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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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Biochemical analysis of selected revertant clones. (A) Western blotting. At 48-h posttransfection, pΔKADenv–transfected 293T cells were lysed and subjected to reducing SDS-PAGE followed by western blotting with DV-012 to gp120 (upper panel) and mAb C8 to gp41 (lower panel). (B) gp120-gp41 association was determined as for Figure 1B. gp120-shedding index was calculated according to the formula: ([mutant gp120]supernatant x [WT gp120]cell)/([mutant gp120]cell x [WT gp120]supernatant) [12]. (C) Characterization of virions produced by pAD8 infectious clones. Pelleted HIV-1 virions were analysed by Western blotting using DV-012 (upper panel) and pooled IgG from HIV-1-infected persons (lower panel). gp120 and p24 band intensities were determined using a Licor Odyssey scanner.
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Figure 5: Biochemical analysis of selected revertant clones. (A) Western blotting. At 48-h posttransfection, pΔKADenv–transfected 293T cells were lysed and subjected to reducing SDS-PAGE followed by western blotting with DV-012 to gp120 (upper panel) and mAb C8 to gp41 (lower panel). (B) gp120-gp41 association was determined as for Figure 1B. gp120-shedding index was calculated according to the formula: ([mutant gp120]supernatant x [WT gp120]cell)/([mutant gp120]cell x [WT gp120]supernatant) [12]. (C) Characterization of virions produced by pAD8 infectious clones. Pelleted HIV-1 virions were analysed by Western blotting using DV-012 (upper panel) and pooled IgG from HIV-1-infected persons (lower panel). gp120 and p24 band intensities were determined using a Licor Odyssey scanner.

Mentions: The synthesis and processing of the cloned Env glycoproteins were examined by western blot. The gp120-specific polyclonal antibody, DV-012, (Figure 5A, upper) revealed that similar levels of gp160 were expressed for all clones and that cell-associated gp120 was present for WT and His-601-containing clones. Consistent with the shedding defect seen previously (Figure 1B), gp120 was largely absent for WL/KD. The monoclonal antibody (mAb) C8, directed to a linear epitope within the cytoplasmic domain of gp41 [38], revealed similar levels of gp160 and gp41 expression for the Env constructs examined (Figure 5A, lower). Interestingly, the presence of the WL/KD mutation in gp41 resulted in a distinct glycosylation pattern relative to the other clones, suggesting a subtly different structure. The gp120 anchoring ability of the cell-surface expressed revertant Env proteins was analyzed by immunoprecipitation of pulse-chase biosynthetically labelled Env transfected 293T cells. Figure 5B again confirms the gp120 shedding defect of WL/KD and indicates that the subsequent D601H mutation, present in the WL/KH clone was sufficient to partially restore gp120 association levels with no further improvements to association following the addition of 2nd and 3rd site mutations. By contrast, Western blot analysis of viruses pelleted from the supernatants of 293T cells transfected with pAD8 proviral clones revealed gp120 shedding phenotypes for the revertants (Figure 5C). These data indicate that stable gp120-gp41 association is not conferred by the reverting mutations in a cell-free virion context. The apparent discrepancy between cellular and virion Env could be related to the fact that cellular Env was analysed 5 h post synthesis whereas virions were analysed at 48 h posttransfection. It may be that a subtly unstable gp120-gp41 complex becomes more evident over time. Interactions between the cytoplasmic tail of gp41 and the MA domain of Gag in the context of cell free virions have been found to impose structural and functional constraints on the Env ectodomain [39-42]. These data suggest an alternative scenario where WL/KH-containing revertant Envs attain apparently WT gp120-gp41 stability when expressed in the absence of other viral proteins, whereas the corresponding virion-associated glycoprotein complexes are less stable in an Env conformation that is modulated by internal Env-Gag interactions.


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)

Biochemical analysis of selected revertant clones. (A) Western blotting. At 48-h posttransfection, pΔKADenv–transfected 293T cells were lysed and subjected to reducing SDS-PAGE followed by western blotting with DV-012 to gp120 (upper panel) and mAb C8 to gp41 (lower panel). (B) gp120-gp41 association was determined as for Figure 1B. gp120-shedding index was calculated according to the formula: ([mutant gp120]supernatant x [WT gp120]cell)/([mutant gp120]cell x [WT gp120]supernatant) [12]. (C) Characterization of virions produced by pAD8 infectious clones. Pelleted HIV-1 virions were analysed by Western blotting using DV-012 (upper panel) and pooled IgG from HIV-1-infected persons (lower panel). gp120 and p24 band intensities were determined using a Licor Odyssey scanner.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Biochemical analysis of selected revertant clones. (A) Western blotting. At 48-h posttransfection, pΔKADenv–transfected 293T cells were lysed and subjected to reducing SDS-PAGE followed by western blotting with DV-012 to gp120 (upper panel) and mAb C8 to gp41 (lower panel). (B) gp120-gp41 association was determined as for Figure 1B. gp120-shedding index was calculated according to the formula: ([mutant gp120]supernatant x [WT gp120]cell)/([mutant gp120]cell x [WT gp120]supernatant) [12]. (C) Characterization of virions produced by pAD8 infectious clones. Pelleted HIV-1 virions were analysed by Western blotting using DV-012 (upper panel) and pooled IgG from HIV-1-infected persons (lower panel). gp120 and p24 band intensities were determined using a Licor Odyssey scanner.
Mentions: The synthesis and processing of the cloned Env glycoproteins were examined by western blot. The gp120-specific polyclonal antibody, DV-012, (Figure 5A, upper) revealed that similar levels of gp160 were expressed for all clones and that cell-associated gp120 was present for WT and His-601-containing clones. Consistent with the shedding defect seen previously (Figure 1B), gp120 was largely absent for WL/KD. The monoclonal antibody (mAb) C8, directed to a linear epitope within the cytoplasmic domain of gp41 [38], revealed similar levels of gp160 and gp41 expression for the Env constructs examined (Figure 5A, lower). Interestingly, the presence of the WL/KD mutation in gp41 resulted in a distinct glycosylation pattern relative to the other clones, suggesting a subtly different structure. The gp120 anchoring ability of the cell-surface expressed revertant Env proteins was analyzed by immunoprecipitation of pulse-chase biosynthetically labelled Env transfected 293T cells. Figure 5B again confirms the gp120 shedding defect of WL/KD and indicates that the subsequent D601H mutation, present in the WL/KH clone was sufficient to partially restore gp120 association levels with no further improvements to association following the addition of 2nd and 3rd site mutations. By contrast, Western blot analysis of viruses pelleted from the supernatants of 293T cells transfected with pAD8 proviral clones revealed gp120 shedding phenotypes for the revertants (Figure 5C). These data indicate that stable gp120-gp41 association is not conferred by the reverting mutations in a cell-free virion context. The apparent discrepancy between cellular and virion Env could be related to the fact that cellular Env was analysed 5 h post synthesis whereas virions were analysed at 48 h posttransfection. It may be that a subtly unstable gp120-gp41 complex becomes more evident over time. Interactions between the cytoplasmic tail of gp41 and the MA domain of Gag in the context of cell free virions have been found to impose structural and functional constraints on the Env ectodomain [39-42]. These data suggest an alternative scenario where WL/KH-containing revertant Envs attain apparently WT gp120-gp41 stability when expressed in the absence of other viral proteins, whereas the corresponding virion-associated glycoprotein complexes are less stable in an Env conformation that is modulated by internal Env-Gag interactions.

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