Retroviral Gag protein-RNA interactions: Implications for specific genomic RNA packaging and virion assembly

Abstract

Retroviral Gag proteins are responsible for coordinating many aspects of virion assembly. Gag possesses two distinct nucleic acid binding domains, matrix (MA) and nucleocapsid (NC). One of the critical functions of Gag is to specifically recognize, bind, and package the retroviral genomic RNA (gRNA) into assembling virions. Gag interactions with cellular RNAs have also been shown to regulate aspects of assembly. Recent results have shed light on the role of MA and NC domain interactions with nucleic acids, and how they jointly function to ensure packaging of the retroviral gRNA. Here, we will review the literature regarding RNA interactions with NC, MA, as well as overall mechanisms employed by Gag to interact with RNA. The discussion focuses on human immunodeficiency virus type-1, but other retroviruses will also be discussed. A model is presented combining all of the available data summarizing the various factors and layers of selection Gag employs to ensure specific gRNA packaging and correct virion assembly.

Keywords: Assembly; Gag; RNA; Retroviruses; gRNA packaging.

Fig. 1

Retroviral Gag domain organization. The domains present in all retroviruses, matrix (MA), capsid N-terminal domain (CA-NTD), capsid C-terminal domain (CA-CTD), and nucleocapsid (NC), as well as the various spacer peptides (SP) and other peptide sequences (p6 in HIV-1, p2 and p10 in RSV, p4 in BLV, and p12 in MLV) are illustrated. RSV Gag additionally contains the enzymatic protease (PR) domain located at its C-terminus. MA is shown in dark blue, CA-NTD in dark green, CA-CTD in light green, NC in maroon, SP1 and p2 in black, and p6 and PR in grey, while SP2, p10, SP, p4, and p12 are all shown in light blue.

Fig. 2

Proposed retroviral RNA Psi (Ψ) domains. Secondary structures of the known Psi domain of MLV (Shinnick strain) (A), RSV (Prague C strain) (B), and a portion of the HIV-1 Psi domain (NL4-3 strain) (C) are shown. The numbering corresponds to the nt number in the context of the genomic RNAs. The boxes indicate sites of NC interaction that are believed to be involved in recognition of the RNA element for specific packaging. In the case of RSV, the minimal 82-nt packaging element known as µPsi is boxed.

Fig. 3

Working model for specific gRNA packaging in HIV-1. This model attempts to convey the many reported mechanisms that may function together to ensure that viral particle assembly preferentially occurs on Ψ⁺ RNA, while proceeding less efficiently on Ψ⁻ RNA. HIV-1 Gag domains colored as in Fig. 1. Left: Interaction with Ψ⁺ RNA shifts the Gag conformational equilibrium to an extended conformation wherein RNA interacts with the NC domain and the MA remains bound to cellular tRNA. The role of tRNA binding in facilitating the conformational switch of Gag is unclear. Multiple NC interaction sites in close proximity on the RNA (indicated by the asterisks) lead to optimal clustering of Gag and multimerization via CA-CA interactions. The SP1 domain of Gag undergoes a coil-to-helix transition possibly upon Gag-Psi binding and/or Gag multimerization. When Gag reaches the PI(4,5)P₂-containing PM, it retains Ψ⁺ RNA, releasing tRNA to allow MA-PM binding and further assembly. In the cytoplasm, tRNA-MA interactions prevent Gag-RNA complexes from binding non-specific (PI(4,5)P₂-lacking) membranes. Right: In the case of Ψ⁻ RNA interactions, Gag interacts with RNAs (cellular or spliced viral RNAs) in either a bent or extended conformation via both the NC and MA domains. tRNA-MA interactions still prevent Gag assembly on PI(4,5)P₂-lacking membranes. Ψ⁻ RNA lacks a sufficient cluster of NC interaction sites to facilitate Gag multimerization. Gag-Ψ⁻ RNA complexes can interact with PI(4,5)P₂-containing membranes with either the MA domain alone or with both NA binding domains. In this model, higher Gag concentrations are required for the assembly on Ψ⁻ RNA to be as efficient as on Ψ⁺ RNA.