p21 regulates the HIV-1 restriction factor SAMHD1.

Allouch et al. (1) have shown that CDKN1A (p21) restricts HIV-1 replication in monocyte-derived macrophages (MDM) by controlling the expression of the ribonucleotide reductase subunit R2 (RNR2) of the ribonucleotide reductase enzyme that, in turn, controls the intracellular deoxynucleotide (dNTP) pool required for HIV-1 reverse transcription. dNTP levels are also tightly controlled by the dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1), which is constitutively expressed in myeloid and lymphoid cells and is counteracted by the lentiviral virus protein x (Vpx) (reviewed in ref. 2). SAMHD1 is deactivated in proliferating cells by amechanism that requires phosphorylation of SAMHD1 (3). Allouch et al. (1) conclude that p21-driven HIV-1 restriction in macrophages is independent of SAMHD1 because (i) p21 did not affect SAMHD1 expression and (ii) Vpx did not affect p21 expression. Here, we show that M-CSF induces monocyte differentiation into macrophages and cell proliferation (Fig. 1A), and RNA interference of p21 leads to an increase in the number of proliferating cells (Fig. 1B). Macrophages become susceptible to HIV-1 replication (Fig. 1C) because SAMHD1 is inactivated as measured by specific SAMHD1 phosphorylation at residue T592 (Fig. 1D). Delivery of simian immunodeficiency virus (SIV) mac Vpxinduced SAMHD1 degradation (Fig. 1E) and subsequently increased virus infection (Fig. 1F) (2, 4, 5). Of importance, siRNA-induced down-regulation of p21 (Fig. 2A) strongly enhanced the phosphorylation of SAMHD1 (Fig. 2 B and C), followed by an increase in HIV-1 proviral DNA formation (Fig. 2 D and E) and virus infection (Fig. 2 F and G), without affecting the overall SAMHD1 expression (Fig. 2 B and C). Our results strongly suggest that p21 positively correlates with the degree of SAMHD1-mediated HIV-1 restriction. Allouch et al. (1) did not assess SAMHD1 deactivation by phosphorylation and, therefore, were unable to exclude a role for SAMHD1 in p21-mediated HIV-1 restriction. In their study, up-regulation of p21 by immobilized Ig (IVIg) and siRNA down-regulation of RNR2 in the presence or absence of Vpx were also used to suggest that p21-mediated regulation of HIV-1 was independent of SAMHD1 expression. However, Allouch et al. did not evaluate the effect of p21 downregulation in the presence or absence of SAMHD1. Here, we show that the increased HIV-1 replication observed after SAMHD1 degradation is not affected or further enhanced by down-regulation of p21 (Fig. 2H), suggesting that the effect of p21 was indeed dependent on SAMHD1 expression. Nevertheless, these results do not formally exclude the possibility of a dual effect of p21 through RNR2 and through SAMHD1. p21 is a cyclin-dependent kinase inhibitor (1) controlling cell-cycle progression through activation of cyclin-CDK1/2 complexes. Recently, it has been shown that phosphorylation of SAMHD1 by CDK1 leads to SAMHD1 inactivation (3). Thus, cell-cycle control must include a coordinated regulation of RNR2 and SAMHD1 function and dNTP availability. In this scenario, CDK activity may be the underlying mechanism explaining p21-mediated control of RNR2 and SAMHD1 (Fig. 2I). ACKNOWLEDGMENTS. This work was funded through the Ministerio de Economía y Comercio BFU201231569, SAF2010-21617-C02, and PI13/01083.