The Mechanism Of Rotavirus-encoded Small RNA 1755 Activating Autophagy And Regulating Itself Replication

The interaction between the host cell and the virus is important to reveal the mechanism of proliferation of the virus.In the process of viral infection,the virus can encode the expression of the microRNA molecule to regulate its genes and host genes,promoting the replication of the virus and avoiding the host's immune surveillance.Research shows that in the process of viral infection,the virus can hijack the autophagy membrane system of the host cell to replicate the virus itself.There are several regulatory processes and factors involved in the process,but the exact mechanism of the hijacking is not yet clear.Rotavirus(Rotavirus,RV)is a genetic component segments of double-stranded RNA virus,is called enterovirus,Rotavirus,in infants and young children diarrhea caused by has important status in etiology and epidemiology.Papers around the RV replication mechanism,especially to infected host cells after the regulation mechanism of autophagy and the relationship of viral replication ability carried on the thorough careful research.This study is of great significance not only to reveal the molecular mechanism of virus replication,but also to investigate the mechanism of pathogenesis and drug target screening.In this study we adopt the method of the depth of the small RNA sequencing,the wild strains isolated from endemic areas ZTR68(G1P[8])infected host cells MA104 batches(monkey kidney cell line)after the screening of differentially expressed miRNAs,found the small RNA molecules chr-1755 expression is significantly higher than the other,thus speculated that the molecule may play a major role in the RV replication,named it the vsRNA1755.To confirm the molecule in different type of virus and whether exist in different cells expressing universal,experiment to choose the other four different types G(G2,and G3,G4,G9)of the RV and two types of cells HT-29 optimization colon cancer cell line(people),Caco-2(cancer of colorectal adenomas)were compared,found vsRNA1755 all have high expression.A series of regulatory mechanisms after the G1 type ZTR68 strain infected MA104 cells were studied.The correlation was found in the virus's early replication ability and the vsRNA1755 relationship.Through Blsat comparison and in vitro transfection NSP4 gene analysis shows that vsRNA1755 comes from the RV nonstructural protein NSP4 enterotoxin(encoding genes associated with viral RNA replication)genes,are encoded by NSP4 gene coil-coiled area small RNA molecules,biosynthesis and AGO 1 participation by Dicer-1.To find the downstream target gene of vsRNA1755,a miRanda software forecast reveals that the insulin-like Growth Factor 1 Receptor,IGF1R,is a potential target for vsRNA1755.We use dual luciferase report system,by in vitro transfection vsRNA1755 and using vsRNA1755 simulation objects(mimics)expression in transfection cell detection,prove the existence of the relationship between target and cut IGF1R vsRNA1755 can express.After get the relationship between the target genes,we on vsRNA1755 biological function are discussed in this paper,considering the IGF1R is the starting component of PI3K/Akt pathway and closely related to the activation of autophagy pathway,we experiment on vsRNA1755 and the mechanism of autophagy research.By autophagy flow detection kit(specificity)and electron microscopy morphology inspection,found that when the transfection autophagy protein markers LC3 again after the RV infection,LC3-I converted to LC3-?,that have taken place in the RV infection of cells after clear autophagy.The tracer results of green fluorescent protein(EGFP)and LC3 showed that RV and LC3 had a co-located relationship,which further validates that RV infection is closely related to autophagy.Morphological observations showed that after infection,the infection occurred in small bodies,with a significant increase in the number of infections between 4 and 6 hours,and the subsequent increase in the number of virus particles.When the virus replicates and the virus replicates,the virus replication is prompted by the use of autophagy 3-MA.Autophagy regulatory mechanism in research vsRNA1755 experiment,we use the molecular simulation objects(mimics)transfection cells tested multiple autophagy regulatory proteins(ATG5,ATG12,P62)expression,found that autophagy related proteins ATG5 and ATG12 expression,autophagy substrate P62 expression.The protein-printing experiment showed that after expressing vsRNA1755,the autophagy specific substrate P62 protein was significantly reduced.By adding the PI3K/Akt pathway inhibitor,LY294002,to detect the infection and copy number of the virus,the results show that the PI3K/Akt pathway inhibits the replication of the virus.To vsRNA1 755 with target gene IGF1R qRT-PCR and protein imprinting testing,the results suggest that the expression of the RV early in infection can cut IGF1R,speculation vsRNA1755 cut IGF1R through inhibition of PI3K/Akt/mTOR signaling pathway activation,trigger,activate autophagy,to achieve viral replication.The results of the pathway were also confirmed by the increase in external transfection mimics.To sum up,the RV infection after the host cell,by the RV NSP4 gene encoding of small RNA molecules(vsRNA1755)1755,caused by inhibiting IGF1R express PI3K/Akt/mTOR signaling pathways downstream of the deactivation,lift its inhibition of autophagy pathways,activate cell autophagy,viruses hijack a cell autophagy pathway to promote its own replication.Research results not only provides the main basis for revealing the RV infection mechanism,as well as interaction between virus and host cells and future targeted drug targets,more accurate design provides the theory basis of meaningful.