Research On The Interaction Between Outer Capsid Proteins Of Grass Carp Reovirus And Pathway Involved In Viral Cellular Entry

Grass carp reovirus（GCRV）is one of the main viral pathogens with high lethality and infectivity,which causes the grass carp hemorrhagic disease.GCRV belongs to the aquareovirus and is one of the most virulent strains in the genus.The virus mainly divides into three kinds of genotype,GCRV 873 as the representative strain ofⅠtype,GCRV HZ08 as the representative strain ofⅡtype and HGDRV（GCRV 104）as the representative strain ofⅢtype.Our research uses modern molecular biology technology to verify that interaction between GCRV outer capsid protein VP5 and VP7,which are responsible for adsorbed host cell.The main GCRV proliferation dynamics and pathway involved in viral cellular entry.The main contents of this research are as follows:1.Identification and analysis of the interaction structure between the outer capsid protein VP5 and VP7 of GCRV.GCRV outer capsid protein VP5 and VP7 are located at the outermost layer of the virus particles to form the heterogeneous dimer,which responsible for identifying the adsorbed host cells.In the research of this chapter,the yeast two-hybrid system was used to preliminarily verify the interaction between VP5 and VP7,and we expressed VP5 and VP7 proteins by prokaryotic expression and baculovirus expression system respectively.The Dot-blot experiment further confirmed the interaction between VP5and VP7.In order to confirm the structural sites of interaction between VP5 and VP7proteins preliminarily,the coded VP5 and VP7 protein genes were truncated respectively.We tested the interaction between truncated and full-length in yeast two-hybrid experiment.The results shows that the interection between VP5 truncated protein and VP7 full-length protein all existed.However,there is no interaction between VP7 truncated protein and VP5 full-length protein.So we inferred that the interaction between VP5 and VP7 protein exist many binding sites in VP5.2.The proliferation kinetics of two genotypes grass carp reovirus.The different genotypes of GCRV,GCRV JX01 and GCRV 104,were used to compare proliferation kinetics of the host cells infection.By the same infection conditions,GCRV JX01 and GCRV 104 were infected respectively.According to the virus titer of the supernatant at different time points and the reproduction of virus particles in the host cells,studying on the proliferation kinetics of the two genotypes of the GCRV.The results showed that GCRV 104 infect the host in the process of cell proliferation,which is slower than that of GCRV JX01.According to previous research on the GCRV JX01 endocytic pathway,our research of this chapter makes a preliminary discussion on the GCRV 104 pathway involved in viral cellular entry.The inhibitors NH₄Cl and Dynasore were used to screen the corresponding pathway,and we find that the inhibition effect on GCRV 104 of NH₄CL is very obvious,Dynasore also has obvious inhibitory effect on GCRV 104.We come to the conclusion that GCRV 104 in the process of infecting the host cell with the low pH environment,and depends on the role of the initiation protein dynamin.3.Research on the pathway involved in viral cellular entry of typeⅢgrass carp reovirus（GCRV 104）.We futher study on the pathway involved in viral cellular entry of typeⅢgrass carp reovirus（GCRV 104）in this chapter.The 11 inhibitors were selected to carry out the pathway screening experiment.The cell were infected by GCRV 104 under the treatment of different inhibitors,and the inhibitory effect of different inhibitors were analyzed by the quantitative analysis of the virus titer.We screening preliminarily four inhibitors has significant inhibitory effect on GCRV 104 infection.The clathrin-mediated endocytosis inhibitors,CPZ and Pistop2.Rottlerin of PKC inhibitor and Wortmannin of PI3K inhibitor.To further confirm the inhibitory of these four inhibitors for GCRV 104 infection,GCRV 104 into the cell level experiments were carried out.The quantification of viral particles and the expression of viral proteins in the host cells were used to test and analyze.The inhibitors CPZ and Pistop2 which are related to the clathrin-mediate endocytic pathway（CME）,they has obvious inhibitory effect on entry of GCRV JX01 and GCRV 104.This result consistent with previous research on the pathway involved in viral cellular entry of typeⅠgrass carp reovirus（GCRV JX01）.Thus we confirmed that type Ⅲ grass carp reovirus（GCRV 104）invaded the host cells involved in clathrin-mediated endocytosis pathway.The two inhibitors of Rottlerin and Wortmannin also has a significant inhibitory effect on the entry of type Ⅲ grass carp reovirus（GCRV 104）into the host cell in the cell level experiment.These two inhibitors has inhibition of PKC and PI3K respectively.Therefore,it is also explained that the PKC and PI3K is involved in the process of type Ⅲ grass carp reovirus（GCRV 104）into the host cell.From the viral infection and entry level of the test to suggest that theⅢtype of GCRV might invade host cells via clathrin-mediate endocytosis in a pH-dependent manner.Additionally,Dynamin,PKC and PI3K are critical for efficient viral entry.