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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 virus infection.RNAs derived from the WT and E H244A mutant DENV1 WP infectious clones were electroporated into BHK cells. Cells were cultured for 3 d and infected cells were detected by immunofluorescence. In parallel, cells were cultured at 28°C for 6 d and progeny virus in the culture medium was quantitated using infectious center assays on indicator BHK cells. Progeny virus titers are shown in the box below each fluorescence image. Results are given for two independent infectious clones of H244A, indicated as (2) and (4). Bar represents 30 µm.
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ppat-1001157-g007: DENV E H244A mutation inhibits virus infection.RNAs derived from the WT and E H244A mutant DENV1 WP infectious clones were electroporated into BHK cells. Cells were cultured for 3 d and infected cells were detected by immunofluorescence. In parallel, cells were cultured at 28°C for 6 d and progeny virus in the culture medium was quantitated using infectious center assays on indicator BHK cells. Progeny virus titers are shown in the box below each fluorescence image. Results are given for two independent infectious clones of H244A, indicated as (2) and (4). Bar represents 30 µm.

Mentions: Since the E H244A mutation disrupts E protein's interaction with pr, we used this mutation to address the importance of pr in protecting DENV during transport through the exocytic pathway. We introduced the E H244A mutation into the infectious clone of DENV1 WP. WT and mutant viral RNAs were prepared by in vitro transcription and were electroporated into BHK cells. After culture for 3 d at 37°C, both WT and mutant RNA-electroporated cells expressed abundant E protein as detected by immunofluorescence microscopy (Fig. 7). Parallel cultures were incubated for 6 d and progeny virus in the culture media was detected by infectious center assays on indicator BHK cells. WT-infected cells produced infectious progeny virus with a titer of ∼1.5×105 IC/ml. However, two independent infectious clones of the H244A mutant produced no detectable progeny virus, even though the viral RNAs mediated efficient primary infection as shown in Fig. 7. This agrees with previous studies indicating lethal effects of an H244A mutation on DENV2 [43].


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 virus infection.RNAs derived from the WT and E H244A mutant DENV1 WP infectious clones were electroporated into BHK cells. Cells were cultured for 3 d and infected cells were detected by immunofluorescence. In parallel, cells were cultured at 28°C for 6 d and progeny virus in the culture medium was quantitated using infectious center assays on indicator BHK cells. Progeny virus titers are shown in the box below each fluorescence image. Results are given for two independent infectious clones of H244A, indicated as (2) and (4). Bar represents 30 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001157-g007: DENV E H244A mutation inhibits virus infection.RNAs derived from the WT and E H244A mutant DENV1 WP infectious clones were electroporated into BHK cells. Cells were cultured for 3 d and infected cells were detected by immunofluorescence. In parallel, cells were cultured at 28°C for 6 d and progeny virus in the culture medium was quantitated using infectious center assays on indicator BHK cells. Progeny virus titers are shown in the box below each fluorescence image. Results are given for two independent infectious clones of H244A, indicated as (2) and (4). Bar represents 30 µm.
Mentions: Since the E H244A mutation disrupts E protein's interaction with pr, we used this mutation to address the importance of pr in protecting DENV during transport through the exocytic pathway. We introduced the E H244A mutation into the infectious clone of DENV1 WP. WT and mutant viral RNAs were prepared by in vitro transcription and were electroporated into BHK cells. After culture for 3 d at 37°C, both WT and mutant RNA-electroporated cells expressed abundant E protein as detected by immunofluorescence microscopy (Fig. 7). Parallel cultures were incubated for 6 d and progeny virus in the culture media was detected by infectious center assays on indicator BHK cells. WT-infected cells produced infectious progeny virus with a titer of ∼1.5×105 IC/ml. However, two independent infectious clones of the H244A mutant produced no detectable progeny virus, even though the viral RNAs mediated efficient primary infection as shown in Fig. 7. This agrees with previous studies indicating lethal effects of an H244A mutation on DENV2 [43].

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