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In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions.

Zheng A, Umashankar M, Kielian M - PLoS Pathog. (2010)

Bottom Line: Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles.Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway.The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

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DENV E H244A mutation inhibits release of virus-like particles via a low pH-dependent mechanism.A) WT and H244A mutant E proteins are comparably expressed. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E protein expression was detected by immunofluorescence and the nuclei were stained with DAPI. Fluorescence images are shown at the same magnification and exposure time. Bar represents 30 µm. B) WT and H244A mutant E proteins are comparably immunoprecipitated by conformation-specific mAbs. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E proteins in the cell lysates were immunoprecipitated by Sango, a rabbit polyclonal antibody to DIII, and by the mouse mAbs 4G2 and 4E11, as indicated at the top of the panel. Samples were then analyzed by SDS-PAGE and western blot using mouse anti-DENV2 Ab for the Sango samples and Sango for the mAbs samples. Asterisks indicate the positions of the IgG and IgG heavy chain, which cross-react in the western blot. Equivalent sample input was evaluated by western blot for β-actin (lower panel). C) Effect of low pH on WT and H244A VLP production. WT and H244A mutant cells were incubated with tetracycline for 2 h and then in this medium plus 20mM NH4Cl where indicated for a total of 36h. VLP released in the culture media were pelleted by ultracentrifugation, and E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were analyzed by SDS-PAGE and western blot using Sango. 5-fold more culture media from the H244A cells than the WT cells were loaded. Data are representative examples of two or more independent experiments.
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ppat-1001157-g008: DENV E H244A mutation inhibits release of virus-like particles via a low pH-dependent mechanism.A) WT and H244A mutant E proteins are comparably expressed. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E protein expression was detected by immunofluorescence and the nuclei were stained with DAPI. Fluorescence images are shown at the same magnification and exposure time. Bar represents 30 µm. B) WT and H244A mutant E proteins are comparably immunoprecipitated by conformation-specific mAbs. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E proteins in the cell lysates were immunoprecipitated by Sango, a rabbit polyclonal antibody to DIII, and by the mouse mAbs 4G2 and 4E11, as indicated at the top of the panel. Samples were then analyzed by SDS-PAGE and western blot using mouse anti-DENV2 Ab for the Sango samples and Sango for the mAbs samples. Asterisks indicate the positions of the IgG and IgG heavy chain, which cross-react in the western blot. Equivalent sample input was evaluated by western blot for β-actin (lower panel). C) Effect of low pH on WT and H244A VLP production. WT and H244A mutant cells were incubated with tetracycline for 2 h and then in this medium plus 20mM NH4Cl where indicated for a total of 36h. VLP released in the culture media were pelleted by ultracentrifugation, and E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were analyzed by SDS-PAGE and western blot using Sango. 5-fold more culture media from the H244A cells than the WT cells were loaded. Data are representative examples of two or more independent experiments.

Mentions: We established stable HEK 293 cells that inducibly express the DENV1 WT or H244A prM-E proteins. After 36 h induction with tetracycline, both WT and H244A cells show abundant intracellular expression of the DENV1 E protein as detected by immunofluorescence, while the parent cell line is negative for E expression (Fig. 8A). To evaluate whether WT and H244A E proteins were correctly folded, cells were induced for 36 h, lysed, and immunoprecipitated with a rabbit polyclonal antibody to E DIII, and with two conformation-specific mAbs. mAb 4E11 recognizes a discontinuous epitope on DENV E DIII and requires proper DIII disulfide bond formation for recognition [49], [50]. mAb 4G2 recognizes the fusion loop at the tip of flavivirus E DII and its epitope is sensitive to reduction [51]. Expression studies have shown that the 4G2 epitope is not formed if the E protein is expressed in the absence of prM [52], indicating that this epitope is particularly useful for diagnostic tests of prM's chaperone interaction with E (see also reference [37]). As shown in Fig. 8B, lysates from cells induced to express prM plus WT or H244A E proteins showed strong reactivity with all three antibodies. Quantitation of multiple experiments confirmed that WT and H244A E proteins were comparably recognized by the 4E11 and 4G2 mAbs. Thus, by these criteria H244A E protein interacts with prM protein and is correctly folded. This result also agrees with our finding that truncated H244A E protein expressed with prM in the S2 cell system was fully active in low pH-dependent membrane binding and trimerization, suggesting correct folding (Fig. 6).


In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions.

