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Identification of the interaction of VP1 with GM130 which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

Li X, Xia Y, Huang S, Liu F, Ying Y, Xu Q, Liu X, Jin G, Papasian CJ, Chen J, Fu M, Huang X - Sci Rep (2015)

Bottom Line: Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans.Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells.Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, School of Medicine, Nanchang University, Nanchang, Jiangxi, China.

ABSTRACT
Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans. Although the susceptibility of CVB3-induced acute pancreatitis is age-dependent, the underlying mechanisms remain unclear. Here we identified the host factor Golgi matrix protein 130 (GM130) as a novel target of CVB3 during CVB3-induced acute pancreatitis. The viral protein VP1 interacted with GM130, disrupted GM130-GRASP65 complexes, and caused GM130 degradation, which may lead to disruption of the Golgi ribbon and development of acute pancreatitis in mice. Interestingly, the expression level of GM130 in mouse pancreas was age-dependent, which was nicely correlated with the age-associated susceptibility of CVB3-induced acute pancreatitis. Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells. Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

No MeSH data available.


Related in: MedlinePlus

Mapping the regions of VP1 and GM130 that are required for their interaction.(a) The regions of VP1 interacting with GM130 were mapped by a Y2H assay. Truncation/deletion mutants of VP1 in pGBKT7 vector were used as the bait plasmids. These were tested for interaction with GM130 prey cloned into pGADT7 vector by α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids, and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate VP1 prey constructs that interacted with GM130, while “–” or hollow bars indicate constructs that did not interact with GM130. (b) Molecular Modeling of CVB3 VP1 protein (GenBank accession number: M88483) on the basis of the published crystal structure of CVB3, as predicted by SWISS-Model server and Discovery Studio Visualizer software. The region of VP1 that binds to GM130 is colored in red. (c) The regions of GM130 interacting with VP1 were mapped by an Y2H assay. The prey proteins, the truncation/deletion mutants of GM130, were tested against the bait VP1. Shown are the results of Y2H α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate GM130 prey constructs that interacted with VP1, while “–” or hollow bars indicate constructs that did not interact with VP1.
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f4: Mapping the regions of VP1 and GM130 that are required for their interaction.(a) The regions of VP1 interacting with GM130 were mapped by a Y2H assay. Truncation/deletion mutants of VP1 in pGBKT7 vector were used as the bait plasmids. These were tested for interaction with GM130 prey cloned into pGADT7 vector by α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids, and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate VP1 prey constructs that interacted with GM130, while “–” or hollow bars indicate constructs that did not interact with GM130. (b) Molecular Modeling of CVB3 VP1 protein (GenBank accession number: M88483) on the basis of the published crystal structure of CVB3, as predicted by SWISS-Model server and Discovery Studio Visualizer software. The region of VP1 that binds to GM130 is colored in red. (c) The regions of GM130 interacting with VP1 were mapped by an Y2H assay. The prey proteins, the truncation/deletion mutants of GM130, were tested against the bait VP1. Shown are the results of Y2H α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate GM130 prey constructs that interacted with VP1, while “–” or hollow bars indicate constructs that did not interact with VP1.

Mentions: To further characterize the interaction between the two proteins, we attempted to map the regions of VP1 that interacted with GM130 utilizing the Y2H approach. A series of VP1 truncation/deletion mutants in pGBKT7 vector were co-transformed with pGADT7-GM130 vector into yeast cells. Yeast transformants positive for prey-bait interactions were selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal and assayed for α-galactosidase activity. The VP1 truncation segment spanning amino acids 61–284 interacted with GM130 while segments spanning amino acids 3–197 and 126–284 failed to interact with GM130 (Fig. 4a,b).


Identification of the interaction of VP1 with GM130 which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

Li X, Xia Y, Huang S, Liu F, Ying Y, Xu Q, Liu X, Jin G, Papasian CJ, Chen J, Fu M, Huang X - Sci Rep (2015)

Mapping the regions of VP1 and GM130 that are required for their interaction.(a) The regions of VP1 interacting with GM130 were mapped by a Y2H assay. Truncation/deletion mutants of VP1 in pGBKT7 vector were used as the bait plasmids. These were tested for interaction with GM130 prey cloned into pGADT7 vector by α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids, and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate VP1 prey constructs that interacted with GM130, while “–” or hollow bars indicate constructs that did not interact with GM130. (b) Molecular Modeling of CVB3 VP1 protein (GenBank accession number: M88483) on the basis of the published crystal structure of CVB3, as predicted by SWISS-Model server and Discovery Studio Visualizer software. The region of VP1 that binds to GM130 is colored in red. (c) The regions of GM130 interacting with VP1 were mapped by an Y2H assay. The prey proteins, the truncation/deletion mutants of GM130, were tested against the bait VP1. Shown are the results of Y2H α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate GM130 prey constructs that interacted with VP1, while “–” or hollow bars indicate constructs that did not interact with VP1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551966&req=5

f4: Mapping the regions of VP1 and GM130 that are required for their interaction.(a) The regions of VP1 interacting with GM130 were mapped by a Y2H assay. Truncation/deletion mutants of VP1 in pGBKT7 vector were used as the bait plasmids. These were tested for interaction with GM130 prey cloned into pGADT7 vector by α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids, and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate VP1 prey constructs that interacted with GM130, while “–” or hollow bars indicate constructs that did not interact with GM130. (b) Molecular Modeling of CVB3 VP1 protein (GenBank accession number: M88483) on the basis of the published crystal structure of CVB3, as predicted by SWISS-Model server and Discovery Studio Visualizer software. The region of VP1 that binds to GM130 is colored in red. (c) The regions of GM130 interacting with VP1 were mapped by an Y2H assay. The prey proteins, the truncation/deletion mutants of GM130, were tested against the bait VP1. Shown are the results of Y2H α-Galactosidase assays performed on yeast colonies cotransformed with the prey and bait plasmids and selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal. “+” or gray sticks indicate GM130 prey constructs that interacted with VP1, while “–” or hollow bars indicate constructs that did not interact with VP1.
Mentions: To further characterize the interaction between the two proteins, we attempted to map the regions of VP1 that interacted with GM130 utilizing the Y2H approach. A series of VP1 truncation/deletion mutants in pGBKT7 vector were co-transformed with pGADT7-GM130 vector into yeast cells. Yeast transformants positive for prey-bait interactions were selected on plates lacking adenine, histidine, leucine, and tryptophan with X-α-Gal and assayed for α-galactosidase activity. The VP1 truncation segment spanning amino acids 61–284 interacted with GM130 while segments spanning amino acids 3–197 and 126–284 failed to interact with GM130 (Fig. 4a,b).

Bottom Line: Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans.Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells.Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, School of Medicine, Nanchang University, Nanchang, Jiangxi, China.

ABSTRACT
Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans. Although the susceptibility of CVB3-induced acute pancreatitis is age-dependent, the underlying mechanisms remain unclear. Here we identified the host factor Golgi matrix protein 130 (GM130) as a novel target of CVB3 during CVB3-induced acute pancreatitis. The viral protein VP1 interacted with GM130, disrupted GM130-GRASP65 complexes, and caused GM130 degradation, which may lead to disruption of the Golgi ribbon and development of acute pancreatitis in mice. Interestingly, the expression level of GM130 in mouse pancreas was age-dependent, which was nicely correlated with the age-associated susceptibility of CVB3-induced acute pancreatitis. Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells. Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

No MeSH data available.


Related in: MedlinePlus