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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 showing Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR, H132R, R155H, and HIV-1 Gag∆p6 with RSV MΨ, RSV 167, HIV-1 Ψ, and HIV-1 TARpolyA. Values of three or four trials performed in each case are shown with the height of the bar indicating the mean value.
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viruses-08-00256-f003: Bar graphs showing Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR, H132R, R155H, and HIV-1 Gag∆p6 with RSV MΨ, RSV 167, HIV-1 Ψ, and HIV-1 TARpolyA. Values of three or four trials performed in each case are shown with the height of the bar indicating the mean value.

Mentions: The chimeric H132R Gag construct interacted with RSV MΨ with a highly specific Kd(1M) = 7.2 × 10−5 M, which was very similar to RSV Gag∆PR, whereas the Zeff was slightly higher at ~5 (versus ~4) (Figure 3 and Table 2). Furthermore, similar to RSV Gag∆PR, H132R interacted with RSV 167 nonspecifically with a Kd(1M) ~ 1.9 M and a large Zeff of ~9. These data suggest that despite the presence of a heterologous HIV-1 MA domain, RSV Gag was still able to distinguish Ψ from non-Ψ RNA. In the reciprocal experiment, R155H maintained a specific Kd(1M) = 3.1 × 10−5 M with HIV-1 Ψ RNA and a low Zeff of ~4, while the chimera’s interaction with HIV-1 TARpolyA was characterized by a nonspecific Kd(1M) ~ 2.6 M and high Zeff of ~9 (Figure 3 and Table 2). Thus, HIV-1 Gag was also capable of accommodating another retroviral MA domain while still retaining the ability to differentiate Ψ from non-Ψ RNA. Taken together, these data indicate an important, but non-specific role for MA in Ψ recognition, with RNA specificity likely arising from the identity of the NC domain.


Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag
Bar graphs showing Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR, H132R, R155H, and HIV-1 Gag∆p6 with RSV MΨ, RSV 167, HIV-1 Ψ, and HIV-1 TARpolyA. Values of three or four 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-f003: Bar graphs showing Kd(1M) values (A) and Zeff values (B) determined from salt-titration assays with RSV Gag∆PR, H132R, R155H, and HIV-1 Gag∆p6 with RSV MΨ, RSV 167, HIV-1 Ψ, and HIV-1 TARpolyA. Values of three or four trials performed in each case are shown with the height of the bar indicating the mean value.
Mentions: The chimeric H132R Gag construct interacted with RSV MΨ with a highly specific Kd(1M) = 7.2 × 10−5 M, which was very similar to RSV Gag∆PR, whereas the Zeff was slightly higher at ~5 (versus ~4) (Figure 3 and Table 2). Furthermore, similar to RSV Gag∆PR, H132R interacted with RSV 167 nonspecifically with a Kd(1M) ~ 1.9 M and a large Zeff of ~9. These data suggest that despite the presence of a heterologous HIV-1 MA domain, RSV Gag was still able to distinguish Ψ from non-Ψ RNA. In the reciprocal experiment, R155H maintained a specific Kd(1M) = 3.1 × 10−5 M with HIV-1 Ψ RNA and a low Zeff of ~4, while the chimera’s interaction with HIV-1 TARpolyA was characterized by a nonspecific Kd(1M) ~ 2.6 M and high Zeff of ~9 (Figure 3 and Table 2). Thus, HIV-1 Gag was also capable of accommodating another retroviral MA domain while still retaining the ability to differentiate Ψ from non-Ψ RNA. Taken together, these data indicate an important, but non-specific role for MA in Ψ recognition, with RNA specificity likely arising from the identity of the NC domain.

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.