Mechanism Of Vpx/Vpr Counteracting Host Restriction Factors By Hijacking CRL4 (DCAF1) E3 Ubiquitin Ligase

The Vpr and its paralog Vpx are accessory proteins encoded by different HIV/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 of degradating host restriction factors by recruiting CRL4(DCAF1)E3 ligase.Selective disruption of viral CRL4(DCAF1)E3 ligase could be a promising antiviral strategy.Recently,we have determined that post-translational modification(neddylation)of Cullin-4 is required for the activation of Vpx-CRL4(DCAF1)E3 ligase and found MLN4924 as a first-in-class cancer drug by inhibiting the Nedd8-activating enzyme(NAE)blocks Vpx/Vpr-induced SAMHD1 degradation and blocks Vpx-dependent macaque simian immunodeficiency virus(SIVmac)or HIV-1 replication in myeloid cells.However,the mechanism of Vpx/Vpr-CRL4(DCAF1)E3 ligase assembly is still poorly understood.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 their substrates.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 or Vprmediated HLTF degradation.Importantly,altering the intracellular zinc concentration by treatment with the zinc chelator,N,N,N,N-tetrakis-(2-pyridylmethyl)ethylenediamine(TPEN)potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac 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 G2 cell 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-HIV drug development.