetics and fidelity

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014.HIV-1 replication is restricted in macrophages as compared to activated CD4+ T cells. Recently, SAMHD1 has been identified as a HIV-1 restriction factor that is counteracted by Vpx. Here we show that HIV-1 infection of monocyte-derived macrophages (MDM) is restricted due to active hydrolyzation of dNTP by SAMHD1, reducing the cellular dNTP pool below the Km and Kd of HIV-1 reverse transcriptase (RT), possibly slowing proviral DNA synthesis. MDMs treatment by exogenous dNs or VLPs containing Vpx elevated cellular dNTP pools, suggesting that both affect similar pathway. Together, these observations showed that SAMHD1 is actively involved in regulation of dNTPs and contributes to the low dNTP concentration found in macrophages. RT can synthesize proviral DNA in non-dividing cells like macropages due to its tight dNTP binding affinity. A structural model shows that the A114 occupies a dNTP binding pocket. Thus, we tested whether increasing size of the side chain at position 114 will reduce RT dNTP binding affinity by reducing space in the pocket. Indeed, our biochemical data demonstrate that the residue 114 mutant RTs have reduced polymerase activity at low dNTP concentrations. Pre-steady state kinetic study also shows that the A114C RT mutant has reduced dNTP binding affinity (Kd), but without altering catalysis (kpol). Compared to WT, the A114C mutant RT showed similar incorporation capability of acydNTPs lacking the sugar ring. These data support that A114 plays a key role in the tight dNTP binding affinity of RT and a unique HIV-1 infectivity of macrophages. RT incorporates rNTPs frequently under macrophage dNTP conditions. Thus we investigated the mechanistic impact of rNMP embedded in DNA templates on RT-mediated DNA synthesis. Our study showed that under simulated macrophage dNTP concentrations, rNMP induced pausing of RT and delayed DNA synthesis kinetics. Although RT template/primer (T/P) binding affinity was not altered, its dNTP incorporation kinetics was significantly reduced at one nucleotide upstream and downstream of the rNMP site. RT becomes more error prone with an elevated mismatch extension capability but not enhanced misinsertion capability. These data show that rNMPs embedded in DNA templates may influence HIV-1 reverse transcription kinetics and simultaneously viral mutagenesis