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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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Replication of representative WLKD-1 (A) and WLKD-2 (B) clones in U87.CD4.CCR5 cells. Viruses produced in 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. Reverse transcriptase activity in culture supernatants was measured at days 3, 7, 10 and 14. The mean RT activity ± standard deviation of triplicate samples is shown. (C) Single-cycle infectivity was determined in U87.CD4.CCR5 cells infected with Env-pseudotyped luciferase reporter viruses at 48-h postinfection. Luciferase activity was normalized against the RT activity present in each virus inoculum. The mean RLU ± standard errors of 3 independent assays are presented here. **, P < 0.01, unpaired t test assuming unequal variances. (D) Serial 10-fold dilutions of Env-pseudotyped luciferase reporter viruses were added to U87.CD4.CCR5 target cells and luciferase activity determined 48 h later. The mean RLU ± standard deviations from a representative experiment are presented.
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Figure 3: Replication of representative WLKD-1 (A) and WLKD-2 (B) clones in U87.CD4.CCR5 cells. Viruses produced in 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. Reverse transcriptase activity in culture supernatants was measured at days 3, 7, 10 and 14. The mean RT activity ± standard deviation of triplicate samples is shown. (C) Single-cycle infectivity was determined in U87.CD4.CCR5 cells infected with Env-pseudotyped luciferase reporter viruses at 48-h postinfection. Luciferase activity was normalized against the RT activity present in each virus inoculum. The mean RLU ± standard errors of 3 independent assays are presented here. **, P < 0.01, unpaired t test assuming unequal variances. (D) Serial 10-fold dilutions of Env-pseudotyped luciferase reporter viruses were added to U87.CD4.CCR5 target cells and luciferase activity determined 48 h later. The mean RLU ± standard deviations from a representative experiment are presented.

Mentions: The dominant genotypes were reconstructed in the context of the pAD8 proviral clone. In the case of WLKD-1, cell-free virus-initiated replication in U87.CD4.CCR5 cells was partially restored by D601H in the DSR (WL/KH) and was optimised further by D674E in the MPER (WL/KH/DE) (Figure 3A). The addition of L85M to WL/KH/DE did not improve replication any further. Interestingly, the WL/KH/DG combination was replication-incompetent. Gly-674 can arise via an A-to-G mutation in the 2nd position of the Asp and Glu codons but in a WL/KH context appears to be an evolutionary dead-end. These data suggest that D601H and D674E can act synergistically to suppress the original replication defect. For WLKD-2, step-wise improvements in replication competence were observed with WL/KH/DN, ΔTW/WL/KH/DN and WL/KH, respectively (Figure 3B). Thus D674N is inhibitory to cell-free virus initiated replication in the context of WL/KH with ΔTW partially relieving this inhibition. The G145E V1 mutation observed at days 30–40 did not confer a replication advantage to WL/KH/DN. Interestingly, the replication competence of WLKD-2 genotypes were inferior to those derived from the WLKD-1 culture even though revertant virus emerged in the WLKD-2 culture first, suggesting that additional mechanisms of reversion were operating in this culture.


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)

Replication of representative WLKD-1 (A) and WLKD-2 (B) clones in U87.CD4.CCR5 cells. Viruses produced in 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. Reverse transcriptase activity in culture supernatants was measured at days 3, 7, 10 and 14. The mean RT activity ± standard deviation of triplicate samples is shown. (C) Single-cycle infectivity was determined in U87.CD4.CCR5 cells infected with Env-pseudotyped luciferase reporter viruses at 48-h postinfection. Luciferase activity was normalized against the RT activity present in each virus inoculum. The mean RLU ± standard errors of 3 independent assays are presented here. **, P < 0.01, unpaired t test assuming unequal variances. (D) Serial 10-fold dilutions of Env-pseudotyped luciferase reporter viruses were added to U87.CD4.CCR5 target cells and luciferase activity determined 48 h later. The mean RLU ± standard deviations from a representative experiment are presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643854&req=5

Figure 3: Replication of representative WLKD-1 (A) and WLKD-2 (B) clones in U87.CD4.CCR5 cells. Viruses produced in 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. Reverse transcriptase activity in culture supernatants was measured at days 3, 7, 10 and 14. The mean RT activity ± standard deviation of triplicate samples is shown. (C) Single-cycle infectivity was determined in U87.CD4.CCR5 cells infected with Env-pseudotyped luciferase reporter viruses at 48-h postinfection. Luciferase activity was normalized against the RT activity present in each virus inoculum. The mean RLU ± standard errors of 3 independent assays are presented here. **, P < 0.01, unpaired t test assuming unequal variances. (D) Serial 10-fold dilutions of Env-pseudotyped luciferase reporter viruses were added to U87.CD4.CCR5 target cells and luciferase activity determined 48 h later. The mean RLU ± standard deviations from a representative experiment are presented.
Mentions: The dominant genotypes were reconstructed in the context of the pAD8 proviral clone. In the case of WLKD-1, cell-free virus-initiated replication in U87.CD4.CCR5 cells was partially restored by D601H in the DSR (WL/KH) and was optimised further by D674E in the MPER (WL/KH/DE) (Figure 3A). The addition of L85M to WL/KH/DE did not improve replication any further. Interestingly, the WL/KH/DG combination was replication-incompetent. Gly-674 can arise via an A-to-G mutation in the 2nd position of the Asp and Glu codons but in a WL/KH context appears to be an evolutionary dead-end. These data suggest that D601H and D674E can act synergistically to suppress the original replication defect. For WLKD-2, step-wise improvements in replication competence were observed with WL/KH/DN, ΔTW/WL/KH/DN and WL/KH, respectively (Figure 3B). Thus D674N is inhibitory to cell-free virus initiated replication in the context of WL/KH with ΔTW partially relieving this inhibition. The G145E V1 mutation observed at days 30–40 did not confer a replication advantage to WL/KH/DN. Interestingly, the replication competence of WLKD-2 genotypes were inferior to those derived from the WLKD-1 culture even though revertant virus emerged in the WLKD-2 culture first, suggesting that additional mechanisms of reversion were operating in this culture.

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