Replication of HIV in macrophages : effects of SAMHD1 on dNTP Levels, viral mutagenesis, and antiviral efficacy

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014.Lentiviruses such as HIV are unique in that they replicate in both dividing CD4+ T cells and non-dividing macrophages. Activated CD4+ T cells progress through S phase and have an abundant amount of dNTPs to replicate genomic DNA, whereas macrophages remain in the resting phase and do not require high levels of dNTPs. Another factor that maintains low dNTP levels in macrophages is a protein called SAM domain and HD domain-containing protein 1 (SAMHD1), which hydrolyzes dNTPs into dNs. SAMHD1 was previously characterized as a dGTP dependent triphosphohydrolase, but with several assays in vitro we have shown that the ribonucleotide GTP is the primary allosteric activator. SAMHD1 is an antiretroviral host factor that restricts viral replication by limiting the dNTP substrate availability for reverse transcriptase (RT). HIV-2 counteracts SAMHD1 by expressing an accessory protein vpx, which targets it for proteasomal degradation. This results in an increase in cellular dNTPs and rapid completion of viral DNA synthesis. However, HIV-1 does not employ a mechanism to degrade SAMHD1, and therefore replicates, although at low levels, even with the limited dNTPs found in macrophages. SAMHD1 is specific for dNTPs and is not able to hydrolyze rNTPs; therefore the concentration of rNTPs is in the millimolar range in both dividing and non-dividing cell types such that RNA polymerases can continuously synthesize RNA. This creates a large disparity between dNTPs and rNTPs, particularly in macrophages, during HIV-1 replication. Consequently, HIV-1 RT frequently incorporates rNMPs in the viral genome possibly contributing to the high mutation rate of HIV-1. SAMHD1 also selects dNTPs over the triphosphate form of nucleoside reverse transcriptase inhibitors (NRTI-TPs), which are analogous in structure to cellular dNTPs, but lack a 3' OH group on the ribose moiety. Although SAMHD1 does not decrease the concentration of NRTI-TPs by direct hydrolysis, degradation of SAMHD1 in macrophages significantly reduces drug efficacy. This is caused by the increase in cellular dNTPs upon degradation of SAMHD1, which out compete NRTI-TPs for the active site of RT. These findings suggest that NRTI-TPs are not as effective at inhibiting HIV-2 as they are at inhibiting HIV-1 infection in macrophages