Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a "Ψ" packaging signal located in the gRNA 5'-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5'-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag-RNA interactions.

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 5'-untranslated region (5'UTR) 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.

Fluorescence anisotropy-based salt-titration approach to characterize protein-nucleic acid interactions.

Many proteins bind nucleic acids (NA) via cationic residues that interact electrostatically with the anionic phosphate backbone of RNA or DNA. These electrostatic interactions are often insensitive to NA sequence and structure, but confer strong salt dependence to the binding interactions. In contrast, salt-independent non-electrostatic contacts reflect more specific binding interactions. Proteins with multiple cationic NA-binding domains connected by flexible linkers, such as the HIV-1 Gag polyprotein, may bind different NA molecules in distinct ways. For example, Gag binding to the Psi-packaging signal of the HIV-1 RNA genome optimizes the specific non-electrostatic binding component of this protein-RNA interaction. In contrast, Gag binding to a non-psi RNA optimizes the electrostatic interactions at the expense of specific contacts. Here, we describe a fluorescence anisotropy-based salt-titration approach that allows complete characterization of both electrostatic and non-electrostatic binding components for any protein-NA complex in a quantitative manner within a single assay.

Mechanistic differences between nucleic acid chaperone activities of the Gag proteins of Rous sarcoma virus and human immunodeficiency virus type 1 are attributed to the MA domain.

Host cell tRNAs are recruited for use as primers to initiate reverse transcription in retroviruses. Human immunodeficiency virus type 1 (HIV-1) uses tRNA(Lys3) as the replication primer, whereas Rous sarcoma virus (RSV) uses tRNA(Trp). The nucleic acid (NA) chaperone function of the nucleocapsid (NC) domain of HIV-1 Gag is responsible for annealing tRNA(Lys3) to the genomic RNA (gRNA) primer binding site (PBS). Compared to HIV-1, little is known about the chaperone activity of RSV Gag. In this work, using purified RSV Gag containing an N-terminal His tag and a deletion of the majority of the protease domain (H6.Gag.3h), gel shift assays were used to monitor the annealing of tRNA(Trp) to a PBS-containing RSV RNA. Here, we show that similar to HIV-1 Gag lacking the p6 domain (GagΔp6), RSV H6.Gag.3h is a more efficient chaperone on a molar basis than NC; however, in contrast to the HIV-1 system, both RSV H6.Gag.3h and NC have comparable annealing rates at protein saturation. The NC domain of RSV H6.Gag.3h is required for annealing, whereas deletion of the matrix (MA) domain, which stimulates the rate of HIV-1 GagΔp6 annealing, has little effect on RSV H6.Gag.3h chaperone function. Competition assays confirmed that RSV MA binds inositol phosphates (IPs), but in contrast to HIV-1 GagΔp6, IPs do not stimulate RSV H6.Gag.3h chaperone activity unless the MA domain is replaced with HIV-1 MA. We conclude that differences in the MA domains are primarily responsible for mechanistic differences in RSV and HIV-1 Gag NA chaperone function. Importance: Mounting evidence suggests that the Gag polyprotein is responsible for annealing primer tRNAs to the PBS to initiate reverse transcription in retroviruses, but only HIV-1 Gag chaperone activity has been demonstrated in vitro. Understanding RSV Gag's NA chaperone function will allow us to determine whether there is a common mechanism among retroviruses. This report shows for the first time that full-length RSV Gag lacking the protease domain is a highly efficient NA chaperone in vitro, and NC is required for this activity. In contrast to results obtained for HIV-1 Gag, due to the weak nucleic acid binding affinity of the RSV MA domain, inositol phosphates do not regulate RSV Gag-facilitated tRNA annealing despite the fact that they bind to MA. These studies provide insight into the viral regulation of tRNA primer annealing, which is a potential target for antiretroviral therapy.