title: HIV-1 Vif N-terminal motif is required for recruitment of Cul5 to suppress APOBEC3.
abstract: BACKGROUND
  HIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-β). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C-terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide.
  
  
  RESULTS
  Here, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25-30 (25VXHXMY30) reduced the ability for Vif to bind Cul5, but not CBF-β or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-β-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding.
  
  
  CONCLUSIONS
  Taken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.
authors.forename: Sean L
authors.initials: SL
authors.lastname: Evans
authors.forename: Arne
authors.initials: A
authors.lastname: Schön
authors.forename: Qimeng
authors.initials: Q
authors.lastname: Gao
authors.forename: Xue
authors.initials: X
authors.lastname: Han
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhou
authors.forename: Ernesto
authors.initials: E
authors.lastname: Freire
authors.affiliation: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N, Wolfe Street, Baltimore, MD 21205, USA. xfyu@jhsph.edu.
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Yu
mesh_terms: D000071480:APOBEC-3G Deaminase; D044842:Cullin Proteins / Q000378:metabolism*; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D043525:Cytosine Deaminase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology*; D006801:Humans; D057131:Immune Evasion; D025941:Protein Interaction Mapping; D059748:Proteolysis; D054320:vif Gene Products, Human Immunodeficiency Virus
chemical_list: C496871:CUL5 protein, human; D044842:Cullin Proteins; D054320:vif Gene Products, Human Immunodeficiency Virus; C514847:vif protein, Human immunodeficiency virus 1; C492906:APOBEC3F protein, human; D043525:Cytosine Deaminase; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase