Abstract
BackgroundThe HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-β is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul5 E3 ligase complex.ResultsThe results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-β to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-β interaction. (2) Introduction of both the Vif SLQ → AAA mutant (VifΔSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL → AAA mutant (VifΔPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-β binding. (3) EloB-EloC but not CBF-β could greatly enhance the folding of full-length Vif in Escherichia coli. (4) The over-expression of EloB or the N-terminal ubiquitin-like (UbL) domain of EloB could significantly improve the stability of Vif/VifΔSLQ/VifΔPPL through the region between residues 9 and 14.ConclusionOur results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-β to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization.
Highlights
The Human immunodeficiency virus type 1 (HIV-1) accessory factor Virus Infectivity Factor (Vif) is necessary for efficient viral infection in non-permissive cells
Our results indicate that the Vif interaction with EloB-EloC may contribute to recruitment of CBF-β to Vif, demonstrating that the EloB C-teminus may play a role in improving Vif function and that the over-expression of EloB results in Vif stabilization
The N-terminal region of Vif contains the main sites involved in binding of CBF-β and APOBEC3 proteins [17,20,21,22,23,24,25], while its C-terminal domain contains a so-called suppressor of cytokine signaling (SOCS)-box motif, which is responsible for binding to the EloB-EloC complex [26,27,28], and a conserved HCCH zinc coordination site that mediates selective binding to Cul5 [29,30,31,32]
Summary
The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloBEloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. We comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul E3 ligase complex. The N-terminal region of Vif contains the main sites involved in binding of CBF-β and APOBEC3 proteins [17,20,21,22,23,24,25], while its C-terminal domain contains a so-called SOCS-box motif, which is responsible for binding to the EloB-EloC complex [26,27,28], and a conserved HCCH zinc coordination site that mediates selective binding to Cul5 [29,30,31,32]. EloB and EloC are known as regulatory subunits, whereas Cul functions as a scaffold protein [13]
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