Characterizing the Protein/RNA Interactions in the Initial Events of HIV-1 Assembly

Abstract

Transcription of the Human Immunodeficiency Virus type 1 (HIV-1) proviral DNA results in viral RNA (vRNA) with its monomeric form translated into the Gag polyprotein and its dimeric form selectively packaged into progeny viruses. vRNA constitutes less than 1% of cellular RNAs, yet the nucleocapsid (NC) region of Gag specifically recognizes the 5’-untranslated region (5’-UTR) of dimeric vRNA for effective packaging. In this study, we seek to biochemically characterize the protein/RNA interactions that mediate selective packaging of vRNA during the initial events of HIV-1 assembly. We have identified 20 NC binding sites on the dimeric 5’-UTR Core Encapsidation Signal (CES). Our crosslinking data showed binding of Gag derivatives to CES results in the formation of higher-ordered protein oligomers including hexamers. Negative stain Electron Microscopy (EM) further revealed these hexamers have a ring-like structure with a size similar to the capsid (CA) hexamer formed during viral assembly. To study how Gag interacts with the CES, we isolated a single stable hexamer by fusing a hexameric scaffolding protein, CCHex, to NC. Gel shift data reveals this CCHex-NC protein forms a 1:1 protein:RNA complex with CES. Competition binding assay shows CCHex-NC preferentially binds to dimeric CES. We plan to further elucidate the atomic structure of this protein/RNA complex using cryo-EM. Our results suggest clustering of Gag on the 5’-UTR increases the local protein concentration to promote the formation of Gag hexamers, which serves as the nucleation site to initiate selective genome packaging and viral assembly.