A Cyclin-Binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation

• Type of Material: Article
• Authors: Corine St. Gelais, Ohio State University; Sun Hee Kim, Ohio State University; Victoria V. Maksimova, Ohio State University; Olga Buzovetsky, Yale University; Kirsten M. Knecht, Yale University; Caitlin Shepard, Emory University; Baek Kim, Emory University; Yong Xiong, Yale University; Li Wu, Ohio State University
• Language: English
• Date: 2018-03-01
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2018 American Society for Microbiology.
• Final Published Version (URL): http://dx.doi.org/10.1128/JVI.01787-17
• Title of Journal or Parent Work: Journal of Virology
• ISSN: 0022-538X
• Volume: 92
• Issue: 6
• Grant/Funding Information: This work was supported by a grant (AI104483) from the National Institutes of Health (NIH) to L.W. L.W. is also supported in part by NIH grants (AI120209 and GM128212).; C.S. and B.K. are supported by NIH grants GM104198 and AI049781. O.B. was supported by NIH T32 grant GM007223 and a National Science Foundation Graduate Research Fellowship, and K.K. was supported by NIH T32 grant GM008283.
• Keywords: infection; cyclin binding; Life Sciences & Biomedicine; HIV-1; SAMHD1; STRUCTURAL BASIS; dNTPase; structure; restriction; IMMUNODEFICIENCY-VIRUS TYPE-1; DOWN-REGULATION; CHAPERONE-MEDIATED AUTOPHAGY; MAMMALIAN-CELLS; Science & Technology; CD4(+) T-CELLS; RECOGNITION MOTIF; SEZARY-SYNDROME PATIENTS; GENE-EXPRESSION; ALPHA-SYNUCLEIN; Virology
• Research Categories: Biophysics, Medical; Health Sciences, Pharmacy

Abstract

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.