Investigating Competition between the HIV-1 Proteins Rev and Gag for Stem 1 of the Rev Response Element

Abstract

HIV replication requires the export of full length RNA genome transcripts from the nucleus to the cytoplasm, where the genome is packaged. Host surveillance mechanisms prevent such RNA export, so HIV uses Rev, a viral protein translated from fully spliced transcripts to enter the nucleus and bind to a highly conserved RNA element of the HIV genome, the Rev Response Element (RRE), which is retained on un/incompletely spliced RNA. The Rev-RRE complex binds to host export machinery, and exits the nucleus to the cytoplasm, where unspliced RNA is available for translation or packaging. Rev binds to the RRE on two stem II binding sites and on one purine-rich bulge in stem 1. The RRE has been shown to also be bound by Gag ?a viral protein involved in genome packaging? at the same RRE stem I binding region as Rev. To understand the interaction and biological relevance between Rev and Gag on RRE stem 1, we use a peptide containing the RNAbinding, arginine-rich motif (ARM) of Rev, a protein containing the nucleocapsid (NC) domain of Gag, and truncated RRE stem I fragments in EMSA and ITC studies. Some RNA fragments include mutations that allow us to probe for specific protein binding sites. We find that NC displays tighter binding affinity to our stem 1 constructs than the Rev ARM peptide, which may suggest that Gag binds to the RRE in the cytoplasm and displaces Rev from the stem 1 binding site. This interaction may be biologically relevant and may represent a link between nuclear export of the genome and subsequent genome packaging. Future studies are needed to more accurately explore this competitive interaction, such as the usage of full-length RNA constructs, which would contain more Rev binding sites, and full-length protein constructs that would more accurately represent physiological interactions.