The Influence Of Down-regulated Autophagy-related Beclin1 On HCMV Latent Infection

Objectives:Human cytomegalovirus(HCMV),a ubiquitous virus,belongs to the herpes virus ?subfamily.It infects as high as 90%of adults around the world.In our country,more than 95%of pregnant women have infected with HCMV.Although immunocompetent individuals infected with HCMV does not have clinical symptoms,but after infection,HCMV can establish latent infection in the body.When the host's immune function becomes to be limited,it can lead to severe clinical consequences,such as hepatitis,encephalitis,and even death.In congenital HCMV infection,about 10%-15%of the infected newborns have clinical symptoms and are characterized by jaundice hepatitis,liver and spleen enlargement,pneumonia and other serious clinical consequences.However,among the asymptomatic infected children,10%to 17%will end up with deafness or neurodevelopmental abnormalities.Autophagy is a kind of degradation pathways by the use of the lysosome,and aging organelles or misfolded proteins are degraded into small molecular amino acid which is used to provide the energy for the host cell.So,autophagy not only can resist pathogens invasion,but also provides energy for cells for maintaining the stability of internal environment.In recent years,more and more studies about the relationship between HCMV and autophagy have been reported.But these studies were conducted on HCMV proliferation infection model,recently rare studies about the relationship between autophagy level change and HCMV latent infection have been reported.Whether or not the autophagy level change influences HCMV latency is needed to be determined.This may help understand the mechanisms of HCMV latent infection and provide new targets for the clinical treatment of HCMV.Methods:This study includes two parts:(1)establishment of HCMV latent model:human acute mononuclear leukemia cells(human acute monocytic leukaemia cell line,THP-1)infected with HCMV Towne strain were used as the experimental group,the uninfected THP-1 cells as control group,after 1 d,3 d,5 d,7 d,9 d post infection,removed the nutrient solution and collected the cells.Then DNA was extracted for detection of HCMV DNA copies with use of fluorescence quantitative polymerase chain reaction(PCR);RNA was extracted through trizol method and then detected HCMV UL122,UL123 and UL138 gene expression levels by using reverse transcription polymerase chain reaction(RT-PCR)method;Total proteins were obtained through lysis of cells,the protein levels of HCMV IE and UL138 were detected with western blot.At last,the establishment of HCMV latency was confirmed.(2)The influence of down-regulated autophagy-related Beclinl on HCMV latency:Beclinl RNAi was built,the effective carrier was screened and concentration was also confirmed.The experiment were classified into three groups:THP-1 infected with HCMV Towne strain as control group,THP-1 infected with HCMV Towne strains +blank carrier as the blank control group,THP-1 infected with HCMV Towne strains +Beclinl RNAi carrier as experimental group.The number of autophagosomes or autolysosomes were observed and calculated by transmission electron microscope(TEM);RT-PCR method was used for detecting the mRNA levels of Beclinl,Atg5,Atg7,LC3,UL122,UL123,UL138;Western blot was used for detecting protein levels of Beclinl,Atg5,Atg7,LC3,IE,UL138 in these three groups.The autophagy levels were confirmed and whether or not HCMV latency can be reactivated were also determined when the level of Beclinl was down-regulated.Results:HCMV latent model was successfully established.The result of fluorescence quantitative PCR showed that at 1 day,3 days,5 days post infection(pi),HCMV DNA copies in THP-1 cells infected with HCMV Towne strain increased gradually,at 5 days,7 days,9 days pi,DNA copies were reduced gradually;At 1 day and 3 days pi,the mRNA level of UL122 gene were expressed,after 5 days pi,the mRNA level was reduced and could not be detected.And the mRNA expression level of UL123 gene were too low to be detected;At early infection,the mRNA expression level of HCMV latency-related UL138 gene was also not detected.Then the expression level increased as times went by.The results of western blot showed that IE protein were detected at 1 d,3 d,not at 5d,7d pi.But there has always been UL138 protein was expressed.HCMV latency could not be reactivated.The result through TEM method showed that the number of normal group of autophagosomes or autolysosomes in five randomly selected cells among these four groups were 3 for normal THP-1 cells,10 for control group,9 for the blank control group,6 for the experimental group.So,compared with normal THP-1 cells,autophagosomes or autolysosomes were increased.But compared with the blank control group and the control group,the autophagosomes or autolysosomes were reduced.Compared with the blank control and the control group,the transcription and protein expression levels of autophagy-related genes Beclinl,Atg5,Atg7 and LC3 in experimental group significantly decreased;But no significant difference among these three groups in the transcription and protein expression levels of replication-associated gene UL122 and the latency-related gene UL138.The result of fluorescence quantitative PCR showed that HCMV DNA copies in experimental group were almost the same at early infection,but when the latency was established,the DNA copies decreased abruptly when compared with the blank control group and the control group.What is more,the HCMV DNA copies still could be detected after the establishment of latency.Conclusion:(1)Successful establishment of HCMV latency in THP-1 cells.(2)Down-regulation of Beclinl may not affect the state of HCMV latency.