The methods of using engineered RNase P catalytic RNA (termed as M1GS RNA) for in vitro and in vivo in trans-cleavage of target viral mRNA are described in this chapter. Detailed information is focused on (1) mapping accessible regions of target viral mRNA in infected cells, (2) generation and in vitro cleavage assay of the customized M1GS ribozyme, (3) stable expression of M1GS RNAs and evaluation of its antiviral activity in cultured cells. Using these methods, we have constructed functional M1GS ribozyme that can cleave an overlapping region of the mRNAs coding for the human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin in vitro. Further study has demonstrated that, in cultured human cells expressing the functional M1GS ribozyme and infected with HCMV, more than 85% reduction in the expression of CSP and assemblin and a 4,000-fold reduction in viral growth were achieved. Our study provided the direct evidence that the customized M1GS ribozyme can be used as an effective gene-targeting agent for in trans-cleavage of viral genes and inhibition of viral growth in cultured cells.
"Moreover, M1 RNA is able to cleave a target RNA sequence efficiently if an additional small RNA is covalently linked to the 3′ end of M1 RNA. The new sequence-specific ribozyme is named M1GS ribozyme (Figure 1A) [7,8], and any RNA could in principle be targeted by a custom-designed M1GS for specific cleavage. When introduced into human cells, M1GS ribozyme can function independently from the endogenous human RNase P to cleave a targeting sequence that base pairs with the guide sequence . "
[Show abstract][Hide abstract] ABSTRACT: Background
Hepatitis C virus (HCV) is a human pathogen causing chronic liver disease in about 200 million people worldwide. However, HCV resistance to interferon treatment is one of the important clinical implications, suggesting the necessity to seek new therapies. It has already been shown that some forms of the catalytic RNA moiety from E. coli RNase P, M1 RNA, can be introduced into the cytoplasm of mammalian cells for the purpose of carrying out targeted cleavage of mRNA molecules. Our study is to use an engineering M1 RNA (i.e. M1GS) for inhibiting HCV replication and demonstrates the utility of this ribozyme for antiviral applications.
By analyzing the sequence and structure of the 5′ untranslated region of HCV RNA, a putative cleavage site (C67-G68) was selected for ribozyme designing. Based on the flanking sequence of this site, a targeting M1GS ribozyme (M1GS-HCV/C67) was constructed by linking a custom guide sequence (GS) to the 3′ termini of catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli through an 88 nt-long bridge sequence. In vitro cleavage assays confirmed that the engineered M1GS ribozyme cleaved the targeted RNA specifically. Moreover, ~85% reduction in the expression levels of HCV proteins and >1000-fold reduction in viral growth were observed in supernatant of cultured cells that transfected the functional ribozyme. In contrast, the HCV core expression and viral growth were not significantly affected by a “disabled” ribozyme (i.e. M1GS-HCV/C67*). Moreover, cholesterol-conjugated M1GS ribozyme (i.e. Chol-M1GS-HCV/C67) showed almost the same bioactivities with M1GS-HCV/C67, demonstrating the potential to improve in vivo pharmacokinetic properties of M1GS-based RNA therapeutics.
Our results provide direct evidence that the M1GS ribozyme can function as an antiviral agent and effectively inhibit gene expression and multiplication of HCV.
[Show abstract][Hide abstract] ABSTRACT: Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but the molecular mechanisms of their antiviral activity remain unclear. The review discusses the well-known RNases that possess established antiviral effects, including both intracellular RNases (RNase L, MCPIP1 protein, and eosinophil-associated RNases) and exogenous RNases (RNase A, BS-RNase, onconase, binase, and synthetic RNases). Attention is paid to two important, but not always obligatory, aspects of molecules of RNases that have antiviral properties, i.e., catalytic activity and ability to dimerize. The hypothetic scheme of virus elimination by exogenous RNases that reflects possible types of interaction of viruses and RNases with a cell is proposed. The evidence for RNases as classical components of immune defense and thus perspective agents for the development of new antiviral therapeutics is proposed.
[Show abstract][Hide abstract] ABSTRACT: Многие рибонуклеазы (РНКазы) способны подавлять репродукцию РНК-содержащих вирусов в культурах инфицированных клеток и лабораторных животных, однако молекулярные механизмы противовирусного действия РНКаз изучены недостаточно. В представленном обзоре рассмотрены наиболее изученные внутриклеточные РНКазы, обладающие противовирусной активностью (РНКаза L, белок MCPIP1, РНКазы эозинофилов), а также РНКазы, применяемые экзогенно (РНКаза А, BS-РНКаза, онконаза, биназа, синтетические РНКазы). Особое внимание уделено двум важным, хотя и не всегда обязательным свойствам этих РНКаз: каталитической активности и способности к димеризации. Предложена схема элиминации вируса экзогенными РНКазами, отражающая возможные типы взаимодействия РНКаз с вирусом и клеткой. Приведены доказательства того, что РНКазы, как классические компоненты иммунной защиты, представляют значительный интерес для разработки новых противовирусных препаратов.
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