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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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Long-term culture of W596L/K601D virus. (A) Wild type and W596L/K601D-mutated HIV-1AD8 virus stocks produced by transfected 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. The cell-free virus obtained at day 10 was filtered through a 0.45 μm nitrocellulose filter, normalized according to RT activity and used to infect fresh U87.CD4.CCR5 cells. Viruses were subjected to 5 sequential passages in total. (B) Infection of U87.CD4.CCR5 cells was initiated with VSV G-pseudotyped WT and W596L/K601D mutant viruses. The cells were trypsinized 24-h later to remove residually adsorbed viruses. The passaging procedure described in A was then followed. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) Reversion pathways in WLKD-1 and WLKD-2 cultures. The env region was PCR amplified from proviral DNA isolated at days 10, 20, 30, 40 and 50, cloned into pΔKAD8env and sequenced. Upper case lettering connected by a bold horizontal line denotes a major evolutionary pathway, while lower case lettering connected via thin horizontal lines denotes a minor pathway. Lower case lettering only: low-frequency genotypes arising at the specified days.
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Figure 2: Long-term culture of W596L/K601D virus. (A) Wild type and W596L/K601D-mutated HIV-1AD8 virus stocks produced by transfected 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. The cell-free virus obtained at day 10 was filtered through a 0.45 μm nitrocellulose filter, normalized according to RT activity and used to infect fresh U87.CD4.CCR5 cells. Viruses were subjected to 5 sequential passages in total. (B) Infection of U87.CD4.CCR5 cells was initiated with VSV G-pseudotyped WT and W596L/K601D mutant viruses. The cells were trypsinized 24-h later to remove residually adsorbed viruses. The passaging procedure described in A was then followed. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) Reversion pathways in WLKD-1 and WLKD-2 cultures. The env region was PCR amplified from proviral DNA isolated at days 10, 20, 30, 40 and 50, cloned into pΔKAD8env and sequenced. Upper case lettering connected by a bold horizontal line denotes a major evolutionary pathway, while lower case lettering connected via thin horizontal lines denotes a minor pathway. Lower case lettering only: low-frequency genotypes arising at the specified days.

Mentions: Viruses derived from 2 independent HIV-1AD8 proviral clones carrying WL/KD were subjected to long-term culture in U87.CD4.CCR5 cells with serial passaging of cell-free virus onto fresh cells every 10 days. Evidence of replication was not observed for either clone, even after 50 days of culture (Figure 2A). The markedly diminished gp120-anchoring ability of WL/KD gp41 was assumed to have blocked viral entry and therefore reverse transcription, which is required for the generation of suppressor mutations. Mutant WL/KD HIV-1 particles were therefore pseudotyped with vesicular stomatitis virus glycoprotein G (VSV G) in trans in order to initiate HIV-1 envelope glycoprotein (Env)-independent infection via the endosomal pathway. Twenty-four h after infection, the U87.CD4.CCR5 cells were extensively washed and trypsinized to remove residual adsorbed virus prior to further culture for 10 days. The sequential passaging of the resultant cell-free virus in U87.CD4.CCR5 cells led to restored infectivity after 47 and 30 days in WL/KD cultures 1 and 2 (WLKD-1 and WLKD-2), respectively (Figure 2B).


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)

Long-term culture of W596L/K601D virus. (A) Wild type and W596L/K601D-mutated HIV-1AD8 virus stocks produced by transfected 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. The cell-free virus obtained at day 10 was filtered through a 0.45 μm nitrocellulose filter, normalized according to RT activity and used to infect fresh U87.CD4.CCR5 cells. Viruses were subjected to 5 sequential passages in total. (B) Infection of U87.CD4.CCR5 cells was initiated with VSV G-pseudotyped WT and W596L/K601D mutant viruses. The cells were trypsinized 24-h later to remove residually adsorbed viruses. The passaging procedure described in A was then followed. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) Reversion pathways in WLKD-1 and WLKD-2 cultures. The env region was PCR amplified from proviral DNA isolated at days 10, 20, 30, 40 and 50, cloned into pΔKAD8env and sequenced. Upper case lettering connected by a bold horizontal line denotes a major evolutionary pathway, while lower case lettering connected via thin horizontal lines denotes a minor pathway. Lower case lettering only: low-frequency genotypes arising at the specified days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3643854&req=5

Figure 2: Long-term culture of W596L/K601D virus. (A) Wild type and W596L/K601D-mutated HIV-1AD8 virus stocks produced by transfected 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. The cell-free virus obtained at day 10 was filtered through a 0.45 μm nitrocellulose filter, normalized according to RT activity and used to infect fresh U87.CD4.CCR5 cells. Viruses were subjected to 5 sequential passages in total. (B) Infection of U87.CD4.CCR5 cells was initiated with VSV G-pseudotyped WT and W596L/K601D mutant viruses. The cells were trypsinized 24-h later to remove residually adsorbed viruses. The passaging procedure described in A was then followed. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) Reversion pathways in WLKD-1 and WLKD-2 cultures. The env region was PCR amplified from proviral DNA isolated at days 10, 20, 30, 40 and 50, cloned into pΔKAD8env and sequenced. Upper case lettering connected by a bold horizontal line denotes a major evolutionary pathway, while lower case lettering connected via thin horizontal lines denotes a minor pathway. Lower case lettering only: low-frequency genotypes arising at the specified days.
Mentions: Viruses derived from 2 independent HIV-1AD8 proviral clones carrying WL/KD were subjected to long-term culture in U87.CD4.CCR5 cells with serial passaging of cell-free virus onto fresh cells every 10 days. Evidence of replication was not observed for either clone, even after 50 days of culture (Figure 2A). The markedly diminished gp120-anchoring ability of WL/KD gp41 was assumed to have blocked viral entry and therefore reverse transcription, which is required for the generation of suppressor mutations. Mutant WL/KD HIV-1 particles were therefore pseudotyped with vesicular stomatitis virus glycoprotein G (VSV G) in trans in order to initiate HIV-1 envelope glycoprotein (Env)-independent infection via the endosomal pathway. Twenty-four h after infection, the U87.CD4.CCR5 cells were extensively washed and trypsinized to remove residual adsorbed virus prior to further culture for 10 days. The sequential passaging of the resultant cell-free virus in U87.CD4.CCR5 cells led to restored infectivity after 47 and 30 days in WL/KD cultures 1 and 2 (WLKD-1 and WLKD-2), respectively (Figure 2B).

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