Dissecting The Regulatory Mechanisms Of Human Cytomegalovirus Replication And Nucleic Acid Metabolism

Human cytomegalovirus(HCMV)is a slow-growing beta-herpesvirus.Its genetic material is double-stranded DNA.HCMV infection is generally asymptomatic in healthy individuals.However,it can lead to severe infectious complications and even death in immunocompromised individuals.HCMV is also a seriously infectious pathogen causing congenital diseases in newborns.HCMV has a very large genome and owns strict species specificity,which makes it very difficult to exploring HCMV pathogenic mechanism.Until now,there is no licensed vaccine for HCMV.Our peoject focus on two aspects: Dissecting the relationship between virus and its hosts,as well as exploring functions of HCMV viral genes.1.Explore the relationship between DDX21 and HCMV replication.DDX21 is a kind of RNA helicase,it can regulates RNA metabolism and the growth of some RNA viruses.We aimed to investigate the relationship between DDX21 and HCMV replication.Firstly,we found that the protein and mRNA levels of DDX21 gradually decreased in mock infected cells,but HCMV infection could prevent their reduction.And the results of viral growth analysis and RT-qPCR showed that DDX21 knockdown significantly inhibited HCMV growth and viral late gene transcription and expression.It has been reported that DDX21 knockout results in the accumulation of R-loops,which could restrain RNA polymerase II(Pol II)elongation and inhibit the transcription of certain genes.Our results of DNA-RNA immunoprecipitation found that DDX21 knockdown promoted the accumulation of R-loops on the viral late gene.And we observed virus growth and viral late gene transcription could be partially rescued while co-expression with RNase H.Taken all together,these results revealed that DDX21 knockdown could result in the accumulation of R-loops on the viral late gene,which inhibited its transcription and subsequently suppressed HCMV replication.2.Investigate the regulatory mechanism of HCMV UL24 and UL43 on HCMV replication.The US22 family of human cytomegalovirus has 12 members.They encode viral tegument proteins that are very important components of the virions.UL24 and UL43 are two members of this family.And the viral proteins they encode,pUL24 and pUL43,interact with each other.By mass spectrometry,we found interacting proteins respectively with pUL24 and pUL43: Dicer and TRBP.We verified the results that pUL24 and pUL43 could interact with TRBP and Dicer by immunoprecipitation and also found existence of endogenous interactions.Due to Dicer and TRBP regulate the biogenesis of miRNAs,next,we detected whether knockout of UL24 and UL43 affect the expression of viral miRNAs.We conducted RNA-seq and found that knockout of these two genes reduced the expression of miR-UL59.Expect for the experimental virus,we also found that knockout of these two genes on the clinical virus down-regulated the expression of miR-UL59.These results indicated that knockout of UL24 and UL43 could reduce the expression of miR-UL59.It has been reported that the target gene of miR-UL59 is ULBP1,which is one of ligands of NKG2 D.NKG2D is a kind of receptor expressed on a variety of immune cells and can recognize different MHC I related ligands,including MIC and ULBP proteins.Infection or stress responses can induce the expression of NKG2 D ligands,resulting in the activation of effector cells and finally killing ligand-related target cells.Therefore,we speculate whether UL24 and UL43 affect the expression of ULBP1 by regulating miR-UL59,which prevent recognition of infected cells by NK cells and thus facilitate viral immune evasion.Our results also showed that the expression of miR-UL59 decreased and the mRNA levels of ULBP1 increased after knockout of these two genes.And next we will examine whether infection with double deletion virus could affect NK cells mediated cytotoxicity.