The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

Julien R C Bergeron,Stephen J Matthews,Hendrik Huthoff,Rebecca L Beavil,Dennis A Veselkov,Michael H Malim,Mark R Sanderson,Peter J Simpson,Bryan R Cullen

doi:10.1371/journal.ppat.1000925

Julien R C Bergeron, Stephen J Matthews + Show 7 more

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https://doi.org/10.1371/journal.ppat.1000925

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Abstract

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.

Highlights

The cullin-RING ubiquitin ligases form one of the largest family of E3 ubiquitin ligases, and members are implicated in many cellular functions such as the cell cycle, signal transduction, transcription, circadian clock, development, DNA replication and protein quality control [1]
A faint band could be observed in the recovered fraction for the DPPL protein; since we have demonstrated above that this mutation abrogates the interaction between HIV-1 viral infectivity factor (Vif) and the Cterminal helix of elongin B (EloB), it appears that in the context of overexpressed protein minimal binding to cullin 5 (Cul5) is still possible for this protein
We have reported a dissection of the interactions between the HIV-1 Vif protein and the elongin B and C (EloBC) heterodimer

Summary

Introduction

The cullin-RING ubiquitin ligases form one of the largest family of E3 ubiquitin ligases, and members are implicated in many cellular functions such as the cell cycle, signal transduction, transcription, circadian clock, development, DNA replication and protein quality control [1]. All members of this family are composed of a cullin E3 core and a specificity factor, which will select the appropriate substrate for ubiquitylation [2]. It has been shown that one such specificity factor, SOCS3, does not bind to EloBC when purified separately, but requires co-expression to form a SOCS3-EloBC ternary complex [7]

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