Functional equivalence of retroviral MA domains in facilitating Psi RNA binding specificity by Gag

Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The “psi” (ψ) element within the 5ʹ-untranslated region (5ʹUTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for ψ versus non-ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1) Gag bound to ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA) domain was less effective at discriminating ψ from non-ψ RNA. We now find that Rous sarcoma virus (RSV) Gag also effectively discriminates RSV ψ from non-ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate ψ RNAs. Using ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating ψ RNA selectivity by Gag, as well as ψ elements that promote this selectivity.