Inhibition Of Human Cytomegalovirus Maturation By Activation Of Liver X Receptor

Objective: Human cytomegalovirus(HCMV),a member of the herpesvirus family,is a large complex enveloped virus which widespread in the population.The protein expression of the virus itself,the replication of the genome,the assembly and maturation of the virus particles are completely dependent on host cell mechanism.Liver X receptor(LXR)is a widely existed transcription factor in cells,which plays an important role in cell growth,development,differentiation and metabolism.LXR is activated by the ligand,which regulates the transcription of target genes through two different mechanisms: transcriptional activation and transcriptional inhibition.Always on the research of the virus replication mechanism,mainly focused on the virus biological function of its coding protein,and influence on viral replication,the host immune system and the lack of effect on virus infection of the host cell metabolism and virus infection of the host cell metabolism product changes on the impact of viral replication and a series of key problems of research.Whether activated LXR regulates viral gene transcription closely related to virus particle assembly and maturation through transcriptional regulation mechanism and inhibits the maturation process of virus particles,reduces the maturation number of virus particles and inhibits the infection ability of virus particles has not been clearly confirmed.To understand the inhibition mechanism of activated liver X receptor on maturation and infectivity of virus particles,we plan to study the effects of liver X receptor activated by its agonist,GW3965,on maturation and infectivity of virus particles,identify the viral target genes that are regulated by the activated liver X receptor,discover the inhibition mechanism of the activated liver X receptor on transcriptions of the virus target genes,and demonstrate the inhibition effects of virus target genes on virus titers.Finally,discover the inhibition mechanism of the activated liver X receptor on maturation and infectivity of virus particles.Methods: 1.HFF cells were cultured by serum-free starvation method for 24 h and then treated with LXR agonist,while DMSO was added in the control group.HCMV experimental strain Towne Bac was infected 1h later,fresh medium was replaced,and the concentration of LXR agonist was maintained in the experimental group.The cells were crushed by ultrasound at different time points(2,24,48,72,96,120hpi)after infection,and the progeny virus particles were harvested.The viral infection activity was titrated by TCID50 method,and the DNA content was detected by real-time fluorescence quantitative PCR.2.Cells were collected at different time points(24,48,72hpi)after infection,and total RNA was extracted.HCMV gene RNA content was detected by real-time fluorescence quantitative PCR.3.72 h post infection(72hpi),the total protein was extracted,and the expression levels of important virus-encoded proteins and housekeep protein GAPDH were detected by western blot.At the same time,the cells and all the culture medium were collected,the purified virus particles were obtained by ultracentrifugation,and the expression level of virus encoded protein in the progeny virus particles was detected by western blotting.4.Multiple fields were selected to count the number of three kinds of capsids in the nucleus and calculate the proportion.There was no significant difference in the number and percentage of A,B,and C capsids between LXR activated and inactived HCMV infected HFF cells.VAC regions of multiple infected cells were randomly selected to count the number of each virus particle.A high proportion of abnormal enveloped virus particles were present in LXR-activated infected HFF.5.The decrease of LXR? content in cells at different time points after infection can mediate the inhibition of ABCA1 expression in cholesterol efflux.LXR activation significantly reduced total cholesterol in infected cells.The cholesterol content of the progeny virus from LXR activated cells decreased significantly.Results: 1.After 72 hpi,activated LXR significantly reduced the viral titer of HCMV progeny by more than 1000 times,and activated LXR only reduced the genome DNA copy number of HCMV progeny virus by less than 10 times.2.At 72 hpi,the transcription level of 9 viral genes was not affected by activated LXR;Activated LXR significantly inhibited the transcription level of at least 20 viral genes.3.At 72 h after infection,activated LXR significantly reduced the protein expression of some viral genes,such as IE86(UL122),pp65(UL83),g B(UL55)and pp28(UL99),but had little effect on the protein expression of pp52(UL44).The expression of proteins encoded by viral genes in the virus particles was consistent with the results of whole cell lysate.4.Multiple scopes were selected to count the number of three kinds of capsids in the nucleus and calculate the proportion.There was no significant difference in the number and percentage of A,B,and C capsids between LXR activated and inactived HCMV infected HFF cells.VAC regions of multiple infected cells were randomly selected to count the number of each virus particle.A high proportion of abnormal enveloped virus particles were present in LXR-activated infected HFF.5.The decrease of LXR? content in cells at different time points after infection can mediate the inhibition of ABCA1 expression in cholesterol efflux.LXR activation significantly reduced total cholesterol in infected cells.The cholesterol content of the progeny virus from LXR activated cells decreased significantly.Conclusion: 1.Activated LXR reduces the number of infective progeny viruses with intact structure and reduces the lipid content in mature virus particles to reduce the infective ability of progeny viruses by affecting the maturity and release process of viruses.2.Inhibition of HCMV titer by LXR activation is a multi-layered process including viral gene transcription,genomic DNA replication,viral particle maturation release and progeny virus structure,etc.,which shows a significant inhibitory effect of LXR activation on HCMV titer.3.Activated LXR plays an obvious inhibitory role in the assembly and maturation of HCMV,which is applied in the development of antiviral drugs and has a certain guiding role in the treatment of HCMV-related diseases.