Limits...
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.


Related in: MedlinePlus

(A) Salt dependence of RSV Gag∆PR, RSV CANC (construct lacking RSV MA, p2, p10, and PR), and RSV MA binding to RSV MΨ (solid curves) and RSV 167 (dashed curves). Data from panel A are re-graphed in log-log plots of the apparent binding affinity (Kd) as a function of NaCl concentration for RSV Gag∆PR (B); RSV CANC (C); and RSV MA (D); Bar graphs of Kd(1M) values (E) and Zeff values (F) were determined by salt-titration assays using the indicated RSV proteins and RNAs. Kd(1M) values describe the nonelectrostatic component of binding and Zeff values describe the electrostatic component of the protein–nucleic acid interactions [46]. 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
getmorefigures.php?uid=PMC5035970&req=5

viruses-08-00256-f002: (A) Salt dependence of RSV Gag∆PR, RSV CANC (construct lacking RSV MA, p2, p10, and PR), and RSV MA binding to RSV MΨ (solid curves) and RSV 167 (dashed curves). Data from panel A are re-graphed in log-log plots of the apparent binding affinity (Kd) as a function of NaCl concentration for RSV Gag∆PR (B); RSV CANC (C); and RSV MA (D); Bar graphs of Kd(1M) values (E) and Zeff values (F) were determined by salt-titration assays using the indicated RSV proteins and RNAs. Kd(1M) values describe the nonelectrostatic component of binding and Zeff values describe the electrostatic component of the protein–nucleic acid interactions [46]. Values of three trials performed in each case are shown with the height of the bar indicating the mean value.

Mentions: To further test whether there are any mechanistic differences in RSV Gag∆PR interaction with Ψ versus non-Ψ RNAs, the salt dependence of the apparent binding affinity was measured, allowing contributions from both electrostatic and nonelectrostatic interactions to be assessed [46]. RSV Gag∆PR binding to RSV MΨ was characterized by a ~1900-fold stronger nonelectrostatic binding component (Kd(1M) = 7.1 × 10−5 M) and 3 fewer electrostatic interactions (Zeff ~ 4) than binding to RSV 167 (Kd(1M) = 1.3 × 10−1 M and Zeff = 7) (Figure 2 and Table 1). These values are comparable to those previously obtained for HIV-1 Gag∆p6 binding to HIV-1 Ψ versus TARpolyA (a non-Ψ RNA) (Table 1) [27]. Interestingly, RSV CANC binds to RSV MΨ with only a ~30-fold stronger nonelectrostatic binding component (Kd(1M) = 1.3 × 10−5 M) and the same number of electrostatic interactions (Zeff = 2.3) as RSV 167 (Kd(1M) = 4.2 × 10−4 M and Zeff = 2.8), suggesting that the MA domain is responsible for the three additional charge contacts made between Gag∆PR and non-Ψ RNA. This finding is consistent with a previous study showing that HIV-1 MA contributes to Ψ versus non-Ψ RNA discrimination by HIV-1 Gag∆p6 (Table 1) [27]. While we cannot rule out a contribution from the p2 and p10 domains not present in the CANC construct, these peptides lack basic character and are unlikely to interact with nucleic acids. To determine whether RSV MA intrinsically contributes to Gag specificity for Ψ RNA, salt-titration binding assays were performed using purified RSV MA. The RSV MA domain lacked binding specificity for RSV MΨ (Kd(1M) = 4.9 × 10+1 M and Zeff ~ 6) and bound RSV 167 with very similar affinity and charge interactions (Kd(1M) = 1.9 × 10+2 M and Zeff ~ 6) (Figure 2 and Table 1). HIV-1 MA generally binds RNA with much higher affinity than RSV MA [16,47,58] but also fails to discriminate between HIV-1 Ψ and TARpolyA RNAs based on FA salt-titration assay results [59]. Taken together, these and previous data suggest that both HIV-1 Gag∆p6 and RSV Gag∆PR interact with Ψ RNA in an NC-only binding mode using more nonelectrostatic contacts and with fewer charge interactions than binding to non-Ψ RNAs. The latter binding interaction involves additional electrostatic contacts, most likely with the MA domain. More importantly, MA is required for Gag-facilitated high-specificity binding to Ψ RNA in both retroviruses.


Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag
(A) Salt dependence of RSV Gag∆PR, RSV CANC (construct lacking RSV MA, p2, p10, and PR), and RSV MA binding to RSV MΨ (solid curves) and RSV 167 (dashed curves). Data from panel A are re-graphed in log-log plots of the apparent binding affinity (Kd) as a function of NaCl concentration for RSV Gag∆PR (B); RSV CANC (C); and RSV MA (D); Bar graphs of Kd(1M) values (E) and Zeff values (F) were determined by salt-titration assays using the indicated RSV proteins and RNAs. Kd(1M) values describe the nonelectrostatic component of binding and Zeff values describe the electrostatic component of the protein–nucleic acid interactions [46]. 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-f002: (A) Salt dependence of RSV Gag∆PR, RSV CANC (construct lacking RSV MA, p2, p10, and PR), and RSV MA binding to RSV MΨ (solid curves) and RSV 167 (dashed curves). Data from panel A are re-graphed in log-log plots of the apparent binding affinity (Kd) as a function of NaCl concentration for RSV Gag∆PR (B); RSV CANC (C); and RSV MA (D); Bar graphs of Kd(1M) values (E) and Zeff values (F) were determined by salt-titration assays using the indicated RSV proteins and RNAs. Kd(1M) values describe the nonelectrostatic component of binding and Zeff values describe the electrostatic component of the protein–nucleic acid interactions [46]. Values of three trials performed in each case are shown with the height of the bar indicating the mean value.
Mentions: To further test whether there are any mechanistic differences in RSV Gag∆PR interaction with Ψ versus non-Ψ RNAs, the salt dependence of the apparent binding affinity was measured, allowing contributions from both electrostatic and nonelectrostatic interactions to be assessed [46]. RSV Gag∆PR binding to RSV MΨ was characterized by a ~1900-fold stronger nonelectrostatic binding component (Kd(1M) = 7.1 × 10−5 M) and 3 fewer electrostatic interactions (Zeff ~ 4) than binding to RSV 167 (Kd(1M) = 1.3 × 10−1 M and Zeff = 7) (Figure 2 and Table 1). These values are comparable to those previously obtained for HIV-1 Gag∆p6 binding to HIV-1 Ψ versus TARpolyA (a non-Ψ RNA) (Table 1) [27]. Interestingly, RSV CANC binds to RSV MΨ with only a ~30-fold stronger nonelectrostatic binding component (Kd(1M) = 1.3 × 10−5 M) and the same number of electrostatic interactions (Zeff = 2.3) as RSV 167 (Kd(1M) = 4.2 × 10−4 M and Zeff = 2.8), suggesting that the MA domain is responsible for the three additional charge contacts made between Gag∆PR and non-Ψ RNA. This finding is consistent with a previous study showing that HIV-1 MA contributes to Ψ versus non-Ψ RNA discrimination by HIV-1 Gag∆p6 (Table 1) [27]. While we cannot rule out a contribution from the p2 and p10 domains not present in the CANC construct, these peptides lack basic character and are unlikely to interact with nucleic acids. To determine whether RSV MA intrinsically contributes to Gag specificity for Ψ RNA, salt-titration binding assays were performed using purified RSV MA. The RSV MA domain lacked binding specificity for RSV MΨ (Kd(1M) = 4.9 × 10+1 M and Zeff ~ 6) and bound RSV 167 with very similar affinity and charge interactions (Kd(1M) = 1.9 × 10+2 M and Zeff ~ 6) (Figure 2 and Table 1). HIV-1 MA generally binds RNA with much higher affinity than RSV MA [16,47,58] but also fails to discriminate between HIV-1 Ψ and TARpolyA RNAs based on FA salt-titration assay results [59]. Taken together, these and previous data suggest that both HIV-1 Gag∆p6 and RSV Gag∆PR interact with Ψ RNA in an NC-only binding mode using more nonelectrostatic contacts and with fewer charge interactions than binding to non-Ψ RNAs. The latter binding interaction involves additional electrostatic contacts, most likely with the MA domain. More importantly, MA is required for Gag-facilitated high-specificity binding to Ψ RNA in both retroviruses.

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.


Related in: MedlinePlus