Host Factor CD63 Promotes Replication Of Astrovirus And Rotavirus In Vitro

ObjectiveTo investigate the effect of host factor CD63 on the replication of human rotavirus(RV)SA11 strain and human astrovirus HAst V-1 strain in vitro.In order to lay the foundation for further study of the molecular pathogenic mechanism of the virus and the interaction of the virus with host factor.Methods1.Detecting the titers of HAst V-1 and RV SA11 virus using the micro-fluorescence stove method.2.The CD63 silencing expression plasmids sh RNA-4038,sh RNA-4041 and the overexpression plasmid pc DNA3.1-CD63-flag(CD63-Flag)were transfected into HAst V-1 host cells Caco2 and RV SA11 host cells CV-1 and MA104,respectively.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression of CD63 m RNA in cells,Detecting the expression of CD63 protein in cells by Western blotting and CD63 silencing expression and CD63 overexpression cell strains were screened.3.The two groups of transfected sh RNA-4038 plasmid group and transfected sh RNA-4041 plasmid group with high interference efficiency at m RNA level and protein level are CD63 Interference Group,the group with the highest expression of m RNA and protein level was pc DNA3.1-CD63-flag as the CD63 overexpression group,CNr was the blank control group,and sh NC was the transfection control group.Five groups of cells were infected with HAst V-1virus with a titer of 5.17 log FFU/ml.The titer of HAst V-1 virus was detected bymicro-immunofluorescence assay and the copy number of the viral gene was detected by q RT-PCR 48 hours later.4.The experiment was divided into CD63 Interference Group(CD63-L),CD63 overexpression cell group(CD63-H),transfection control group(Sh NC)and normal control cell group(CNr).The sh RNA-4038,sh RNA-4041,and overexpression plasmid CD63-Flag were transfected into different host cells of the virus with transfection technology in vitro.The virus infected the cells after transfection 48 hours.The copy number of the viral gene was detected by q RT-PCR,and the expression of viral protein was detected by Western blotting.The effect of CD63 expression on the replication of RV-SA11 viruses in vitro was clarified.Results1.After sh RNA-4038 and sh RNA-4041 plasmids were transfected into ascovirus host cell Caco2,CD63 expression was significantly inhibited.Compared with control group Sh NC,the inhibition efficiency of sh RNA-4038 and sh RNA-4041 at the m RNA level were respectively 91.3% and74.6%;proteinlevel inhibition efficiency was 83.7% and 68.4%,respectively.After Caco2 were transfected into pc DNA3.1-CD63-flag plasmid,compared with thecontrol group Sh NC,the m RNA and protein expression of CD63 increasedsignificantly(P<0.001).2.HAst V-1 virus with a titer of 5.17 log FFU/ml was used to infect CD63high-expressing cell lines and low-expressing cell lines,respectively.Compared with the normal cell control group,the test result was HAst V-1 virus droplets transfected with sh RNA-4038 plasmid group Degree decreased by 2.06 log FFU/ml,and the titer of HAst V-1 virus in the sh RNA-4041 plasmid group decreased by 1.88 log FFU/ml.After one-way analysis of variance,the difference was statistically significant(P<0.001).The titer of HAst V-1 virus in the transfected pc DNA3.1-CD63-flag plasmid group increased by 1.19 log FFU/ml,and the cells inthe control group had no significant change.The difference was statistically significant by single factor analysis of variance(P<0.01).3.After infection of cells with HAst V-1 virus at a titer of 5.17 log FFU /ml,HAst V-1 virus gene copy number in the sh RNA-4038 plasmid group decreased by 85.4%,and HAst V-1 virus gene copy number in the sh RNA-4041 plasmid group decreased.69.6%;the number of copies of virus gene in the pc DNA3.1-CD63-flag plasmid group transfected significantly increased,and the virus expression in the control group did not change significantly(P<0.05).4.After rotavirus host cells CV-1 and MA104 were transfected with sh RNA-4038 and sh RNA-4041 plasmids,the expression of CD63 was significantlyinhibited,and the inhibitory efficiency of m RNA and protein levels in CV-1 cells was 69.7% and 68.5%,respectively.The inhibition efficiency of m RNA and protein levels in MA104 cells was 76.4% and 69.8%,respectively.After rotavirus host cells CV-1 and MA104 were transfected with pc DNA3.1(+)-CD63-flag plasmid,the expression of CD63 m RNA and protein was significantly increased(P<0.05).5.After infecting cells with SA11 virus with a titer of 8 log FFU / ml,the number of virus gene copies in the CD63-L group decreased by 41.1%(CV-1)and 35.7%(MA104);viral protein expression levels decreasedby 51.6%(CV-1)And 54.5%(MA104);the virus gene copy number and protein expression level of the CD63-H group increased significantly,while the virus expression in the control group had no significant change(P<0.05).ConclusionsThe over expression of host factor CD63 can promote the replication of astrovirus and rotavirus.After CD63 inhibits expression,virus replication is significantly reduced.The expression of host factor CD63 is positively correlated with the replication of astrovirus and rotavirus.