Study On The Interactions Of Human Cytomegalovirus Proteins UL80.5 And UL84 With Host Celluar Factors And The Inhibition Of Herpes Simplex Virus 1 Gene Expression And Replication By EGS

Human cytomegalovirus (HCMV) and Herpes simplex virus (HSV-1) are members of herpesvirus family, and both have a high infection rate in population. The HCMV antiviral drugs mainly target the DNA polymerase UL54 and easy to develop drug resistance. In order to find new antiviral strategy, the interactions between viral proteins and cellular factors during the processes of capsid assembly and immune escape have been studied. HSV-1 normally persist a subclinical latent infection, immediate early gene ICP4 is the key to regulate virus gene expression and cause clinical symptoms. We studied EGSs that targeted ICP4 gene mRNA, and tested its effect as a new antiviral strategy on inhibiting HSV-1 gene expression and poliferation.HCMV assembly protein precursor UL80.5 plays a key role in capsid formation. It intracts with several viral capsid proteins, initiates and modulates the whole process. In this study we found that SUMOylation E2 ligase UBC9 interacts with UL80.5. The SUMOylation of UL80.5 further has been confirmed, and the SUMOylaition site is lysine 371 residue. The SUMOylation of UL80.5 inhibited its interaction with major capsid protein UL86 in competitive Co-IP assay. SUMOylation-deficient UL80.5 slightly inhibited viral growth but was not essential. These result showed that HCMV may use SUMOylation system of host cells to enhance viral growth, but this enhancement is not very important.HCMV UL84 protein is essential for the initiation of lytic replication by altering the conformation of an RNA stem loop structure within oriLyt. Other than the function of initiating DNA replication, the structure analysis of UL84 opens up possibilities such as the regulation of gene expression and RNA transport. In this study, we found that UL84 interacted with FHL2 in Y2H and Co-IP assays, and coexisted with it in the nucleus. The interaction domain (400-586aa) of UL84 with FHL2 contains the walker B motif of DExD/H-box family. UL84 inhibited the promoter of IFN?,and FHL2 enhanced the promoter of IFN? in Dual-Luciferase Assay, which showed antagonistic influences on IFN? expression.In this study, we constructed several EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. External guide sequences (EGS) are RNA sequence which can target specific mRNA to form a tertiary structure similar to pre-tRNA and recruit intracellular ribonuclease P (RNase P) to degrade that mRNA. The EGS variant C468-A generated from selection procedure induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS SER-A generated from a natural pre-tRNA. In HSV-1 infected and expressing the C468-A or SER-A EGS cells, a decrease of around 97% and 75% in the level of ICP4 gene expression and an inhibition of around 7,000-and 500-fold in viral growth were observed respectively.