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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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Spread of cell-associated virus. U87.CD4.CCR5 cells were inoculated with VSV G-pseudotyped HIV-1 particles [50,000 (A) or 20,000 (B) cpm of RT activity per inoculum] and then trypsinized 24-h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) As for B except that 1 μM C34 peptide was maintained in the culture following the trypsinization step. Effects of D674E and D674N mutations on viral spread initiated by cell-free (D) and cell-associated (E) virus in 10-day U87.CD4.CCR5 cultures. The results shown represent the mean p24 content ± standard deviation of duplicate samples obtained from the cultures.
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Figure 4: Spread of cell-associated virus. U87.CD4.CCR5 cells were inoculated with VSV G-pseudotyped HIV-1 particles [50,000 (A) or 20,000 (B) cpm of RT activity per inoculum] and then trypsinized 24-h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) As for B except that 1 μM C34 peptide was maintained in the culture following the trypsinization step. Effects of D674E and D674N mutations on viral spread initiated by cell-free (D) and cell-associated (E) virus in 10-day U87.CD4.CCR5 cultures. The results shown represent the mean p24 content ± standard deviation of duplicate samples obtained from the cultures.

Mentions: The functional advantages conferred by D601H and D674E were less obvious in the single cycle infectivity assay when compared to 14-day replication experiments (compare Figure 3A, B and C). This apparent discrepancy may be explained by the fact that only a single cycle of infection mediated by cell-free virus occurs in the reporter assay, whereas multiple rounds of infection mediated by both cell-free and cell-associated virus occur in the replication assay [36,37]. We therefore inoculated U87.CD4.CCR5 cells with HIV-1-VSV G pseudotyped particles, reasoning that the highly fusogenic nature of VSV G will normalize the cellular entry of cell-free WT and revertant viruses in the first 24 hours of infection, thereby enabling an assessment of virus production following multiple rounds of cell-cell and cell-free viral transmission. At 24-h postinfection, the cells were trypsinized to remove residual surface-adsorbed virus, replated and then cultured for a further 10 days. Virus production was elevated for WL/KH/DE with respect to WT at day 7 and approached WT levels at day 10, whereas WL/KH, WL/KH/DN and ΔTW/WL/KH/DN replication was almost identical to WT over the 10-day culture (Figure 4A). A low level of reverse transcriptase (RT) activity was observed for WL/KD, which is likely due to a combination of virus production by cells infected by VSV G-pseudotypes in the initial 24 h plus low-level cell-cell spread. The results were confirmed in an experiment employing smaller inocula (20,000 cpm RT activity-equivalents of VSV G-HIV-1 pseudotypes) but virus production was delayed to day 10 in this case (Figure 4B). Notably, the presence of D674N was not inhibitory to replication when combined with WL/KH in this infection system. We found that viral spread in VSV G-HIV-1 pseudotype initiated cultures was blocked by the C34 fusion inhibitor peptide, consistent with viral spread being HIV-1 Env dependent (Figure 4C). These data suggest that the D601H pseudoreversion and 2nd (and 3rd) site mutations optimise viral spread mediated by cell-associated virus.


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)

Spread of cell-associated virus. U87.CD4.CCR5 cells were inoculated with VSV G-pseudotyped HIV-1 particles [50,000 (A) or 20,000 (B) cpm of RT activity per inoculum] and then trypsinized 24-h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) As for B except that 1 μM C34 peptide was maintained in the culture following the trypsinization step. Effects of D674E and D674N mutations on viral spread initiated by cell-free (D) and cell-associated (E) virus in 10-day U87.CD4.CCR5 cultures. The results shown represent the mean p24 content ± standard deviation of duplicate samples obtained from the cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Spread of cell-associated virus. U87.CD4.CCR5 cells were inoculated with VSV G-pseudotyped HIV-1 particles [50,000 (A) or 20,000 (B) cpm of RT activity per inoculum] and then trypsinized 24-h later to remove residual adsorbed virus. The cells were then replated and cultured for a further 10 days. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) As for B except that 1 μM C34 peptide was maintained in the culture following the trypsinization step. Effects of D674E and D674N mutations on viral spread initiated by cell-free (D) and cell-associated (E) virus in 10-day U87.CD4.CCR5 cultures. The results shown represent the mean p24 content ± standard deviation of duplicate samples obtained from the cultures.
Mentions: The functional advantages conferred by D601H and D674E were less obvious in the single cycle infectivity assay when compared to 14-day replication experiments (compare Figure 3A, B and C). This apparent discrepancy may be explained by the fact that only a single cycle of infection mediated by cell-free virus occurs in the reporter assay, whereas multiple rounds of infection mediated by both cell-free and cell-associated virus occur in the replication assay [36,37]. We therefore inoculated U87.CD4.CCR5 cells with HIV-1-VSV G pseudotyped particles, reasoning that the highly fusogenic nature of VSV G will normalize the cellular entry of cell-free WT and revertant viruses in the first 24 hours of infection, thereby enabling an assessment of virus production following multiple rounds of cell-cell and cell-free viral transmission. At 24-h postinfection, the cells were trypsinized to remove residual surface-adsorbed virus, replated and then cultured for a further 10 days. Virus production was elevated for WL/KH/DE with respect to WT at day 7 and approached WT levels at day 10, whereas WL/KH, WL/KH/DN and ΔTW/WL/KH/DN replication was almost identical to WT over the 10-day culture (Figure 4A). A low level of reverse transcriptase (RT) activity was observed for WL/KD, which is likely due to a combination of virus production by cells infected by VSV G-pseudotypes in the initial 24 h plus low-level cell-cell spread. The results were confirmed in an experiment employing smaller inocula (20,000 cpm RT activity-equivalents of VSV G-HIV-1 pseudotypes) but virus production was delayed to day 10 in this case (Figure 4B). Notably, the presence of D674N was not inhibitory to replication when combined with WL/KH in this infection system. We found that viral spread in VSV G-HIV-1 pseudotype initiated cultures was blocked by the C34 fusion inhibitor peptide, consistent with viral spread being HIV-1 Env dependent (Figure 4C). These data suggest that the D601H pseudoreversion and 2nd (and 3rd) site mutations optimise viral spread mediated by cell-associated virus.

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