Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages.

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.