HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

Abstract

BackgroundHIV-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.ResultsHere, 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.ConclusionsTaken 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.

Cellular Requirements for Bovine Immunodeficiency Virus Vif-Mediated Inactivation of Bovine APOBEC3 Proteins

“…In the HIV-1 Vif recruitment of the E3 ubiquitin complex, HIV-1 Vif has been proposed to bind ELOB/C at its C terminus, followed by CBF-␤ at its N terminus, inducing structural changes at both termini, and, consequently, to bind CUL5, which requires residues in both the N and C termini of the protein to assemble a functional ubiquitin ligase (29). Here, BIV Vif seems to have different steps for assembling a functional ubiquitin ligase with ELOB/C and CUL2.…”

“…Previous studies have shown that bovine A3Z2Z3 (btA3Z2Z3) can restrict HIV-1 infection (11,(28)(29)(30). Thus, we performed a series of single-cycle HIV-1 infectivity assays to test whether BIV Vif can overcome the antiviral activity of btA3Z2Z3.…”

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif

“…18,22 Also, Vif interacts with EloB/C through the B/C box, whose previously determined 144 SLQ 146 motif 5,12,13 contributes most significantly to the interaction. Surprisingly, although several regions of Vif have been identified to be critical for Cul5 binding, [23][24][25][26][27][28][29] only one a-helix is utilized for direct interaction with Cul5, but the importance of the Vif HCCH domain 9,23,24,30,31 and zinc binding [25][26][27] have been verified by the structure of the complex. The essential requirement of EloB/ C for the Vif-induced E3 complex assembly 5 has also been demonstrated by the binding of Vif and Cul5 to the complex, which facilitates their interaction with each other.…”

Challenges in HIV Vaccine Research for Treatment and Prevention

“…The effects of anti-Tat Abs on the immunological, virological, and clinical outcome of HIV-infected subjects were recently assessed in a prospective observational study (ISS OBS T-003, NCT01029548) conducted in asymptomatic drug-naïve HIV-infected adult volunteers (84). A significant association between the presence of anti-Tat Abs and a slower disease progression was found.…”