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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.


Related in: MedlinePlus

Rous sarcoma virus (RSV) RNA, and RSV and human immunodeficiency virus type 1 (HIV-1) protein constructs used in this work. (A) Predicted secondary structures of RSV MΨ (top) and RSV 167 (bottom) derived from nucleotides (nt) 156–315 and 1249–1409 of the RSV Prague C genome, respectively. Four mutant MΨ constructs are indicated by the boxed nt and arrows: UGCG to GAGA, GG to CC, A197G, and a triple mutation AGC to UCA. Additional nt at the 5′ and 3′ ends of each RNA that are not present in the RSV genome are shown in gray; (B) RSV and HIV-1 Gag constructs used in this work, with full-length wild-type Gag shown at the top of each set for comparison. Matrix (MA), capsid (CA) and nucleocapsid (NC), as well as various spacer peptides (SP, SP1, and SP2), the HIV-1 p6 domain, and the RSV p2, p10, and protease domain (PR) comprise these constructs. In the case of the two chimeric constructs, H132R and R155H, the residues at the junctions are explicitly shown. H132R contains the 132-residue HIV-1 MA in place of RSV MA in the context of RSV Gag, and R155H contains the 155-residue RSV MA in place of HIV-1 MA in the context of HIV-1 Gag.
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viruses-08-00256-f001: Rous sarcoma virus (RSV) RNA, and RSV and human immunodeficiency virus type 1 (HIV-1) protein constructs used in this work. (A) Predicted secondary structures of RSV MΨ (top) and RSV 167 (bottom) derived from nucleotides (nt) 156–315 and 1249–1409 of the RSV Prague C genome, respectively. Four mutant MΨ constructs are indicated by the boxed nt and arrows: UGCG to GAGA, GG to CC, A197G, and a triple mutation AGC to UCA. Additional nt at the 5′ and 3′ ends of each RNA that are not present in the RSV genome are shown in gray; (B) RSV and HIV-1 Gag constructs used in this work, with full-length wild-type Gag shown at the top of each set for comparison. Matrix (MA), capsid (CA) and nucleocapsid (NC), as well as various spacer peptides (SP, SP1, and SP2), the HIV-1 p6 domain, and the RSV p2, p10, and protease domain (PR) comprise these constructs. In the case of the two chimeric constructs, H132R and R155H, the residues at the junctions are explicitly shown. H132R contains the 132-residue HIV-1 MA in place of RSV MA in the context of RSV Gag, and R155H contains the 155-residue RSV MA in place of HIV-1 MA in the context of HIV-1 Gag.

Mentions: A hallmark of retroviruses is their ability to reverse transcribe single-stranded genomic RNA (gRNA) into double-stranded DNA for subsequent integration into the host cell genome. Following nuclear transcription and export into the cytoplasm, highly selective packaging of the full-length, unspliced gRNA into newly assembled virus particles is achieved, despite the presence of spliced viral RNA and a vast excess of cellular RNA [1,2]. Human immunodeficiency virus type 1 (HIV-1) packages only a single, dimeric copy of its gRNA [3,4,5,6]. Specific HIV-1 gRNA packaging is achieved, in part, via the interaction between the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein and the “psi” (Ψ) packaging signal, located in the gRNA 5′-untranslated region (5′UTR) [7,8]. In addition to the NC domain, HIV-1 Gag consists of matrix (MA), capsid (CA), two spacer peptides, and p6 (Figure 1B). In the context of the polyprotein, MA targets the assembling virus to the plasma membrane (PM) [9], CA is involved in Gag multimerization associated with immature viral particle formation [10,11], NC functions as a nucleic acid chaperone in addition to its role in gRNA packaging [12,13,14,15,16,17,18], and the p6 domain recruits factors required for viral fission from the cell [19,20]. Importantly, Gag’s main structural domains provide functional redundancy to ensure proper viral assembly through Gag–nucleic acid, Gag–Gag, and Gag–membrane interactions [21,22].


Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag
Rous sarcoma virus (RSV) RNA, and RSV and human immunodeficiency virus type 1 (HIV-1) protein constructs used in this work. (A) Predicted secondary structures of RSV MΨ (top) and RSV 167 (bottom) derived from nucleotides (nt) 156–315 and 1249–1409 of the RSV Prague C genome, respectively. Four mutant MΨ constructs are indicated by the boxed nt and arrows: UGCG to GAGA, GG to CC, A197G, and a triple mutation AGC to UCA. Additional nt at the 5′ and 3′ ends of each RNA that are not present in the RSV genome are shown in gray; (B) RSV and HIV-1 Gag constructs used in this work, with full-length wild-type Gag shown at the top of each set for comparison. Matrix (MA), capsid (CA) and nucleocapsid (NC), as well as various spacer peptides (SP, SP1, and SP2), the HIV-1 p6 domain, and the RSV p2, p10, and protease domain (PR) comprise these constructs. In the case of the two chimeric constructs, H132R and R155H, the residues at the junctions are explicitly shown. H132R contains the 132-residue HIV-1 MA in place of RSV MA in the context of RSV Gag, and R155H contains the 155-residue RSV MA in place of HIV-1 MA in the context of HIV-1 Gag.
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viruses-08-00256-f001: Rous sarcoma virus (RSV) RNA, and RSV and human immunodeficiency virus type 1 (HIV-1) protein constructs used in this work. (A) Predicted secondary structures of RSV MΨ (top) and RSV 167 (bottom) derived from nucleotides (nt) 156–315 and 1249–1409 of the RSV Prague C genome, respectively. Four mutant MΨ constructs are indicated by the boxed nt and arrows: UGCG to GAGA, GG to CC, A197G, and a triple mutation AGC to UCA. Additional nt at the 5′ and 3′ ends of each RNA that are not present in the RSV genome are shown in gray; (B) RSV and HIV-1 Gag constructs used in this work, with full-length wild-type Gag shown at the top of each set for comparison. Matrix (MA), capsid (CA) and nucleocapsid (NC), as well as various spacer peptides (SP, SP1, and SP2), the HIV-1 p6 domain, and the RSV p2, p10, and protease domain (PR) comprise these constructs. In the case of the two chimeric constructs, H132R and R155H, the residues at the junctions are explicitly shown. H132R contains the 132-residue HIV-1 MA in place of RSV MA in the context of RSV Gag, and R155H contains the 155-residue RSV MA in place of HIV-1 MA in the context of HIV-1 Gag.
Mentions: A hallmark of retroviruses is their ability to reverse transcribe single-stranded genomic RNA (gRNA) into double-stranded DNA for subsequent integration into the host cell genome. Following nuclear transcription and export into the cytoplasm, highly selective packaging of the full-length, unspliced gRNA into newly assembled virus particles is achieved, despite the presence of spliced viral RNA and a vast excess of cellular RNA [1,2]. Human immunodeficiency virus type 1 (HIV-1) packages only a single, dimeric copy of its gRNA [3,4,5,6]. Specific HIV-1 gRNA packaging is achieved, in part, via the interaction between the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein and the “psi” (Ψ) packaging signal, located in the gRNA 5′-untranslated region (5′UTR) [7,8]. In addition to the NC domain, HIV-1 Gag consists of matrix (MA), capsid (CA), two spacer peptides, and p6 (Figure 1B). In the context of the polyprotein, MA targets the assembling virus to the plasma membrane (PM) [9], CA is involved in Gag multimerization associated with immature viral particle formation [10,11], NC functions as a nucleic acid chaperone in addition to its role in gRNA packaging [12,13,14,15,16,17,18], and the p6 domain recruits factors required for viral fission from the cell [19,20]. Importantly, Gag’s main structural domains provide functional redundancy to ensure proper viral assembly through Gag–nucleic acid, Gag–Gag, and Gag–membrane interactions [21,22].

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