The HIV-1 restriction factor SAM domain– and HD domain–containing protein 1 (SAMHD1)1,2 is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool3,4,5. However, phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels6,7,8, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1 for defects in RNase or dNTPase activity, we identify SAMHD1 point mutants that cause loss of one or both functions. The RNase-positive and dNTPase-negative SAMHD1D137N mutant is able to restrict HIV-1 infection, whereas the RNase-negative and dNTPase-positive SAMHD1Q548A mutant is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of cell infection. SAMHD1 silencing in macrophages and CD4+ T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in cells and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.
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We are grateful to the members of our laboratory for discussion and technical help. We thank D. Littman (New York University School of Medicine) for HIV-1-GFP and HCMV-VSV-G, N. Manel (Institut Curie) for pLaiΔenvGFP3 and A. Cimarelli (University of Lyon) for pSIV3+ and pSIV3+ΔVpx. This work was supported by the US National Institutes of Health (R01 A1087390 and R21 AI102824 to F.D.-G. and GM104198 and AI049781 to B.K.), the Korean Institute for Basic Science (EM1402 to D.B.), the Korean Basic Science Research Program (2011-0014523 to D.B.), the Korean Creative Research Initiative Program (Research Center for Antigen Presentation, 2006-0050689 to K.A.) and BK21 plus fellowship to J.C., S.-Y.K. and M.S. from a National Research Foundation grant funded by the Ministry of Education, Science, and Technology of Korea.
The authors declare no competing financial interests.
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Ryoo, J., Choi, J., Oh, C. et al. The ribonuclease activity of SAMHD1 is required for HIV-1 restriction. Nat Med 20, 936–941 (2014). https://doi.org/10.1038/nm.3626
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