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Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Bar graphs of Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR and RSV 167, WT MΨ, and MΨ RNA mutants. Values of three trials performed in each case are shown with the height of the bar indicating the mean value.
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viruses-08-00256-f004: Bar graphs of Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR and RSV 167, WT MΨ, and MΨ RNA mutants. Values of three trials performed in each case are shown with the height of the bar indicating the mean value.

Mentions: We next wanted to investigate the Ψ RNA elements required for specific recognition by Gag. In RSV, it was previously reported that mutations A197G and UGCG to GAGA (nt 217–220) in RSV µΨ (nt 156–237, Figure 1A) lead to a 1500 and 10,000-fold loss in binding affinity to RSV NC, respectively [30,44]. A separate report demonstrated that mutations GG to CC (nt 214 and 215) and a triple mutant of nt 195, 199, and 200 in RSV MΨ (hereafter referred to as MΨ Triple) (Figure 1A) lead to 20- and 50-fold losses in gRNA packaging efficiency, respectively [30]. We first tested direct binding by RSV Gag∆PR for these RSV MΨ RNA mutant constructs. All Kd values were measured to be between ~13 and 18 nM (Table S3); thus, differences in binding affinity alone were unable to explain the previously reported results. To further examine how these mutations affect RSV Gag selectivity for Ψ RNA, FA salt titration assays were performed. RSV Gag∆PR bound both RSV MΨ A197G and MΨ UGCG to GAGA with specific Kd(1M) values of ~10−5 M and low Zeff, values (~3) that were essentially indistinguishable from binding to WT Ψ (Figure 4 and Table 3). Based on the Kd(1M) values, binding to these mutant RNAs was actually more specific (~1.5–6-fold) than binding to WT Ψ RNA.


Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag
Bar graphs of Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR and RSV 167, WT MΨ, and MΨ RNA mutants. Values of three trials performed in each case are shown with the height of the bar indicating the mean value.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035970&req=5

viruses-08-00256-f004: Bar graphs of Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR and RSV 167, WT MΨ, and MΨ RNA mutants. Values of three trials performed in each case are shown with the height of the bar indicating the mean value.
Mentions: We next wanted to investigate the Ψ RNA elements required for specific recognition by Gag. In RSV, it was previously reported that mutations A197G and UGCG to GAGA (nt 217–220) in RSV µΨ (nt 156–237, Figure 1A) lead to a 1500 and 10,000-fold loss in binding affinity to RSV NC, respectively [30,44]. A separate report demonstrated that mutations GG to CC (nt 214 and 215) and a triple mutant of nt 195, 199, and 200 in RSV MΨ (hereafter referred to as MΨ Triple) (Figure 1A) lead to 20- and 50-fold losses in gRNA packaging efficiency, respectively [30]. We first tested direct binding by RSV Gag∆PR for these RSV MΨ RNA mutant constructs. All Kd values were measured to be between ~13 and 18 nM (Table S3); thus, differences in binding affinity alone were unable to explain the previously reported results. To further examine how these mutations affect RSV Gag selectivity for Ψ RNA, FA salt titration assays were performed. RSV Gag∆PR bound both RSV MΨ A197G and MΨ UGCG to GAGA with specific Kd(1M) values of ~10−5 M and low Zeff, values (~3) that were essentially indistinguishable from binding to WT Ψ (Figure 4 and Table 3). Based on the Kd(1M) values, binding to these mutant RNAs was actually more specific (~1.5–6-fold) than binding to WT Ψ RNA.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.