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Forced virus evolution reveals functional crosstalk between the disulfide bonded region and membrane proximal ectodomain region of HIV-1 gp41.

Khasawneh AI, Laumaea A, Harrison DN, Bellamy-McIntyre AK, Drummer HE, Poumbourios P - Retrovirology (2013)

Bottom Line: In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site.The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored.The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virus Fusion Laboratory, Burnet Institute, Prahran, VIC 3004, Australia.

ABSTRACT

Background: The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site.

Results: The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER.

Conclusions: The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus.

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Amino acid changes at position 674 modeled on the dodecylphosphocholine-associated MPER peptide (PDB entry 2PV6). The Asp-674 (A), Glu-674 (B), and Asn-674 (C) models were produced with Swiss Model and drawn with Pymol. The N- and C-terminal helical segments are shown in purple and magenta respectively, while Phe-673 that forms part of the hinge region is in yellow. The aromatic layer and Ile-675, which are associated with the hydrophobic phase of the lipid are indicated. The N-terminus is to the left.
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Figure 8: Amino acid changes at position 674 modeled on the dodecylphosphocholine-associated MPER peptide (PDB entry 2PV6). The Asp-674 (A), Glu-674 (B), and Asn-674 (C) models were produced with Swiss Model and drawn with Pymol. The N- and C-terminal helical segments are shown in purple and magenta respectively, while Phe-673 that forms part of the hinge region is in yellow. The aromatic layer and Ile-675, which are associated with the hydrophobic phase of the lipid are indicated. The N-terminus is to the left.

Mentions: Nuclear magnetic resonance studies of synthetic MPER peptides suggest that in membranes, the MPER is a metastable L-shaped structure comprised of N- and C-terminal helical segments that are connected via a hinge composed of Phe-673, which is buried in the lipid phase, and a polar residue at position 674, which is solvent-exposed (Figure 8) (PDB entry, 2PV6 [22]. The C-terminal helix is likely to interact with the transmembrane domain and cholesterol in the lipid phase via the Leu-679-Trp-Tyr-Ile-Lys cholesterol recruitment motif [45-48], while the N-terminal helix represents a more flexible segment that might be in a metastable state and contains 3 Trp residues that are critical for membrane fusion [25]. Molecular modeling predicts that the original Asp side chain at position 674 will hydrogen bond via Oδ1 with the backbone amides of Asn-677 and Ile-675 in 17 of 17 MPER conformers (Figure 8A), thereby conferring rigidity to the interhelical hinge. By contrast, an additional methyl group within the Glu-674 sidechain moves the terminal carboxylate out of hydrogen bonding range in 15/17 conformers, consistent with hinge flexibility (Figure 8B). The boost to the infectivity of WL/KH cell-free virus upon addition of D674E may therefore be related to increased MPER flexibility. Interestingly, the addition of D674E to W596L/K601H only marginally improved gp120-association for cell-free virions whereas single-cycle infectivity was improved some 20-fold. It may be that increased MPER hinge flexibility due to D674E relieves or corrects a structural constraint on WL/KH-containing Env to enable stronger gp120-gp41 association within the virion glycoprotein complex, thereby enhancing entry function. It is also possible that D674E directly enhances an MPER-related function in virus-cell fusion [25,49]. For example, greater flexibility in the MPER may facilitate its relocation from the lipid-polar head group interfacial region of the envelope for interaction with the fusion peptide-proximal segment during terminal clasp formation at the membrane-proximal end of the 6-helix bundle during the initiation of fusion [28,30]. Alternatively, the membrane perturbing properties of the MPER may be enhanced by D674E [26,50-52].


Forced virus evolution reveals functional crosstalk between the disulfide bonded region and membrane proximal ectodomain region of HIV-1 gp41.

Khasawneh AI, Laumaea A, Harrison DN, Bellamy-McIntyre AK, Drummer HE, Poumbourios P - Retrovirology (2013)

Amino acid changes at position 674 modeled on the dodecylphosphocholine-associated MPER peptide (PDB entry 2PV6). The Asp-674 (A), Glu-674 (B), and Asn-674 (C) models were produced with Swiss Model and drawn with Pymol. The N- and C-terminal helical segments are shown in purple and magenta respectively, while Phe-673 that forms part of the hinge region is in yellow. The aromatic layer and Ile-675, which are associated with the hydrophobic phase of the lipid are indicated. The N-terminus is to the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643854&req=5

Figure 8: Amino acid changes at position 674 modeled on the dodecylphosphocholine-associated MPER peptide (PDB entry 2PV6). The Asp-674 (A), Glu-674 (B), and Asn-674 (C) models were produced with Swiss Model and drawn with Pymol. The N- and C-terminal helical segments are shown in purple and magenta respectively, while Phe-673 that forms part of the hinge region is in yellow. The aromatic layer and Ile-675, which are associated with the hydrophobic phase of the lipid are indicated. The N-terminus is to the left.
Mentions: Nuclear magnetic resonance studies of synthetic MPER peptides suggest that in membranes, the MPER is a metastable L-shaped structure comprised of N- and C-terminal helical segments that are connected via a hinge composed of Phe-673, which is buried in the lipid phase, and a polar residue at position 674, which is solvent-exposed (Figure 8) (PDB entry, 2PV6 [22]. The C-terminal helix is likely to interact with the transmembrane domain and cholesterol in the lipid phase via the Leu-679-Trp-Tyr-Ile-Lys cholesterol recruitment motif [45-48], while the N-terminal helix represents a more flexible segment that might be in a metastable state and contains 3 Trp residues that are critical for membrane fusion [25]. Molecular modeling predicts that the original Asp side chain at position 674 will hydrogen bond via Oδ1 with the backbone amides of Asn-677 and Ile-675 in 17 of 17 MPER conformers (Figure 8A), thereby conferring rigidity to the interhelical hinge. By contrast, an additional methyl group within the Glu-674 sidechain moves the terminal carboxylate out of hydrogen bonding range in 15/17 conformers, consistent with hinge flexibility (Figure 8B). The boost to the infectivity of WL/KH cell-free virus upon addition of D674E may therefore be related to increased MPER flexibility. Interestingly, the addition of D674E to W596L/K601H only marginally improved gp120-association for cell-free virions whereas single-cycle infectivity was improved some 20-fold. It may be that increased MPER hinge flexibility due to D674E relieves or corrects a structural constraint on WL/KH-containing Env to enable stronger gp120-gp41 association within the virion glycoprotein complex, thereby enhancing entry function. It is also possible that D674E directly enhances an MPER-related function in virus-cell fusion [25,49]. For example, greater flexibility in the MPER may facilitate its relocation from the lipid-polar head group interfacial region of the envelope for interaction with the fusion peptide-proximal segment during terminal clasp formation at the membrane-proximal end of the 6-helix bundle during the initiation of fusion [28,30]. Alternatively, the membrane perturbing properties of the MPER may be enhanced by D674E [26,50-52].

Bottom Line: In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site.The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored.The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virus Fusion Laboratory, Burnet Institute, Prahran, VIC 3004, Australia.

ABSTRACT

Background: The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site.

Results: The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER.

Conclusions: The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus.

Show MeSH
Related in: MedlinePlus