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Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.

Price AJ, Jacques DA, McEwan WA, Fletcher AJ, Essig S, Chin JW, Halambage UD, Aiken C, James LC - PLoS Pathog. (2014)

Bottom Line: The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation.NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency.These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, United Kingdom.

ABSTRACT
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

No MeSH data available.


Related in: MedlinePlus

CPSF6 and NUP153 interact both within and between CA monomers.Detailed views of the interactions between the ligands and the binding site within hexameric capsid are shown. In each case two adjacent monomers of the hexamer are colored gray and teal, with the side chains of specific contacting residues displayed and labelled in standard text. Potential hydrogen bonds with ligands are indicated by dashed lines. The upper panels (A and B) focus on interactions that occur in the first binding site (within one monomer), while the lower panels (C and D), show interactions in the second binding site (with the second monomer). CPSF6 is shown in yellow and NUP153 in pink, with important ligand residues labelled in bold and italic text.
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ppat-1004459-g005: CPSF6 and NUP153 interact both within and between CA monomers.Detailed views of the interactions between the ligands and the binding site within hexameric capsid are shown. In each case two adjacent monomers of the hexamer are colored gray and teal, with the side chains of specific contacting residues displayed and labelled in standard text. Potential hydrogen bonds with ligands are indicated by dashed lines. The upper panels (A and B) focus on interactions that occur in the first binding site (within one monomer), while the lower panels (C and D), show interactions in the second binding site (with the second monomer). CPSF6 is shown in yellow and NUP153 in pink, with important ligand residues labelled in bold and italic text.

Mentions: CPSF6 and NUP153 have been identified as disparate host cofactors that nevertheless share a common binding site on HIV-1 CA. However, whilst they share interactions within the first, monomeric, binding site they make distinct interactions to the second site, in the context of hexameric CA (Figure 4). Within one monomer, CPSF6 interacts with N53, L56, N57, M66, Q67, L69, K70, I73, N74, A77, S102, A105, G106, T107, T108 and Y130 (Figure 5A). Of these interactions, binding and restriction studies have confirmed an important role for N57, M66, K70, N74 and T107 [21]. Meanwhile, NUP153 interacts with residues N53, L56, N57, Q63, M66, Q67, L69, K70, I73, A105, G106, T107 and Y130 (Figure 5B). There was no observed interaction between NUP153 and CA N74, supporting findings that mutation N74D specifically abolishes CPSF6 binding [20], [21]. The overlap in residue usage reflects similarities in the way in which CPSF6 and NUP153 engage capsid within the monomeric binding site. Previously, we have shown that CPSF6 residue F321, which occupies the same pocket as the phenyl rings of PF74 and BI-2, is critical for interaction of CPSF6 with capsid [21]. We confirmed that CPSF6 F321 is critical for hexamer binding, as mutation to alanine abolishes interaction (Supplementary Figure S2). In NUP153, an equivalent interaction is mediated by F1417, which superposes closely with CPSF6 F321 (Figure 3A and Figure 5A and B). ITC binding experiments confirm that this NUP153 residue is essential, as F1417A has no measurable binding to hexamer (Supplementary Figure S2). The importance of F321 in CPSF6 and F1417 in NUP153 is in part to orient the main-chain for hydrogen bond interactions with the side-chain of N57 (Figure 5A and B). This most likely explains why capsid mutant N57A was identified as critical for co-immunoprecipitation of NUP153 with CA NTD and for restriction by a TRIM-NUP153 fusion [20]. Within the monomer binding site, NUP153 makes unique contacts with Q63, while CPSF6 makes unique contacts with N74, A77, S102 and T108 (Figure 5A and B). The unique interaction of CPSF6 with N74 explains why mutation N74D prevents binding to CPSF6 but allows co-immunoprecipitation with NUP153 and remains sensitive to TRIM-NUP153 restriction [20].


Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.

Price AJ, Jacques DA, McEwan WA, Fletcher AJ, Essig S, Chin JW, Halambage UD, Aiken C, James LC - PLoS Pathog. (2014)

CPSF6 and NUP153 interact both within and between CA monomers.Detailed views of the interactions between the ligands and the binding site within hexameric capsid are shown. In each case two adjacent monomers of the hexamer are colored gray and teal, with the side chains of specific contacting residues displayed and labelled in standard text. Potential hydrogen bonds with ligands are indicated by dashed lines. The upper panels (A and B) focus on interactions that occur in the first binding site (within one monomer), while the lower panels (C and D), show interactions in the second binding site (with the second monomer). CPSF6 is shown in yellow and NUP153 in pink, with important ligand residues labelled in bold and italic text.
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ppat-1004459-g005: CPSF6 and NUP153 interact both within and between CA monomers.Detailed views of the interactions between the ligands and the binding site within hexameric capsid are shown. In each case two adjacent monomers of the hexamer are colored gray and teal, with the side chains of specific contacting residues displayed and labelled in standard text. Potential hydrogen bonds with ligands are indicated by dashed lines. The upper panels (A and B) focus on interactions that occur in the first binding site (within one monomer), while the lower panels (C and D), show interactions in the second binding site (with the second monomer). CPSF6 is shown in yellow and NUP153 in pink, with important ligand residues labelled in bold and italic text.
Mentions: CPSF6 and NUP153 have been identified as disparate host cofactors that nevertheless share a common binding site on HIV-1 CA. However, whilst they share interactions within the first, monomeric, binding site they make distinct interactions to the second site, in the context of hexameric CA (Figure 4). Within one monomer, CPSF6 interacts with N53, L56, N57, M66, Q67, L69, K70, I73, N74, A77, S102, A105, G106, T107, T108 and Y130 (Figure 5A). Of these interactions, binding and restriction studies have confirmed an important role for N57, M66, K70, N74 and T107 [21]. Meanwhile, NUP153 interacts with residues N53, L56, N57, Q63, M66, Q67, L69, K70, I73, A105, G106, T107 and Y130 (Figure 5B). There was no observed interaction between NUP153 and CA N74, supporting findings that mutation N74D specifically abolishes CPSF6 binding [20], [21]. The overlap in residue usage reflects similarities in the way in which CPSF6 and NUP153 engage capsid within the monomeric binding site. Previously, we have shown that CPSF6 residue F321, which occupies the same pocket as the phenyl rings of PF74 and BI-2, is critical for interaction of CPSF6 with capsid [21]. We confirmed that CPSF6 F321 is critical for hexamer binding, as mutation to alanine abolishes interaction (Supplementary Figure S2). In NUP153, an equivalent interaction is mediated by F1417, which superposes closely with CPSF6 F321 (Figure 3A and Figure 5A and B). ITC binding experiments confirm that this NUP153 residue is essential, as F1417A has no measurable binding to hexamer (Supplementary Figure S2). The importance of F321 in CPSF6 and F1417 in NUP153 is in part to orient the main-chain for hydrogen bond interactions with the side-chain of N57 (Figure 5A and B). This most likely explains why capsid mutant N57A was identified as critical for co-immunoprecipitation of NUP153 with CA NTD and for restriction by a TRIM-NUP153 fusion [20]. Within the monomer binding site, NUP153 makes unique contacts with Q63, while CPSF6 makes unique contacts with N74, A77, S102 and T108 (Figure 5A and B). The unique interaction of CPSF6 with N74 explains why mutation N74D prevents binding to CPSF6 but allows co-immunoprecipitation with NUP153 and remains sensitive to TRIM-NUP153 restriction [20].

Bottom Line: The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation.NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency.These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, United Kingdom.

ABSTRACT
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

No MeSH data available.


Related in: MedlinePlus