Effect Of Human Cytomegalovirus Infection On Autophagy In MRC-5 Cells

Objective: Human cytomegalovirus(HCMV)-a member of the Herpesviri dae family,is widespread in human populations,with up to 90% of some populations seropositive for the virus.Although HCMV infection is usually asymptomatic in healthy individuals,it is a major cause of morbidity and mortality in immunocompro-mised individuals,such as AIDS patients or bone marrow and solid organ transplant recipients,and it can cause life-threatening infections that compromise long-term graft function.Moreover,HCMV congenital infection is the most common cause of virus-induced birth defects,causing the damage of multi-system organ.Autophagy plays a crucial role in many cellular activities including development,differentiation,survival,and homoeostasis.In addition to being a response to internal and external stress,autophagy also shapes cellular immunity.It serves as an essential component of host defense against viral infection,by orchestrating pathogen degradation(xenophagy),innate immune signaling and certain aspects of adaptive immunity.Viruses have evolved a variety of strategies to evade autophagic attack and manipulate the autophagic machinery for their own benefit.In this research,we will focus on the relation between autophagy and HCMV infection,and how HCMV modulates autophagy.Method: 1.Detect the changes of autophagy level at the different stages of HCMV infection in MRC-5 cells(human fetal lung fibroblast),and the expression level of the downstream proteins of m TOR signaling pathway.The MOI of 1 HCMVvaccinated MRC-5 cells(human fetal lung fibroblast).After 12 h,24h,36 h,48h,60 h,detected respectively,as follows:1.1 Through the following method to detected autophagy: The positive cells of acridine orange fluorescent particle were detected through flow cytometry.The expression of autophagy marker,LC3 was detected by western blot.1.2 The detection of the expression levels of the key molecule of m TOR signaling pathway: Using Western Blot to detect the expression of m TOR signaling pathway molecules p-p70S6K(phosphorylated p70S6K),p-4E-BP1(phosphorylated 4E-BP1).2.To investigate the effect of the host cellular autophagy on the virus replication,we will detect the number of HCMV DNA copy by fluorescence quantitative PCR.Result: The expression of Beclin1?LC3-II?LC3-I protein in MRC-5 cells infected with HCMV was increase and then gradually decreased.Meanwhile,the expression of4E-BP-1-P and p70S6K-P was decreased and then increased.Consistantly,the result shows that autophagy was promoted and then inhibited by IF.The number of HCMV DNA copy was increased and then decreased.Conclusion: The above results indicate that HCMV infection induces firstly,and then inhibits the autophagy in MRC-5 cells.And m TOR signaling pathway may be involved in it.