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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

Inhibition of HIV-1 infection by BI-2, PF74 and PF74 derivatives.(A) Titration of PF74 or indicated derivatives onto HeLa cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vector. For each titration, infectivity is normalised to the level in the absence of inhibitor (100%). (B) Correlation between the IC90 values derived from (A) and the Kd values for purified hexameric capsid as calculated by ITC (Figure 8). The values are highly correlative with a Pearson correlation coefficient of 0.9928 and a P-value of 0.0007. (C) Titration of PF74 or BI-2 onto cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vectors, normalized as in (A). (D) Data from (C) but with infectivity plotted against drug concentration divided by affinity to hexamer as calculated by ITC. (E) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors 48 h post-infection and levels of reverse transcription (RT) 4 h post-infection under conditions of PF74 inhibition. For both measures, data are normalized to the values obtained in the absence of inhibitor. (F) Data from (E), except plotted against the calculated level of CA occupancy by PF74. The occupancy was calculated assuming that there are 1500 free sites per capsid and that number of free sites = 1500(1−([PF74]/Kd[PF74])) (G) Infectivity and reverse transcription in cells infected in the presence of PF74 (wash) or when removed after four hours (wash out). Reverse transcription was then measured after a further 4 h, while infectivity was determined 48 h post-infection. (H) Levels of reverse transcription 4 h post-infection of HeLa cells in the presence of PF74 and its derivatives at 20× IC9o drug concentrations.
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ppat-1004459-g009: Inhibition of HIV-1 infection by BI-2, PF74 and PF74 derivatives.(A) Titration of PF74 or indicated derivatives onto HeLa cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vector. For each titration, infectivity is normalised to the level in the absence of inhibitor (100%). (B) Correlation between the IC90 values derived from (A) and the Kd values for purified hexameric capsid as calculated by ITC (Figure 8). The values are highly correlative with a Pearson correlation coefficient of 0.9928 and a P-value of 0.0007. (C) Titration of PF74 or BI-2 onto cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vectors, normalized as in (A). (D) Data from (C) but with infectivity plotted against drug concentration divided by affinity to hexamer as calculated by ITC. (E) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors 48 h post-infection and levels of reverse transcription (RT) 4 h post-infection under conditions of PF74 inhibition. For both measures, data are normalized to the values obtained in the absence of inhibitor. (F) Data from (E), except plotted against the calculated level of CA occupancy by PF74. The occupancy was calculated assuming that there are 1500 free sites per capsid and that number of free sites = 1500(1−([PF74]/Kd[PF74])) (G) Infectivity and reverse transcription in cells infected in the presence of PF74 (wash) or when removed after four hours (wash out). Reverse transcription was then measured after a further 4 h, while infectivity was determined 48 h post-infection. (H) Levels of reverse transcription 4 h post-infection of HeLa cells in the presence of PF74 and its derivatives at 20× IC9o drug concentrations.

Mentions: The PF74 derivatives described above also provided an opportunity to test the correlation between compound affinity and potency in infection experiments. As noted earlier, there is a discrepancy in the literature between the published affinity of PF74 and its potency and also between the potency of PF74 and BI-2. When tested, all PF74 derivatives with weaker affinity to hexamer had a correspondingly weaker ability to inhibit HIV-1 infection (Figure 9A). Plotting the IC90 for infection against compound affinity gives a Pearson correlation coefficient of 0.9928 and a P-value of 0.0007, demonstrating that the relationship between affinity to hexamer and potency is highly significant (Figure 9B). These results also suggest that a different affinity for capsid is the reason why PF74 is a more potent inhibitor than BI-2 (Figure 9C). Indeed, when data are normalized using our measurements for drug affinity to hexamer it is apparent that the higher affinity of PF74 completely accounts for the difference between the compounds (Figure 9D).


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)

Inhibition of HIV-1 infection by BI-2, PF74 and PF74 derivatives.(A) Titration of PF74 or indicated derivatives onto HeLa cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vector. For each titration, infectivity is normalised to the level in the absence of inhibitor (100%). (B) Correlation between the IC90 values derived from (A) and the Kd values for purified hexameric capsid as calculated by ITC (Figure 8). The values are highly correlative with a Pearson correlation coefficient of 0.9928 and a P-value of 0.0007. (C) Titration of PF74 or BI-2 onto cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vectors, normalized as in (A). (D) Data from (C) but with infectivity plotted against drug concentration divided by affinity to hexamer as calculated by ITC. (E) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors 48 h post-infection and levels of reverse transcription (RT) 4 h post-infection under conditions of PF74 inhibition. For both measures, data are normalized to the values obtained in the absence of inhibitor. (F) Data from (E), except plotted against the calculated level of CA occupancy by PF74. The occupancy was calculated assuming that there are 1500 free sites per capsid and that number of free sites = 1500(1−([PF74]/Kd[PF74])) (G) Infectivity and reverse transcription in cells infected in the presence of PF74 (wash) or when removed after four hours (wash out). Reverse transcription was then measured after a further 4 h, while infectivity was determined 48 h post-infection. (H) Levels of reverse transcription 4 h post-infection of HeLa cells in the presence of PF74 and its derivatives at 20× IC9o drug concentrations.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004459-g009: Inhibition of HIV-1 infection by BI-2, PF74 and PF74 derivatives.(A) Titration of PF74 or indicated derivatives onto HeLa cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vector. For each titration, infectivity is normalised to the level in the absence of inhibitor (100%). (B) Correlation between the IC90 values derived from (A) and the Kd values for purified hexameric capsid as calculated by ITC (Figure 8). The values are highly correlative with a Pearson correlation coefficient of 0.9928 and a P-value of 0.0007. (C) Titration of PF74 or BI-2 onto cells infected with VSV-G pseudotyped GFP-encoding HIV-1 vectors, normalized as in (A). (D) Data from (C) but with infectivity plotted against drug concentration divided by affinity to hexamer as calculated by ITC. (E) Titres of VSV-G pseudotyped GFP-encoding HIV-1 vectors 48 h post-infection and levels of reverse transcription (RT) 4 h post-infection under conditions of PF74 inhibition. For both measures, data are normalized to the values obtained in the absence of inhibitor. (F) Data from (E), except plotted against the calculated level of CA occupancy by PF74. The occupancy was calculated assuming that there are 1500 free sites per capsid and that number of free sites = 1500(1−([PF74]/Kd[PF74])) (G) Infectivity and reverse transcription in cells infected in the presence of PF74 (wash) or when removed after four hours (wash out). Reverse transcription was then measured after a further 4 h, while infectivity was determined 48 h post-infection. (H) Levels of reverse transcription 4 h post-infection of HeLa cells in the presence of PF74 and its derivatives at 20× IC9o drug concentrations.
Mentions: The PF74 derivatives described above also provided an opportunity to test the correlation between compound affinity and potency in infection experiments. As noted earlier, there is a discrepancy in the literature between the published affinity of PF74 and its potency and also between the potency of PF74 and BI-2. When tested, all PF74 derivatives with weaker affinity to hexamer had a correspondingly weaker ability to inhibit HIV-1 infection (Figure 9A). Plotting the IC90 for infection against compound affinity gives a Pearson correlation coefficient of 0.9928 and a P-value of 0.0007, demonstrating that the relationship between affinity to hexamer and potency is highly significant (Figure 9B). These results also suggest that a different affinity for capsid is the reason why PF74 is a more potent inhibitor than BI-2 (Figure 9C). Indeed, when data are normalized using our measurements for drug affinity to hexamer it is apparent that the higher affinity of PF74 completely accounts for the difference between the compounds (Figure 9D).

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