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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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Cell-cell fusion activities of revertant Envs. 293T effectors (cotransfected with pΔKAD8env plus pCAG-T7) were cocultured with BHK-21 targets cotransfected with pT4luc and pcCCR5 vectors for 18 h and then lysed and assayed for luciferase activity. The data presented here are the means ± standard errors of 3 independent assays. *, P < 0.05, unpaired t test assuming unequal variances.
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Figure 6: Cell-cell fusion activities of revertant Envs. 293T effectors (cotransfected with pΔKAD8env plus pCAG-T7) were cocultured with BHK-21 targets cotransfected with pT4luc and pcCCR5 vectors for 18 h and then lysed and assayed for luciferase activity. The data presented here are the means ± standard errors of 3 independent assays. *, P < 0.05, unpaired t test assuming unequal variances.

Mentions: We next examined the membrane fusion activities of selected revertant Env sequences in a cell-to-cell fusion assay. In this context, Env is expressed in the absence of other viral proteins and is therefore not subjected to the conformational constraints that may be imposed by matrix-gp41 cytoplasmic tail interactions present in virus [39-42]. The assay was conducted at limiting Env concentrations (0.25 μg pΔKADenv) to enable detection of subtle changes in fusion function. Consistent with the cell-free virus infectivity data, WL/KD blocked cell-cell fusion, WL/KH exhibited partially restored fusion function and D674N and D674G mutations were inhibitory in a WL/KH context (Figure 6). However, in contrast to the infectivity data, D674E did not enhance fusogenicity when added to WL/KH. These data suggest that the functional interaction between Leu-596, His-601 and Glu-674 largely operates in the context of assembled virions transmitted via the cell-cell route and the conformational constraints imposed by Gag-gp41 cytoplasmic tail interactions [39-42].


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)

Cell-cell fusion activities of revertant Envs. 293T effectors (cotransfected with pΔKAD8env plus pCAG-T7) were cocultured with BHK-21 targets cotransfected with pT4luc and pcCCR5 vectors for 18 h and then lysed and assayed for luciferase activity. The data presented here are the means ± standard errors of 3 independent assays. *, P < 0.05, unpaired t test assuming unequal variances.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Cell-cell fusion activities of revertant Envs. 293T effectors (cotransfected with pΔKAD8env plus pCAG-T7) were cocultured with BHK-21 targets cotransfected with pT4luc and pcCCR5 vectors for 18 h and then lysed and assayed for luciferase activity. The data presented here are the means ± standard errors of 3 independent assays. *, P < 0.05, unpaired t test assuming unequal variances.
Mentions: We next examined the membrane fusion activities of selected revertant Env sequences in a cell-to-cell fusion assay. In this context, Env is expressed in the absence of other viral proteins and is therefore not subjected to the conformational constraints that may be imposed by matrix-gp41 cytoplasmic tail interactions present in virus [39-42]. The assay was conducted at limiting Env concentrations (0.25 μg pΔKADenv) to enable detection of subtle changes in fusion function. Consistent with the cell-free virus infectivity data, WL/KD blocked cell-cell fusion, WL/KH exhibited partially restored fusion function and D674N and D674G mutations were inhibitory in a WL/KH context (Figure 6). However, in contrast to the infectivity data, D674E did not enhance fusogenicity when added to WL/KH. These data suggest that the functional interaction between Leu-596, His-601 and Glu-674 largely operates in the context of assembled virions transmitted via the cell-cell route and the conformational constraints imposed by Gag-gp41 cytoplasmic tail interactions [39-42].

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