Abstract
Template switching can occur during the reverse transcription of HIV-1. Deoxynucleotide triphosphate (dNTP) concentrations have been biochemically shown to impact HIV-1 reverse transcriptase (RT)-mediated strand transfer. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. Terminally differentiated/nondividing macrophages, which serve as a key HIV-1 reservoir, contain extremely low dNTP concentrations (20-50 nm), which results from the cellular dNTP hydrolyzing sterile α motif and histidine aspartic domain containing protein 1 (SAMHD1) protein, when compared with activated CD4(+) T cells (2-5 μm). In this study, we first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4(+) T cells. Second, SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4(+) T cells. Third, differentiated SAMHD1 shRNA THP-1 cells have a 2-fold increase in HIV-1 template switching efficiency. Fourth, SAMHD1 degradation by Vpx did not alter HIV-1 template switching efficiency in activated CD4(+) T cells. Finally, the HIV-1 V148I RT mutant that is defective in dNTP binding and has DNA synthesis delay promoted RT stand transfer when compared with wild type RT, particularly at low dNTP concentrations. Here, we report that SAMHD1 regulation of the dNTP concentrations influences HIV-1 template switching efficiency, particularly in macrophages.
Highlights
Delaying DNA synthesis kinetics of HIV-1 reverse transcriptase by lowering Deoxynucleotide triphosphate (dNTP) concentrations elevates biochemical RNA template switch
We investigated the effect of the SAMHD1-viral protein X (Vpx) network, which regulates the in vivo cellular dNTP concentration availability, on HIV-1 template switching frequency
HIV-1 Template Switching Frequency in Human Primary Monocyte-derived Macrophages (MDMs) and Activated CD4ϩ T Cells—First, to investigate the effect of the SAMHD1-Vpx network on HIV-1 template switching frequency, we utilized the pHIG NL4-3 HIV-1 vector system, which has been established for investigating the viral mutation rate [35]
Summary
Delaying DNA synthesis kinetics of HIV-1 reverse transcriptase by lowering dNTP concentrations elevates biochemical RNA template switch. Results: Degradation of SAMHD1 by Vpx, which increases dNTP levels, reduces HIV-1 template switching in macrophages, but not in CD4ϩ T cells. Conclusion: Altering cellular dNTP levels directly affects in vivo HIV-1 template switching. Significance: Cellular dNTP abundance controlled by SAMHD1 contributes to HIV-1 template switching in macrophages. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. We first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4؉ T cells. SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4؉ T cells. SAMHD1 degradation by Vpx did not alter HIV-1 template switching effi-
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