The Molecular Mechanism Of Host Protein GM130 In CVB3-induced Pancreatitis

Objective:To investigate the molecular mechanism of the host protein GM130 in CVB3-induced pancreatitis.Methods:1. BALB/c adult mice were inoculated intraperitoneally with 106 plaque forming units (PFU) of CVB3 and sacrificed at day 0, 3, 6 or 9 post infection (p.i.). Viscera Indexes were detected and histopathological studies (Hematoxylin and Eosin stain) were conducted to observe the pathological changes of the pancreas and heart;2. The tissue proteins of pancreas and heart were collected at day 0, 3, 6 and 9 p.i.. WB (Western Blotting) was used to detect the expression of VP1 and GM130 for analysis of CVB3 replication in various tissues and the impact on the expression of GM130 of actue CVB3 infection;3. The level of GM130 protein in the pancreas and heart of both newborn and adult mice were examined by WB and IHF (Immunofluorescence Histochemistry) to characterize the distribution of GM130 in normal BALB/c mice;4. Serial sections from the pancreata at day 0, 3, 6 and 9 days p.i. were stained by IHC (Immunohistochemistry) and IHF to investigate the impact of CVB3 infection on the GM130 and the Golgi ribbon in the BALB/c adult mice;5. Hela cells were infected with CVB3 at MOI fo 5, detected the interaction among the VP1, GM130 and GRASP65 by intracellular Co-immunoprecipitation and confocal microscopy with double immunofluorescent stain, and then analyzed the effect on the Golgi ribbon of the interaction between GM130 and VP1.Results:1. The changes of the viscera indexes and histopathological studies of the pancreas and heart showed that inflammatory cells infiltration was gradually increased in the pancreas and pancreatic acinar structure was gradually destroyed during the early phase of CVB3 infection; And there was no abvious change in the heart, except for only a little inflammatory cell infiltration;2. The virus could replicate in the pancreas during the early phase of CVB3 infection and the level of GM130 protein from the pancreas was observed to decrease gradually with the increase of infection time by WB.3. High expression of GM130 was observed both in the pancreas of adult mice and in the heart of newborn mice by WB and IHF, while there were no expression either in the pancreas of newborn mice or in the heart of adult mice;4. The results of the animal experiment showed that the GM130 was ribbon-like in the normal pancreas and then became disperse during the early phase of infection;5. The results of intracellular Co-IP and confocal microscopy showed that CVB VP1 interacts physically with the cellular partner, GM130, in CVB3-infected Hela cells. The interaction between CVB3 VP1 and GM130 may interfere with GM130-GRASP65 complex and lead to disrupt the Golgi ribbon.Conclusions:1. The host factor GM130 is a tissue- and age-dependent target protein of CVB32. CVB3 VP1 interacts physically with a cellular partner, GM130, in CVB3-infected Hela cells, and the direct interaction between these two proteins leads to disrupt the GM130-GRASP65 complex and the Golgi ribbon which contributes to CVB3-induced acute pancreatitis in adult mice. We define a previously unknown molecular mechanism by which CVB3 causes extensive pancreatic demage during the early phase of infection.