RNase P ribozymes for the studies and treatment of human cytomegalovirus infections.
Ribozymes are promising gene-targeting agents for regulation of gene expression. In our recent studies, RnaseP (M1GS) ribozymes were constructed to target the overlapping region (IE mRNA) of IE1 and IE2 mRNAs of human cytomegalovirus (HCMV) and the mRNA (TK mRNA) coding for thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Our results indicate that RNase P ribozymes efficiently cleaved the IE mRNA and TK mRNA sequences in vitro. Significant inhibitions (approximately 75-85%) of HCMV IE1/IE2 and HSV-1 TK expression were observed in cells that expressed these ribozymes while a reduction of less than 10% was found in cells that did not express the ribozymes or expressed a disabled one that contained mutations abolishing catalytic activity. Ribozyme variants, which cleaved a TK mRNA sequence in vitro more efficiently than the ribozyme derived from the wildtype RNase P sequence, were selected by an in vitro selection system. When the selected ribozymes were expressed in cultured cells, they were more effective in inhibiting viral IE1/IE2 and TK expression and viral growth than the wildtype ribozyme sequence. Our results provide the first direct evidence that RNase P ribozymes are highly effective in inhibiting HCMV gene expression and growth. Moreover, a selection system was developed for generating novel ribozyme variants that cleave a mRNA substrate efficiently in vitro. These results suggest that M1GS ribozyme-mediated inhibition of expression of viral genes can be used as a new approach for the studies of HCMV gene function and the treatment of HCMV infection.
D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000188:drug therapy*; D004722:Endoribonucleases; D015967:Gene Expression Regulation, Viral; D018390:Gene Targeting; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D043262:Ribonuclease P; D013937:Thymidine Kinase / Q000378:metabolism; D015534:Trans-Activators; D014764:Viral Proteins; D014779:Virus Replication
D000998:Antiviral Agents; C088645:IE1 protein, cytomegalovirus; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D015534:Trans-Activators; D014764:Viral Proteins; D013937:Thymidine Kinase; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P