title: Identification of critical regions in human SAMHD1 required for nuclear localization and Vpx-mediated degradation.
abstract: The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.
authors.affiliation: Institute of Virology and AIDS Research, First Hospital of Jilin University, Changchun, Jilin Province, China.
authors.forename: Haoran
authors.initials: H
authors.lastname: Guo
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Wei
authors.forename: Xianjun
authors.initials: X
authors.lastname: Liu
authors.forename: Sean L
authors.initials: SL
authors.lastname: Evans
authors.forename: Weiming
authors.initials: W
authors.lastname: Yang
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Guo
authors.forename: Ke
authors.initials: K
authors.lastname: Zhao
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Zhou
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Yu
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D000076106:SAM Domain and HD Domain-Containing Protein 1; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics* / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
chemical_list: C424166:KPNB1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C513059:VPX protein, Simian immunodeficiency virus; D054334:Viral Regulatory and Accessory Proteins; D028961:beta Karyopherins; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human; D020559:Monomeric GTP-Binding Proteins