Engineered RNase P Ribozymes Effectively Inhibit Human Cytomegalovirus Gene Expression and Replication

Abstract

RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.

RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

“…Active ribozymes V718-A and M1-A were constructed from V718 and M1 RNA, respectively. Control ribozymes M1-C and V718-C, which were generated from an inactive ribozyme mutant C102 in a similar way, were expected to be inactive due to mutations in the catalytic domain [ 27 , 33 ]. These four ribozymes all contained the same guide sequence complementary to the target AP mRNA sequence.…”

“…M1GS-expressing cells were either mock-treated or infected with HCMV at a multiplicity of infection (MOI) of 1. Total RNA and protein samples were prepared at different time points post-infection and studied by Northern and Western blot analyses [ 31 , 32 , 33 ]. To detect IE2 mRNA and protein expression, samples were harvested at 8 and 24 h post-infection, respectively.…”

“…To detect IE2 mRNA and protein expression, samples were harvested at 8 and 24 h post-infection, respectively. To detect the expression of US2 and AP/PR mRNAs, samples were harvested at 48 h post-infection [ 31 , 32 , 33 ]. To detect the protein expression of UL44, UL83, AP, and PR, samples were harvested at 72 h post-infection.…”

“…In control experiments, to detection the 5kb RNA expression, samples were harvested at 8 and 48 h post-infection. To detection the actin protein expression, samples were harvested at 24 and 72 h post-infection [ 31 , 32 , 33 ]. For Northern blot analyses, the prepared RNA samples (30 μg) were separated in denaturing agarose gels (1%) that contained formaldehyde.…”

Genome Sequence of the Multiple-Protease-Producing Strain Geobacillus thermoleovorans N7 , a Thermophilic Bacterium Isolated from Paniphala Hot Spring, West Bengal, India

“…Apart from that, several genes of medically useful enzymes have been found, namely, l -asparaginase, glutaminase, and RNase P. L -asparaginase and glutaminase have anticancer activity and are used to treat acute lymphoblastic leukemia (7). As reported, an engineered RNase P ribozyme variant has been used as a potential antiviral agent in reducing human cytomegalovirus gene expression and growth (8). Xenobiotic-degrading genes encoding for enzymes like catechol-2,3-dioxygenase (EC 1.13.11.2) and nitrilotriacetate monooxygenase (EC 1.14.14.10), which are responsible for the degradation of catechol and nitrilotriacetate, have been found.…”