Zheng A, Umashankar M, Kielian M - PLoS Pathog. (2010)

DENV E H244A mutation inhibits release of virus-like particles via a low pH-dependent mechanism.A) WT and H244A mutant E proteins are comparably expressed. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E protein expression was detected by immunofluorescence and the nuclei were stained with DAPI. Fluorescence images are shown at the same magnification and exposure time. Bar represents 30 µm. B) WT and H244A mutant E proteins are comparably immunoprecipitated by conformation-specific mAbs. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E proteins in the cell lysates were immunoprecipitated by Sango, a rabbit polyclonal antibody to DIII, and by the mouse mAbs 4G2 and 4E11, as indicated at the top of the panel. Samples were then analyzed by SDS-PAGE and western blot using mouse anti-DENV2 Ab for the Sango samples and Sango for the mAbs samples. Asterisks indicate the positions of the IgG and IgG heavy chain, which cross-react in the western blot. Equivalent sample input was evaluated by western blot for β-actin (lower panel). C) Effect of low pH on WT and H244A VLP production. WT and H244A mutant cells were incubated with tetracycline for 2 h and then in this medium plus 20mM NH4Cl where indicated for a total of 36h. VLP released in the culture media were pelleted by ultracentrifugation, and E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were analyzed by SDS-PAGE and western blot using Sango. 5-fold more culture media from the H244A cells than the WT cells were loaded. Data are representative examples of two or more independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958806&req=5

ppat-1001157-g008: DENV E H244A mutation inhibits release of virus-like particles via a low pH-dependent mechanism.A) WT and H244A mutant E proteins are comparably expressed. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E protein expression was detected by immunofluorescence and the nuclei were stained with DAPI. Fluorescence images are shown at the same magnification and exposure time. Bar represents 30 µm. B) WT and H244A mutant E proteins are comparably immunoprecipitated by conformation-specific mAbs. Stable cells inducibly expressing the WT or H244A mutant forms of prM-E were treated with tetracycline for 36 h at 37°C. E proteins in the cell lysates were immunoprecipitated by Sango, a rabbit polyclonal antibody to DIII, and by the mouse mAbs 4G2 and 4E11, as indicated at the top of the panel. Samples were then analyzed by SDS-PAGE and western blot using mouse anti-DENV2 Ab for the Sango samples and Sango for the mAbs samples. Asterisks indicate the positions of the IgG and IgG heavy chain, which cross-react in the western blot. Equivalent sample input was evaluated by western blot for β-actin (lower panel). C) Effect of low pH on WT and H244A VLP production. WT and H244A mutant cells were incubated with tetracycline for 2 h and then in this medium plus 20mM NH4Cl where indicated for a total of 36h. VLP released in the culture media were pelleted by ultracentrifugation, and E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were analyzed by SDS-PAGE and western blot using Sango. 5-fold more culture media from the H244A cells than the WT cells were loaded. Data are representative examples of two or more independent experiments.
Mentions: We established stable HEK 293 cells that inducibly express the DENV1 WT or H244A prM-E proteins. After 36 h induction with tetracycline, both WT and H244A cells show abundant intracellular expression of the DENV1 E protein as detected by immunofluorescence, while the parent cell line is negative for E expression (Fig. 8A). To evaluate whether WT and H244A E proteins were correctly folded, cells were induced for 36 h, lysed, and immunoprecipitated with a rabbit polyclonal antibody to E DIII, and with two conformation-specific mAbs. mAb 4E11 recognizes a discontinuous epitope on DENV E DIII and requires proper DIII disulfide bond formation for recognition [49], [50]. mAb 4G2 recognizes the fusion loop at the tip of flavivirus E DII and its epitope is sensitive to reduction [51]. Expression studies have shown that the 4G2 epitope is not formed if the E protein is expressed in the absence of prM [52], indicating that this epitope is particularly useful for diagnostic tests of prM's chaperone interaction with E (see also reference [37]). As shown in Fig. 8B, lysates from cells induced to express prM plus WT or H244A E proteins showed strong reactivity with all three antibodies. Quantitation of multiple experiments confirmed that WT and H244A E proteins were comparably recognized by the 4E11 and 4G2 mAbs. Thus, by these criteria H244A E protein interacts with prM protein and is correctly folded. This result also agrees with our finding that truncated H244A E protein expressed with prM in the S2 cell system was fully active in low pH-dependent membrane binding and trimerization, suggesting correct folding (Fig. 6).

Bottom Line: Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles.Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway.The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

Show MeSH
Related in: MedlinePlus