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Structure of the low pH conformation of Chandipura virus G reveals important features in the evolution of the vesiculovirus glycoprotein.

Baquero E, Albertini AA, Raux H, Buonocore L, Rose JK, Bressanelli S, Gaudin Y - PLoS Pathog. (2015)

Bottom Line: An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure.Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses.It also reveals the remarkable plasticity of G in terms of local structures.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.

No MeSH data available.


Related in: MedlinePlus

Fusion activity of CHAV-G and VSV-G analyzed in a cell-cell fusion assay.A) BSR cells were transfected with plasmids expressing the full-length G proteins and RABV P-GFP to facilitate the observation of syncytia. At 24 h post-transfection, the cells were incubated for 10 min with cell medium adjusted to the indicated pH, and then replaced by fresh medium at pH 7.4. The cells were kept at 37°C for 1 h before fixation. Nuclei were stained with DAPI. All images are representative examples from at least three independent experiments. The scale is the same for all micrographs. B) Percentage of fluorescent cells having formed a syncytia (containing more than 3 nuclei) as a function of pH for both VSVIND-G and CHAV-G. C) Distribution of the size of syncytia as a function of pH for both VSVIND-G and CHAV-G (at each pH, the examined field corresponded to about 400 transfected fluorescent cells at pH7).
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ppat.1004756.g001: Fusion activity of CHAV-G and VSV-G analyzed in a cell-cell fusion assay.A) BSR cells were transfected with plasmids expressing the full-length G proteins and RABV P-GFP to facilitate the observation of syncytia. At 24 h post-transfection, the cells were incubated for 10 min with cell medium adjusted to the indicated pH, and then replaced by fresh medium at pH 7.4. The cells were kept at 37°C for 1 h before fixation. Nuclei were stained with DAPI. All images are representative examples from at least three independent experiments. The scale is the same for all micrographs. B) Percentage of fluorescent cells having formed a syncytia (containing more than 3 nuclei) as a function of pH for both VSVIND-G and CHAV-G. C) Distribution of the size of syncytia as a function of pH for both VSVIND-G and CHAV-G (at each pH, the examined field corresponded to about 400 transfected fluorescent cells at pH7).

Mentions: We could observe formation of massive syncytia between pH 5.0 and 6.0 for cells expressing either CHAV-G or VSVIND-G. At pH 6.3, syncytia were smaller for both glycoproteins. At pH 6.5, no syncytia were detected for CHAV-G whereas small syncytia (less than 20 nuclei) were still observed for VSVIND-G up to pH 7.0 (Fig. 1).


Structure of the low pH conformation of Chandipura virus G reveals important features in the evolution of the vesiculovirus glycoprotein.

Baquero E, Albertini AA, Raux H, Buonocore L, Rose JK, Bressanelli S, Gaudin Y - PLoS Pathog. (2015)

Fusion activity of CHAV-G and VSV-G analyzed in a cell-cell fusion assay.A) BSR cells were transfected with plasmids expressing the full-length G proteins and RABV P-GFP to facilitate the observation of syncytia. At 24 h post-transfection, the cells were incubated for 10 min with cell medium adjusted to the indicated pH, and then replaced by fresh medium at pH 7.4. The cells were kept at 37°C for 1 h before fixation. Nuclei were stained with DAPI. All images are representative examples from at least three independent experiments. The scale is the same for all micrographs. B) Percentage of fluorescent cells having formed a syncytia (containing more than 3 nuclei) as a function of pH for both VSVIND-G and CHAV-G. C) Distribution of the size of syncytia as a function of pH for both VSVIND-G and CHAV-G (at each pH, the examined field corresponded to about 400 transfected fluorescent cells at pH7).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4372607&req=5

ppat.1004756.g001: Fusion activity of CHAV-G and VSV-G analyzed in a cell-cell fusion assay.A) BSR cells were transfected with plasmids expressing the full-length G proteins and RABV P-GFP to facilitate the observation of syncytia. At 24 h post-transfection, the cells were incubated for 10 min with cell medium adjusted to the indicated pH, and then replaced by fresh medium at pH 7.4. The cells were kept at 37°C for 1 h before fixation. Nuclei were stained with DAPI. All images are representative examples from at least three independent experiments. The scale is the same for all micrographs. B) Percentage of fluorescent cells having formed a syncytia (containing more than 3 nuclei) as a function of pH for both VSVIND-G and CHAV-G. C) Distribution of the size of syncytia as a function of pH for both VSVIND-G and CHAV-G (at each pH, the examined field corresponded to about 400 transfected fluorescent cells at pH7).
Mentions: We could observe formation of massive syncytia between pH 5.0 and 6.0 for cells expressing either CHAV-G or VSVIND-G. At pH 6.3, syncytia were smaller for both glycoproteins. At pH 6.5, no syncytia were detected for CHAV-G whereas small syncytia (less than 20 nuclei) were still observed for VSVIND-G up to pH 7.0 (Fig. 1).

Bottom Line: An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure.Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses.It also reveals the remarkable plasticity of G in terms of local structures.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.

No MeSH data available.


Related in: MedlinePlus