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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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Location and phenotype of WL/KD. (A) Location of WL/KD in the context of the gp120-gp41 ectodomain. gp120 was drawn using the coordinates 3JWD [17] and 2QAD [20]. The gp120 core is colored blue, CD4 binding site (CD4bs) and CCR5-binding site (CCR5bs) in cyan and magenta, respectively, gp41 binding site in green. gp41: the DSR (green) and MPER were drawn using the coordinates 1IM7 [21] and 2PV6 [22]. The N- and C-terminal helical segments of the MPER are colored purple and magenta respectively and the interhelical hinge in yellow. The sidechains of the aromatic/hydrophobic face of the MPER that inserts into the lipid phase of the membrane are indicated. FP: fusion peptide. (B) gp120-gp41 association. Lysates of metabolically labeled WT, K601D, WL/KD, W596L or empty vector (No Env) transfected 293T cells (c) and corresponding culture supernatants (s) were immunoprecipitated with pooled IgG from HIV-1-infected persons and protein G-Sepharose. Proteins were analysed by reducing SDS-PAGE and phosphorimaging. (C) Cell-cell fusion activity. 293T effector cells were cotransfected with pCAG-T7 plus pcDNA3.1-AD8env plasmids and then cocultured (18 h, 37°C) with CD4 plus CCR5-expressing BHK21 cells harboring a luciferase reporter plasmid. The mean relative light units (RLU) of a representative experiment are shown. (D) 14-day replication kinetics in U87.CD4.CCR5 cells. Virus produced in 293T cells was normalised for RT activity and used to infect U87.CD4.CCR5 cells. The RT activity of the culture supernatant was measured at days 3, 7, 10 and 14, postinfection. The mean RT activity ± standard deviation of triplicate samples is shown.
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Figure 1: Location and phenotype of WL/KD. (A) Location of WL/KD in the context of the gp120-gp41 ectodomain. gp120 was drawn using the coordinates 3JWD [17] and 2QAD [20]. The gp120 core is colored blue, CD4 binding site (CD4bs) and CCR5-binding site (CCR5bs) in cyan and magenta, respectively, gp41 binding site in green. gp41: the DSR (green) and MPER were drawn using the coordinates 1IM7 [21] and 2PV6 [22]. The N- and C-terminal helical segments of the MPER are colored purple and magenta respectively and the interhelical hinge in yellow. The sidechains of the aromatic/hydrophobic face of the MPER that inserts into the lipid phase of the membrane are indicated. FP: fusion peptide. (B) gp120-gp41 association. Lysates of metabolically labeled WT, K601D, WL/KD, W596L or empty vector (No Env) transfected 293T cells (c) and corresponding culture supernatants (s) were immunoprecipitated with pooled IgG from HIV-1-infected persons and protein G-Sepharose. Proteins were analysed by reducing SDS-PAGE and phosphorimaging. (C) Cell-cell fusion activity. 293T effector cells were cotransfected with pCAG-T7 plus pcDNA3.1-AD8env plasmids and then cocultured (18 h, 37°C) with CD4 plus CCR5-expressing BHK21 cells harboring a luciferase reporter plasmid. The mean relative light units (RLU) of a representative experiment are shown. (D) 14-day replication kinetics in U87.CD4.CCR5 cells. Virus produced in 293T cells was normalised for RT activity and used to infect U87.CD4.CCR5 cells. The RT activity of the culture supernatant was measured at days 3, 7, 10 and 14, postinfection. The mean RT activity ± standard deviation of triplicate samples is shown.

Mentions: Evidence is accumulating to suggest that the association site formed by the DSR of gp41 and the terminal conserved regions 1 (C1) and 5 (C5) of gp120 [11-13] act as a synapse for gp120-to-gp41 conformational signaling (Figure 1A). For example, the simultaneous introduction of Cys residues to the DSR and to C5 covalently links the gp41-gp120 heterodimer, trapping it in a fusion-inactive state with reduction of the intersubunit disulfide required to activate membrane fusion [14,15]. Furthermore, mutations in the DSR can uncouple CD4-gp120 binding from induction of the gp41 prehairpin intermediate, and can block the initial lipid-mixing or hemifusion phase of the membrane fusion cascade. These findings led to the proposal that the DSR acts as a sensor of receptor-induced conformational changes in gp120 leading to the fusion activation of gp41 [16]. A 7-stranded β-sandwich connecting the gp41-interactive C1 and C5 termini to the inner and outer domains of gp120 [17] also plays a role in mediating association with gp41 [18] and in regulating its activation state [19]. The β-sandwich links together 3 structurally plastic layers that are remodelled by CD4 engagement and coordinates the transmission of this conformational change to the gp41 association site, releasing gp41 from the metastable state.


