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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

Sensitivity of CA mutants to restriction by CPSF6ΔNLS or TRIM-NUP153 or depletion of TNPO3.Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV) or CPSF6ΔNLS (A), control knockdown cells (shControl) or cells depleted of TNPO3 (shTNPO3) (B), control knockdown cells (shControl) or cells expressing empty vector (EV) or TRIM-NUP153 (C). In each case infectivity is plotted as infectious units (IU)/ng p24. The data are representative of two independent experiments, each using three different virus doses.
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ppat-1004459-g006: Sensitivity of CA mutants to restriction by CPSF6ΔNLS or TRIM-NUP153 or depletion of TNPO3.Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV) or CPSF6ΔNLS (A), control knockdown cells (shControl) or cells depleted of TNPO3 (shTNPO3) (B), control knockdown cells (shControl) or cells expressing empty vector (EV) or TRIM-NUP153 (C). In each case infectivity is plotted as infectious units (IU)/ng p24. The data are representative of two independent experiments, each using three different virus doses.

Mentions: Outside of the phenylalanine binding pocket, the two ligands form distinct interactions with hexameric capsid. Both cofactors make interactions across neighbouring hexamers but while CPSF6 contacts the CTD of the second monomer, NUP153 interacts predominantly with the NTD (Figures 3 and 5). The ligands diverge after the shared phenylalanine, with CPSF6 residues 315–319 intercalating between helices 4 and 9 in neighbouring monomers and making contacts with residues Y169, L172, R173, Q179, K182, N183 and T186, including hydrogen bonds with Q179 and K182 (Figure 5C). As these second site contacts are driven by CPSF6 residues that interact via their main-chain or through hydrophobic burial, their importance is difficult to assess through side-chain mutation. However, mutants F316A and P317A both have significantly reduced binding affinity for hexamer as measured by ITC, while the introduction of charged residues by either P317D or G318R abolishes binding completely (Supplementary Figure S2). The dramatic effect of G318 mutation on CPSF6 binding correlates with the reduced ability of NLS deleted CPSF6 mutant G318A to restrict HIV-1 [35]. To complement our studies using different CPSF6 peptides, we tested the sensitivity of interface mutants to restriction by CPSF6ΔNLS. Previously, we have shown that CPSF6 interface mutants N57A, Q67A, K70A, N74D and T107A are all resistant to restriction by CPSF6ΔNLS [21]. Of the remaining interface residues, A77D and T108A were noninfectious whilst S102D and K182R escaped restriction (Figure 6A). Q179P was still sensitive to restriction by CPSF6ΔNLS; however, its hydrogen bond to the carbonyl oxygen of P317 may simply have been replaced by a hydrogen bond with the nearby side-chain of N183, thus maintaining interaction.


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)

Sensitivity of CA mutants to restriction by CPSF6ΔNLS or TRIM-NUP153 or depletion of TNPO3.Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV) or CPSF6ΔNLS (A), control knockdown cells (shControl) or cells depleted of TNPO3 (shTNPO3) (B), control knockdown cells (shControl) or cells expressing empty vector (EV) or TRIM-NUP153 (C). In each case infectivity is plotted as infectious units (IU)/ng p24. The data are representative of two independent experiments, each using three different virus doses.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214760&req=5

ppat-1004459-g006: Sensitivity of CA mutants to restriction by CPSF6ΔNLS or TRIM-NUP153 or depletion of TNPO3.Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors bearing wild type or mutant CA on HeLa cells expressing empty vector (EV) or CPSF6ΔNLS (A), control knockdown cells (shControl) or cells depleted of TNPO3 (shTNPO3) (B), control knockdown cells (shControl) or cells expressing empty vector (EV) or TRIM-NUP153 (C). In each case infectivity is plotted as infectious units (IU)/ng p24. The data are representative of two independent experiments, each using three different virus doses.
Mentions: Outside of the phenylalanine binding pocket, the two ligands form distinct interactions with hexameric capsid. Both cofactors make interactions across neighbouring hexamers but while CPSF6 contacts the CTD of the second monomer, NUP153 interacts predominantly with the NTD (Figures 3 and 5). The ligands diverge after the shared phenylalanine, with CPSF6 residues 315–319 intercalating between helices 4 and 9 in neighbouring monomers and making contacts with residues Y169, L172, R173, Q179, K182, N183 and T186, including hydrogen bonds with Q179 and K182 (Figure 5C). As these second site contacts are driven by CPSF6 residues that interact via their main-chain or through hydrophobic burial, their importance is difficult to assess through side-chain mutation. However, mutants F316A and P317A both have significantly reduced binding affinity for hexamer as measured by ITC, while the introduction of charged residues by either P317D or G318R abolishes binding completely (Supplementary Figure S2). The dramatic effect of G318 mutation on CPSF6 binding correlates with the reduced ability of NLS deleted CPSF6 mutant G318A to restrict HIV-1 [35]. To complement our studies using different CPSF6 peptides, we tested the sensitivity of interface mutants to restriction by CPSF6ΔNLS. Previously, we have shown that CPSF6 interface mutants N57A, Q67A, K70A, N74D and T107A are all resistant to restriction by CPSF6ΔNLS [21]. Of the remaining interface residues, A77D and T108A were noninfectious whilst S102D and K182R escaped restriction (Figure 6A). Q179P was still sensitive to restriction by CPSF6ΔNLS; however, its hydrogen bond to the carbonyl oxygen of P317 may simply have been replaced by a hydrogen bond with the nearby side-chain of N183, thus maintaining interaction.

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