The Influence Of Autophagy Level To HCMV Latent Infections

Objectives:Human cytomegalovirus(HCMV)belongs to the herpes virus ? subfamily.about 30%-90%of the people has HCMV serum antibody in the developed countries.HCMV infection is usually latent which does not have clinical symptoms.But in the low immune population,such as AIDS patients,patients with advanced tumors,etc.HCMV infection can result in serious infections,and even death.Congenital HCMV infection often lead to severe clinical consequences,such as fetal multiple malformations and Late-onset central nervous system damage.Autophagy is a kind of degradation pathways that involves the digestion of aging damaged proteins and organelles via the lysosomal pathway.Autophagy plays an important role in maintaining cell homeostasis,resistance to infection of pathogenic microorganism and parasite.Domestic and foreign research about the relation between HCMV and autophagy is mostly based on proliferation HCMV infection,but the relation about the more common HCMV latent infection and autophagy is no related reports.So study the influence of autophagy level to HCMV latent infections may provide a new way to eliminate HCMV infection and improve the population quality.Methods:This study includes three parts:1.Establishment of HCMV latent infection model:human acute mononuclear leukemia cells(human acute monocytic leukaemia cell line,THP-1)infected with HCMV Towne strain with MOI=5,Extract the total protein and the total RNA of cell respectively at 1 d,2 d,3 d,5 d,7 d,9 d.Using RT-PCR to detect HCMV UL122 and UL138 gene expression.Using western blot to detect the protein levels of UL122 protein(IE)and UL138 protein.Through the above,we can find out the time HCMV become into latency after infected THP-1.At last,we can build stable HCMV latent infection model.2.The influence of autophagy level to HCMV latency:THP-1 infected with HCMV Towne strain with MOI=5,then was allocated to divide into three groups.Autophagy inducer rapamycin and autophagy inhibitor 3-MA were used to THP-1 cells at the same time.THP-1 infected with HCMV as control group,THP-1 infected with HCMV+ rapamycin as experimental group 1,THP-1 infected with HCMV+3-MA as experimental group 2.We extract the total RNA and protein at 2 d,7 d post infection.PCR was used to detect HCMV DNA copy number.RT-PCR method was used to detect the expression levels of UL122 gene,UL138 gene and Beclinl gene.Western blot was used to detect protein levels of UL122 protein(IE),UL138 protein,Beclinl protein and LC3 protein in these three groups.Repeat the above operation,THP-1 infected with HCMV Towne strain with MOI=5,then was allocated to divide into three groups.Rapamycin and 3-MA were used to THP-1 cells at 5 d post infection.THP-1 infectedwith HCMV as control group,THP-1 infected with HCMV+ rapamycin as experimental group 1,THP-1 infected with HCMV+3-MA as experimental group 2.We extract the total protein at 7 d,9 d post infection.Western blot was used to detect protein levels of IE,UL138 protein and Beclinl protein in these three groups.Results:1.DNA electrophoresis results showed that we can detect UL138 gene all the time,but UL122 genes can be detect at 3 d and cannot be detected at 5 d.Western blot results showed that we can detect UL138 protein all the time,but IE can be detected at 3 day and cannot be detected at 5 day.Showed that five days after infected with THP-1,HCMV enter latent infection status.We successfully establish latent infection model in vitro.2.The influence of autophagy level to HCMV latency(1)Influence of early HCMV latent infections?mRNA test results:Rapamycin improved the mRNA level which encoding Beclinl proteins in THP-1 cell,3-MA inhibit the mRNA level which encoding Beclinl proteins.After Changing the host cell autophagy level,UL122 and UL138 expression level in experimental groups were no difference with the control group.?western blot results:Rapamycin increased Beclinl proteins content in THP-1 cell,3-MA reduce the Beclin1 proteins content.After Changing the host cell autophagy level,IE and UL138 proteins in experimental groups were no difference with the control group.?HCMV gene copy number:The change trend of HCMV gene copy number is the same in three groups of THP-1 cell,HCMV gene copy number first increased then decreased.Results show that the change of THP-1 cell autophagy level has no effect on establishment of HCMV latency.(2)The influence of the late of HCMV latent infections? Western blot results show that rapamycin increased Beclin1 proteins content,3-MA reduce the Beclinl proteins content.After Changing the host cell autophagy level,we can detect UL138 proteins in all groups at two time points,but IE was not detected.After building stable HCMV latent infection model in THP-1 cells,Changing THP-1 cell autophagy level not affect the state of HCMV latency.Conclusion:1.Five days after infected with THP-1 with MOI=5,HCMV enter latent infection status.HCMV latent infection model is successfully establish in vitro.2.After building stable HCMV latent infection model in THP-1 cells,Changing THP-1 cell autophagy level not affect the early and late of HCMV latency.