Cellular Mitochondrial Stress Induced By Enterovirus-d68's 3C/3D Protein And The Protein Effect On Viral Infection

Enterovirus-D68(EV-D68)is the small RNA virus family(Picornaviridae)enterovirus genus of a virus,the virus formerly known as Rhinovirus type 87,after the intestinal virus.Enterovirus-D68 The main susceptible crowd for children,the main organ of the invasion is lung,and will cause child bronchiolitis and pneumonia.Before 2010,the world has been a lack of attention to the EV-D68,the number of isolated strains of the world has been a handful,the study of EV-D68 stayed at a lower level,but as the global demand for health level increased,but also due to 2010 years after the United States,Japan,New Zealand and other countries have emerged ev-d68 outbreak,Ev-d68 began to attract global attention.The recent outbreak of ev-d68 in North America,which occurred 2014 years ago,led to the death of many children,and a number of EV-D68 viruses were isolated in China 2010 years later,but no large-scale outbreaks occurred.Mitochondria have been considered as the cell's energy plant for a period of time,providing the necessary energy for a series of cell life activities,but in recent years,as people have studied more deeply,mitochondria have been given a new title-The center of innate immunity.To explore how the mitochondria in cells and cells are affected by the EV-D68 after infection.We use the EV-D68 to infect Hela cells,and through RealTime-PCR,to detect the amount of virus in the cells at different time points in absolutely quantified.The cells of different time points were stained by PI to detect the cell cycle after the virus infection.The mitochondrial morphology of the infected cells was observed by transmission electron microscope.The expression of ATP synthase in mitochondria was detected by Western blot to determine whether mitochondrial function was affected.The JC-1 staining was used to detect whether the homeostasis of mitochondria was affected by virus infection.Detection of mitochondrial DNA(mtDNA)stress in cellular mitochondria after viral infection was detected by immunofluorescence.We found that the EV-D68 virus can proliferate normally in Hela cells by analyzing the results.After viral infection,the number of cells in the infected cells increased in the S phase.After the infection of the cell samples of the transmission electron microscopy observation,we found that after the virus infection in the cells of the mitochondrial morphology changes,mitochondrial membrane gap due to the disappearance of the crest of the phenomenon of fusion expansion.The mitochondria appear to some extent shrink.The expression of ATP synthase in mitochondria of infected cells was detected by Western blot,and the ATP synthase in mitochondria was reduced after virus infection,which showed that the capacity function of mitochondria could be affected after virus infection.After using JC-1 to dye the cells at different points of time after virus infection,we found that the number of JC-1 monomers after viral infection showed an increasing phenomenon,indicating that the viral infection would have an effect on the homeostasis of cell mitochondria.Using immunofluorescence,we observed the cells after viral infection under fluorescence confocal microscopy,and we found the mtDNA of stress after viral infection.In order to explore which protein in the EV-D68 will cause mitochondrial stress,through data access and analysis,we choose VP 1,P1,3C and 3D of these four proteins for analysis and detection,we constructed these four proteins expression plasmid transfection into 293T cells,As a result,we found that the two proteins P1 and VP1 were unrelated to mitochondrial stress,while the 3C and 3D proteins were directly related to mitochondrial stress.We used RealTime-PCR and Western blot to compare the expression of gene levels and protein levels of mitochondrial-associated proteins after infection with whole virus and individual P1 VP1,3C and 3D protein granules.We found that at the gene level,total viral infection and individual plasmid transfection can cause changes in the gene expression of mitochondrial-related proteins,but there is no significant correlation.At the level of protein,similar to total viral infection,both VP1,P1 and 3D proteins can cause a rise in MFN2 expression,and we also find that 3C proteins can reduce MFN1 and MFN2 expression levels.To explore the common effects of ev-d68 total viral infection and individual P1,VP1,3C and 3D protein transfection on innate immunity,we used RealTime-PCR to detect the gene expression of innate immune-related cytokines in infected and transfected cells,we found that the expression of TNF-? and IFN-P gene increased after transfection of 3C protein and 3D protein,which was identical to the whole viral infection.To sum up,EV-D68 infected cells will cause a series of effects on the cell,will have a negative effect on the mitochondria in the cell,will lead to mtDNA produce stress phenomenon.The 3C protein and 3D protein in EV-D68 were the main proteins causing stress in mtDNA,which had an effect on the gene level and expression of protein level of mitochondrial related proteins.These two proteins also play a role in the innate immune response caused by ev-d68 infection.