Engineering of highly active gene targeting RNase P ribozyme against human cytomegalovirus infection

Sequence-specific ribonuclease (RNase) P ribozymes can be engineered in vitro and are promising gene-targeting agents to knock down gene expression. In this study, we applied an RNase P ribozyme variant to hydrolyze the mRNA of human cytomegalovirus (HCMV) major capsid protein (MCP), which is necessary for viral capsid formation and growth. Functional variant R668-F was about 100 times more efficient in slicing the MCP mRNA in vitro than M1-F, the ribozyme with a natural RNase P ribozyme sequence. In R668-F-expressing cells, a decrease of about 98%–99% in the expression of MCP was detected, and the virus production was reduced by 70,000 folds. However, the expression of inactive control ribozymes in cells resulted in a less than 10% decrease in MCP expression and no apparent decrease in HCMV growth. In cells observed with the ribozyme-mediated reduction of MCP expression, HCMV capsid formation and virus growth were inhibited and the expressions of other viral genes were unaffected. These findings provide the first direct evidence that ribozyme R668-F specifically inhibits MCP expression and blocks HCMV growth. Our results further suggest that the engineered RNase P ribozymes, including R668-F, may act as a novel general gene-targeting strategy to treat infections of viruses, including HCMV. • An active sequence-specific ribozyme is engineered from ribonuclease (RNase) P catalytic RNA with novel mutations enhancing its activity. • The engineered RNase P ribozyme represents a gene-targeting agent by cleaving a target mRNA and reducing its expression. • The constructed ribozyme inhibits the major capsid protein expression of human cytomegalovirus and viral growth in cells.