SAMHD1 Specifically Affects the Antiviral Potency of Thymidine Analog HIV Reverse Transcriptase Inhibitors

Abstract

bSterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocytederived macrophages and CD4؉ T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4؉ T cells infected with HIV-2 compared to infection with the HIV-2⌬Vpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes. Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a recently identified human immunodeficiency virus type 1 (HIV-1) host restriction factor that limits retroviral replication at the reverse transcription stage of the viral life cycle (1-5). SAMHD1 functions as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that regulates the intracellular pool of dNTPs (6). It restricts HIV-1 infection in immune cells of myeloid lineage and in quiescent CD4-positive T lymphocytes (1, 2, 4). SAMHD1 reduces cellular dNTP levels to concentrations below the threshold required for reverse transcription of the viral RNA genome into DNA (4, 5). SAMHD1 is counteracted by the retroviral Vpx protein that is encoded by simian immunodeficiency virus (SIV) and HIV-2, but this gene is lacking from the HIV-1 and feline immunodeficiency virus (FIV) genomes (7). However, and despite the lack of Vpx function, HIV-1 is still able to replicate in noncycling myeloid cells, albeit at low levels (7).Most current standard three-drug antiretroviral regimens involve RT inhibitors combined with a protease inhibitor. [EFV], and etravirine). NRTI are phosphorylated to their triphosphate form to act as competitive inhibitors of HIV RT. In contrast, NNRTI bind at...

Cyclin D3-dependent control of the dNTP pool and HIV-1 replication in human macrophages

“…33 Thus, cyclin L2 controls the abundance of SAMHD1 as an alternative mechanism regulating SAMHD1-mediated restriction. We have not observed differences in total SAMHD1 mRNA expression between HIV-1 restricted and susceptible macrophages 34 and SAMHD1 is ubiquitously expressed in monocytes, MDM, and in resting and activated CD4C T cells. 8,19,22,35 While SAMHD1 inactivation through phosphorylation is apparent in HIV-1 susceptible primary cells, regulation of total SAMHD1 expression may be dependent on a fine-tune mechanism irrespective of the cell cycle but similarly controlled by other host factors including cyclin L2.…”

“…Recovered aquose phase was recovered and dried in a SpeedVac. Pellets were resuspended in Tris-HCl buffer (40 mM, pH7.4) and dNTP content determined using a polymerase-based method 19,34,41 with minor modifications. Briefly, 20 mL of reaction mixture contained 5 mL of dNTP extract in 40 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 5 mM dithiothreitol, 0.25 mM oligoprimer, 0.75 mM [8- 3 H]dATP, 12-21 Ci/mmol (or [methyl-3 H]dTTP for the dATP assay) and 1.7 units of Thermo Sequenase DNA Polymerase (GE Healthcare).…”

SAMHD1: At the Crossroads of Cell Proliferation, Immune Responses, and Virus Restriction

“…HIV-2 Vpx induces the degradation of SAMHD1 by targeting it to undergo proteasomal degradation, therefore increasing intracellular dNTP levels and allowing reverse transcription to proceed. In support of this mechanism, SAMHD1-mediated restriction has been shown to be bypassed by simply adding exogenous deoxynucleosides to the culture media [18,41]. …”

“…Nucleoside analogues, such as fludarabine, are a first-line treatment for leukemia [69] and essential components of antiretroviral treatment. Thus, their activity may be strongly affected by SAMHD1 function [41,80].…”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…This minimizes the efficacy of the treatment to such an extent that SAMHD1 expression levels were negatively correlated with Ara-C treatment success in individuals with AML. Additionally, SAMHD1 appears to reduce the efficacy of thymidine based analogs used to treat HIV, and depletion of SAMHD1 from monocyte cells affects the susceptibility of HIV-1 infections to nucleoside reverse transcriptase inhibitors (79, 213, 214). Based on these finding, the development of a robust SAMHD1 inhibitor that can potentiate nucleotide analogue therapeutic regimens should become a priority for SAMHD1 researchers.…”