title: Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly.
abstract: The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or host helicase transcription factor (HLTF), respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that posttranslational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelatorN,N,N'-tetrakis-(2'-pyridylmethyl)ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac (simian immunodeficiency virus of macaques) infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HLTF degradation or Vpr-induced G2cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel anti-human immunodeficiency virus (HIV) drug development.IMPORTANCEThe Vpr and its paralog Vpx are accessory proteins encoded by different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx- or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with its substrate. Selective disruption of Vpx- or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration usingN,N,N'-tetrakis-(2'-pyridylmethyl)ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-dependent cellular protein functions. Therefore, information obtained from this study may be important for novel anti-HIV drug development.
authors.affiliation: School of Life Science, Tianjin University, Tianjin, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.
authors.forename: Haoran
authors.initials: H
authors.lastname: Guo
authors.affiliation: The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang, China.|Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.
authors.forename: Jiaming
authors.initials: J
authors.lastname: Su
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.|Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Rui
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Zheng
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.
authors.forename: Wenying
authors.initials: W
authors.lastname: Gao
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Zhang
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.
authors.forename: Zhaolong
authors.initials: Z
authors.lastname: Li
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.
authors.forename: Guanchen
authors.initials: G
authors.lastname: Liu
authors.affiliation: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Markham
authors.affiliation: First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China wwei6@jlu.edu.cn xyu2@jhu.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: School of Life Science, Tianjin University, Tianjin, China wwei6@jlu.edu.cn xyu2@jhu.edu.|First Hospital of Jilin University, Institute of Virology and AIDS Research, Changchun, Jilin Province, People's Republic of China.|The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang, China.|Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA.
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Yu
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D059565:G2 Phase Cell Cycle Checkpoints; D057809:HEK293 Cells; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D022423:Myeloid Cells / Q000821:virology; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D000076106:SAM Domain and HD Domain-Containing Protein 1; D015302:Simian Immunodeficiency Virus / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D015032:Zinc / Q000378:metabolism; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D053722:Receptors, Interleukin-17
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005029:Ethylenediamines; D020559:Monomeric GTP-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D000076106:SAM Domain and HD Domain-Containing Protein 1; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D054334:Viral Regulatory and Accessory Proteins; D015032:Zinc; D054325:vpr Gene Products, Human Immunodeficiency Virus; C100023:HLTF protein, human; C408922:IL17RB protein, human; D053722:Receptors, Interleukin-17; C513059:VPX protein, Simian immunodeficiency virus; C514912:vpr protein, Human immunodeficiency virus 1; C530533:DCAF1 protein, human; C484280:SAMHD1 protein, human; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
keywords: CRL4 (DCAF1) E3 ligase; Vpr; Vpx; cell cycle arrest; zinc binding