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)

Location and phenotype of WL/KD. (A) Location of WL/KD in the context of the gp120-gp41 ectodomain. gp120 was drawn using the coordinates 3JWD [17] and 2QAD [20]. The gp120 core is colored blue, CD4 binding site (CD4bs) and CCR5-binding site (CCR5bs) in cyan and magenta, respectively, gp41 binding site in green. gp41: the DSR (green) and MPER were drawn using the coordinates 1IM7 [21] and 2PV6 [22]. The N- and C-terminal helical segments of the MPER are colored purple and magenta respectively and the interhelical hinge in yellow. The sidechains of the aromatic/hydrophobic face of the MPER that inserts into the lipid phase of the membrane are indicated. FP: fusion peptide. (B) gp120-gp41 association. Lysates of metabolically labeled WT, K601D, WL/KD, W596L or empty vector (No Env) transfected 293T cells (c) and corresponding culture supernatants (s) were immunoprecipitated with pooled IgG from HIV-1-infected persons and protein G-Sepharose. Proteins were analysed by reducing SDS-PAGE and phosphorimaging. (C) Cell-cell fusion activity. 293T effector cells were cotransfected with pCAG-T7 plus pcDNA3.1-AD8env plasmids and then cocultured (18 h, 37°C) with CD4 plus CCR5-expressing BHK21 cells harboring a luciferase reporter plasmid. The mean relative light units (RLU) of a representative experiment are shown. (D) 14-day replication kinetics in U87.CD4.CCR5 cells. Virus produced in 293T cells was normalised for RT activity and used to infect U87.CD4.CCR5 cells. The RT activity of the culture supernatant was measured at days 3, 7, 10 and 14, postinfection. The mean RT activity ± standard deviation of triplicate samples is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Location and phenotype of WL/KD. (A) Location of WL/KD in the context of the gp120-gp41 ectodomain. gp120 was drawn using the coordinates 3JWD [17] and 2QAD [20]. The gp120 core is colored blue, CD4 binding site (CD4bs) and CCR5-binding site (CCR5bs) in cyan and magenta, respectively, gp41 binding site in green. gp41: the DSR (green) and MPER were drawn using the coordinates 1IM7 [21] and 2PV6 [22]. The N- and C-terminal helical segments of the MPER are colored purple and magenta respectively and the interhelical hinge in yellow. The sidechains of the aromatic/hydrophobic face of the MPER that inserts into the lipid phase of the membrane are indicated. FP: fusion peptide. (B) gp120-gp41 association. Lysates of metabolically labeled WT, K601D, WL/KD, W596L or empty vector (No Env) transfected 293T cells (c) and corresponding culture supernatants (s) were immunoprecipitated with pooled IgG from HIV-1-infected persons and protein G-Sepharose. Proteins were analysed by reducing SDS-PAGE and phosphorimaging. (C) Cell-cell fusion activity. 293T effector cells were cotransfected with pCAG-T7 plus pcDNA3.1-AD8env plasmids and then cocultured (18 h, 37°C) with CD4 plus CCR5-expressing BHK21 cells harboring a luciferase reporter plasmid. The mean relative light units (RLU) of a representative experiment are shown. (D) 14-day replication kinetics in U87.CD4.CCR5 cells. Virus produced in 293T cells was normalised for RT activity and used to infect U87.CD4.CCR5 cells. The RT activity of the culture supernatant was measured at days 3, 7, 10 and 14, postinfection. The mean RT activity ± standard deviation of triplicate samples is shown.
Mentions: Evidence is accumulating to suggest that the association site formed by the DSR of gp41 and the terminal conserved regions 1 (C1) and 5 (C5) of gp120 [11-13] act as a synapse for gp120-to-gp41 conformational signaling (Figure 1A). For example, the simultaneous introduction of Cys residues to the DSR and to C5 covalently links the gp41-gp120 heterodimer, trapping it in a fusion-inactive state with reduction of the intersubunit disulfide required to activate membrane fusion [14,15]. Furthermore, mutations in the DSR can uncouple CD4-gp120 binding from induction of the gp41 prehairpin intermediate, and can block the initial lipid-mixing or hemifusion phase of the membrane fusion cascade. These findings led to the proposal that the DSR acts as a sensor of receptor-induced conformational changes in gp120 leading to the fusion activation of gp41 [16]. A 7-stranded β-sandwich connecting the gp41-interactive C1 and C5 termini to the inner and outer domains of gp120 [17] also plays a role in mediating association with gp41 [18] and in regulating its activation state [19]. The β-sandwich links together 3 structurally plastic layers that are remodelled by CD4 engagement and coordinates the transmission of this conformational change to the gp41 association site, releasing gp41 from the metastable state.

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