Inhibition of hepatitis C virus by an M1GS ribozyme derived from the catalytic RNA subunit of Escherichia coli RNase P

Virology Journal

Gang Li³  Jianguo Wu⁴  Wenjun Zhang⁴  Chengcheng Zhang¹  Zhiwen Huang¹  Xinjun Mao²  Xifang Li¹  Xinliang Mao³ 

[1] Department of Microbiology and Immunology, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, Guangzhou 510006, PR China;Department of Anaesthesia, General Intensive Care and Pain Management, Medical University of Vienna, Vienna A-1090, Austria;Vaccine Institute, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, PR China;State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China

关键词: Antiviral;    5′ UTR;    Hepatitis C virus;    RNase P;    Ribozyme;   

Others  :  804518
DOI  :  10.1186/1743-422X-11-86

 received in 2014-01-29, accepted in 2014-04-30,  发布年份 2014

【 摘 要 】

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.

Results

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.

Conclusion

Our results provide direct evidence that the M1GS ribozyme can function as an antiviral agent and effectively inhibit gene expression and multiplication of HCV.

【 授权许可】

   
2014 Mao et al.; licensee BioMed Central Ltd.

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