The Relationship Between Host Proteasome Subunit Alpha 2 Protein And CVB5 Virus Infection

Coxsackievirus B5 virus is the important pathogens that cause aseptic meningitis and the foot-and-mouth disease.CVB5 epidemiological studies showed that CVB5 popular periodically,every 3 to 6 years,surging trend appears and last for 1 year.However,the pathogenesis of the virus is not yet clear.In terms of the treatment,the methods of specific testing and efficient treatment has not been developed.The study of CVB5 has still limited on the epidemiological investigation,genetic analysis,clinical diagnosis and so on.The protein degradation modificated by the ubiquitin-proteasome pathway(UPP)is a mechanism for controlling the function and availability of regulatory proteins in the cell.It plays a key role in a variety of cell functions,including cell differentiation,cell cycle,apoptosis,DNA repair and antigen processing.Studies have shown that many viruses can evolve different strategies to exploit the ubiquitin-proteasome pathway(UPP)for their own benefit.The proteasome subunit alpha type-2 protein(PSMA2)may participate in the replication of the virus life cycle.Therefore,we use the methods of molecular biology and cell biology to investigate the relationship between CVB5 virus and the host protein in the course of infection.This study consists of three main parts:In this study,we identified the virus subtypes by RT-PCR and DNA sequencing before we use it.Then the Cytodex-1 concentration was used to culture Vero cells and prepare large amounts of CVB5.The virus was harvested?measured the infectious titer and preserved in the-40? refrigerator.To further explore the role of PSMA2 proteins in CVB5 infection,we examine the PSMA2 transcriptional level by RT-PCR in 293T and HCT-116 cells after be treated with CVB5 virus.Results show that the transcriptional level of PSMA2 in 293T and HCT-116 cells was first increasing and then decreasing.Further more,significantly up-regulated at 8h following the stimulation of virus and then have a small decrease,compared with the control group,is still a increasing trend.All of this suggests that the PSMA2 is determined to be an immediately inducible geng responding to viral infection.At the same time we also examine the fellow proteasome subunits PSMC2 transcriptional level by RT-PCR in 293T and HCT-116 cells after be treated with CVB5 virus.Results show that the transcriptional level of PSMC2 were increased in 293T cells and significantly at 20h.However in HCT-116 cells significantly up-regulated at 8h following the stimulation of virus and then have a small decrease,compared with the control group,is still a increasing trend.Shows that viruses also have an impact on PSMC2 gene.We demonstrated the change of PSMA2 protein contents by WB in 293T and HCT-116 cells after be treated with CVB5 virus.Results show that the protein level of PSMA2 in 293T cells was increasing,but was decreasing in HCT-116 cells.Shows that in different types of cells have a different answer to CVB5 virus infection.Successfully build up a pcDNA3.1-PSMA2-2×Flag carrier,and make sure it stably expressing in 293T cells by the means of WB.Transfect the overexpression vector into 293T cells,and use the empty vector and untransfected group as control at the same time.The titer tests experiment show that the CVB5 virus titer rise after PSMA2 protein overexpression.To sum up,PSMA2 protein participate in the CVB5 viral replication process,which lay a theoretical foundation for further study the mechanism of viral infection.