Search

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. We have previously used an in vitro selection procedure to generate EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the mRNA encoding the protease of human cytomegalovirus (HCMV), which is essential for viral capsid formation and replication. The EGS variant was about 35-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 95% in the expression of the protease and a reduction of 4,000-fold in viral growth were observed in HCMV-infected cells that expressed the EGS variant, whereas a reduction of 80% in the protease expression and an inhibition of 150-fold in viral growth were detected in cells that expressed the EGS derived from a natural tRNA sequence. No significant reduction in viral protease expression or viral growth was observed in cells that either did not express an EGS or produced a "disabled" EGS, which carried nucleotide mutations that precluded RNase P recognition. Our results provide direct evidence that engineered EGS variant is highly effective in blocking HCMV expression and growth by targeting the viral protease. Furthermore, these results demonstrate the utility of engineered EGS RNAs in gene targeting applications, including the inhibition of HCMV infection by blocking the expression of virus-encoded essential proteins.

External guide sequences (EGSs), which are RNA molecules derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) essential transcription regulatory factors IE1 and IE2. The EGS variant was approximately 25-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 93% in IE1/IE2 gene expression and a reduction of 3000-fold in viral growth were observed in HCMV-infected cells that expressed the variant, while cells expressing the tRNA-derived EGS exhibited a reduction of 80% in IE1/IE2 expression and an inhibition of 150-fold in viral growth. Our results provide the first direct evidence that EGS variant is highly effective in blocking HCMV gene expression and growth and furthermore, demonstrate the feasibility of developing effective EGS RNA variants for anti-HCMV applications by using in vitro selection procedures.

RNase P complexed with external guide sequence (EGS) represents a novel nucleic-acid-based gene interference approach to modulate gene expression. In this study, a functional EGS RNA was constructed to target the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin. The EGS RNA was shown to be able to direct human RNase P to cleave the target mRNA sequence efficiently in vitro. A reduction of approximately 75%-80% in the mRNA and protein expression levels of both CSP and assemblin and a reduction of 800-fold in viral growth were observed in human cells that expressed the functional EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS that carried nucleotide mutations that precluded RNase P recognition. The action of the EGS is specific as the RNase P-mediated cleavage only reduces the expression of the CSP and assemblin but not other viral genes examined. Further studies of the antiviral effects of the EGS indicate that the expression of the functional EGS has no effect on HCMV genome replication but blocks viral capsid maturation, consistent with the notion that CSP and assemblin play essential roles in HCMV capsid formation. Our study provides the first direct evidence that EGS RNAs effectively inhibit HCMV gene expression and growth. Moreover, these results demonstrate the utility of EGS RNAs in gene therapy applications, including the treatment of HCMV infection by inhibiting the expression of virus-encoded essential proteins.

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.

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.