The Role Of Human Cytomegalovirus Tegument Protein PUL23 In Antiviral Innate Immune & Inhibition Of CMV Infection By Engineered RNase P Ribozymes And EGS

Human cytomegalovirus belongs to ?-herpesvirus subfamily,is an extremely widespread human pathogen.HCMV infects hosts for life and cannot be cleared by immunological processes or antiviral drugs.HCMV causes medically significant disease in immunologically compromised individuals such as AIDS patients,organ transplant recipients and fetus.pUL23 is a viral tegument protein with 33 KD molecular weight.Dunn et al found UL23 deletion mutant exhibited enhanced growth in HFF when they identify the essential role of each 162 ORFs in HCMV genome.However there is nofurther report about pUL23 function.The intracellular innate antiviral response in human cells is an essential component of immunity against virus infection.Viral components are recognized by specific host Pathogen Recognition Receptors(PRRs)which trigger the intracellular signaling cascades and expression of IFN-I and inflammatory cytokine.In this study,we find that IFN?/? expression induced by virus or dsDNA can be up-regulated by pUL23.During HCMV infection,the phosphorylation of cellcular IRF-3 is up-regulated by pUL23,and so as to the nucleus accumulation of IRF-3.TLR-9 have a role in pUL23 function,because inhibition of TLR-9 by shRNA result in a decrease in pUL23 mediaed IFN-I up-regulation.Conversely,viral IE1 expression is down-regulated by pUL23 and virus replication is suppressed in HFF-UL23-Flag cells compares to those in HFF-EGFP cells.As the result,UL23 severs as a positive regulator in intracellular innate antiviral response,and a negative regulator in viral replication.It may assist HCMV long-term persistence by avoiding over-damage of cells induced by the virus.Traditional medicines play a significant role in the treatment of HCMV-infected diseases,but at the same time there are obvious problem such as high toxicity and easy to cause virus resistance.Numerous studies have shown that the engineered ribozyme P(M1GS)and the ribozyme P-based external guide sequence(EGS)can effectively inhibit viral replication by blocking the expression of key genes of the virus.In this study,we used HCMV and MCMV as models respectively to study the application of M1 GS and EGS in the anti-CMV virus field.In the study of M1 GS inhibiting HCMV infection,we employed an engineered variant to target a shared mRNA region of human cytomegalovirus(HCMV)immediate early proteins 1(IE1)and 2(IE2),which are essential for the expression of viral early and late genes as well as viral growth.The functional variant F-R228-IE cleaved the target mRNA sequence in vitro at least 100 times more efficiently than F-M1-IE,the ribozyme derived from the wild type RNase P catalytic RNA sequence.In cultured cells,expression of F-R228-IE resulted in IE1/IE2 expression reduction by 98-99% and in HCMV production reduction by 50,000 folds.In contrast,expression of F-M1-IE resulted in IE1/IE2 expression reduction by less than 80% and in viral production reduction by 200 folds.Studies of the ribozyme-mediated antiviral effects in cultured cells suggest that overall viral early and late gene expression and viral growth were inhibited due to the ribozyme-mediated reduction of HCMV IE1 and IE2 expression.Our results provide direct evidence that engineered RNase P ribozymes,such as F-R228-IE,can serve as a novel class of inhibitors for the treatment and prevention of HCMV infection.Using murine cytomegalovirus(MCMV)as a model system,we examined the antiviral effects of an EGS variant,which was engineered using in vitro selection procedures.EGSs were used to target the shared mRNA region of MCMV essential capsid scaffolding protein(mCSP)and assemblin.In vitro,the EGS variant was 60 times more active in directing RNase P cleavage of the target mRNA than the EGS originating from a natural tRNA.In MCMV-infected cells,the variant reduced mCSP expression by 92% and inhibited viral growth by 8,000-fold.In MCMV-infected mice hydrodynamically transfected with EGS-expressing constructs,the EGS variant was more effective in reducing mCSP expression,decreasing viral production,and enhancing animal survival than the EGS originating from a natural tRNA.These results providedirect evidence that engineered EGS variants with higher targeting activity in vitro are also more effective in reducing gene expression in animals.Furthermore,our findings imply the possibility of engineering potent EGS variants for therapy of viral infections.