Abstract
BackgroundHepatitis 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.ResultsBy 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.ConclusionOur results provide direct evidence that the M1GS ribozyme can function as an antiviral agent and effectively inhibit gene expression and multiplication of HCV.
Highlights
Hepatitis C virus (HCV) is a human pathogen causing chronic liver disease in about 200 million people worldwide
Construction of M1GS ribozymes The 5′ untranslated region (UTR) of HCV genomic RNA consists of 341 nucleotides and folds to six secondary structure domains termed SLI-VI
DNAMAN (v. 6) and RNA structure (v. 4.5) softwares were used to analyze the sequence and secondary structure of HCV 5′ UTR to search for sequences with recognition and cleavage features of M1GS ribozyme as characterized previously, including requirement for a guanosine and a pyrimidine to be the 3′ and 5′ nucleotides adjacent to the site of cleavage, respectively [24]
Summary
Hepatitis C virus (HCV) is a human pathogen causing chronic liver disease in about 200 million people worldwide. 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. 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 [9]. A number of studies have shown that M1GS RNA and RNase P are effective to cleave both viral and cellular mRNAs and block their expression in cultured cells, including inhibition of gene expression of human influenza, herpes viruses, human cytomegalovirus and human immunodeficiency virus [10,11,12,13,14,15]. M1GS-based strategy is a useful method in basic research (e.g. regulation of gene expression) and represents a distinctive therapeutic approach of nucleic-acid-based, sequence-specific interference [16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
