Elucidating the role of Vif-RNA and arms race interfaces in binding and promoting A3G ubiquitination

ABSTRACT

Human APOBEC3G (A3G) is a potent cellular restriction factor against HIV-1. The viral protein Vif hijacks a Cullin5 RING (CRL5) E3 ubiquitin ligase to polyubiquitinate A3G for proteasomal degradation, neutralizing its antiviral potency. An ancient Molecular Arms race between lentiviruses and primate hosts modulates the Vif-A3G protein interface. Recently, we demonstrated in a cryo-EM structure that HIV-1 Vif also uses an RNA oligonucleotide as a molecular glue to bind A3G for polyubiquitination. Vif binds RNA and A3G in a manner that is compatible with polyubiquitination by coenzymes in the CRL5 complex. Vif utilizes RNA-binding to bind conserved residues in A3G, limiting its ability to escape antagonism. We demonstrate that RNA acts as a molecular glue, promoting the complex assembly of Vif and A3G. The role of RNA in promoting A3G ubiquitination is investigated using quantitative pulse-chase assays that monitor A3G mono-ubiquitination. The study of Vif-A3G binding, mediated by different interfaces including the molecular arms race and RNA, is coupled with quantitative ubiquitination assays in this work. A comprehensive understanding of this relationship can further reveal how viral adaptations manifest at the levels of binding and biochemistry.