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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

Structures of PF74 derivatives used in this study and their affinities of binding to CA.Affinities of binding to hexamer as measured by ITC are shown. No binding was detected between PF74_1 and PF74_2 to HIV-1 CA NTD, therefore the interaction with hexamer was not tested.
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ppat-1004459-g008: Structures of PF74 derivatives used in this study and their affinities of binding to CA.Affinities of binding to hexamer as measured by ITC are shown. No binding was detected between PF74_1 and PF74_2 to HIV-1 CA NTD, therefore the interaction with hexamer was not tested.

Mentions: The different PF74 orientations in the monomer versus hexamer structures as well as the resulting new interactions raise the question of what capsid contacts are essential for drug binding. To investigate this we adopted a chemical genetics approach to avoid the pleiotropic effects of capsid mutants. We first attempted to assess the contribution of the 2-methylindole group by removing it either before or after the amine. No binding to CA NTD could be observed by ITC for either compound lacking the 2-methylindole (Figure 8, PF74_1 and PF74_2). This result suggests that hydrogen bonding to N57 is not sufficient for CA interaction and that Q63 and R173 are important for PF74 binding. To dissect the contributions of interaction with Q63 and R173 we replaced the 2-methylindole with pyrrole, furan, thiophene and tetrahydrofuran moieties that have altered aromaticity and hydrogen bond propensities (Figure 8). The pyrrole-containing compound (PF74_3) lacks the 6-membered ring found in 2-methylindole but retains the hydrogen-bond donor. This compound had a 46-fold reduction in affinity to hexamer, indicating that much of the enhanced binding to hexamer was lost (Figure 8; PF74_3). The furan and thiophene (PF74_4 and 5) compounds, which have a hydrogen bond acceptor instead of donor, interacted with hexamer with similarly weakened affinities as the pyrrole (a reduction of 94-fold and 54-fold respectively). This is probably because the electronegative element coordinates a glutamine (Q63), which is able to hydrogen-bond to both hydrogen donors (N) and hydrogen acceptors (O, S). Finally, the tetrahydrofuran derivative has lost aromaticity and therefore the ability to form cation-π interactions and has the weakest affinity for hexamer, reduced by >1000-fold. These results suggest that the binding of PF74 to hexamer is mediated primarily by a stacking interaction with R173.


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)

Structures of PF74 derivatives used in this study and their affinities of binding to CA.Affinities of binding to hexamer as measured by ITC are shown. No binding was detected between PF74_1 and PF74_2 to HIV-1 CA NTD, therefore the interaction with hexamer was not tested.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004459-g008: Structures of PF74 derivatives used in this study and their affinities of binding to CA.Affinities of binding to hexamer as measured by ITC are shown. No binding was detected between PF74_1 and PF74_2 to HIV-1 CA NTD, therefore the interaction with hexamer was not tested.
Mentions: The different PF74 orientations in the monomer versus hexamer structures as well as the resulting new interactions raise the question of what capsid contacts are essential for drug binding. To investigate this we adopted a chemical genetics approach to avoid the pleiotropic effects of capsid mutants. We first attempted to assess the contribution of the 2-methylindole group by removing it either before or after the amine. No binding to CA NTD could be observed by ITC for either compound lacking the 2-methylindole (Figure 8, PF74_1 and PF74_2). This result suggests that hydrogen bonding to N57 is not sufficient for CA interaction and that Q63 and R173 are important for PF74 binding. To dissect the contributions of interaction with Q63 and R173 we replaced the 2-methylindole with pyrrole, furan, thiophene and tetrahydrofuran moieties that have altered aromaticity and hydrogen bond propensities (Figure 8). The pyrrole-containing compound (PF74_3) lacks the 6-membered ring found in 2-methylindole but retains the hydrogen-bond donor. This compound had a 46-fold reduction in affinity to hexamer, indicating that much of the enhanced binding to hexamer was lost (Figure 8; PF74_3). The furan and thiophene (PF74_4 and 5) compounds, which have a hydrogen bond acceptor instead of donor, interacted with hexamer with similarly weakened affinities as the pyrrole (a reduction of 94-fold and 54-fold respectively). This is probably because the electronegative element coordinates a glutamine (Q63), which is able to hydrogen-bond to both hydrogen donors (N) and hydrogen acceptors (O, S). Finally, the tetrahydrofuran derivative has lost aromaticity and therefore the ability to form cation-π interactions and has the weakest affinity for hexamer, reduced by >1000-fold. These results suggest that the binding of PF74 to hexamer is mediated primarily by a stacking interaction with R173.

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