• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.127-140
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• 2019

Since 1990, many nucleic acid expression platforms consisting of DNA or RNA have been developed. However, although RNA expression platforms have been relatively neglected, several such platforms capped at the 5' end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites (IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platforms constructed with IRESs from the 5' untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic region of the cricket paralysis virus (CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5' end of the coxsackievirus B3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms (termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platform showed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platforms prepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.

• Journal of Microbiology
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• 제42권4호
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• pp.361-364
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• 2004

Internal ribosome entry site (IRES) of the hepatitis C virus (HCV) is known to be essential for HCV replication and most conserved among HCV variants. Hence, IRES RNA is a good therapeutic target for RNA-based inhibitors, such as ribozymes. We previously proposed a new anti-HCV modulation strategy based on trans-splicing ribozymes, which can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. To explore this procedure, sites which are accessible to ribozymes in HCV IRES were previously determined by employing an RNA mapping method in vitro. In this study, we evaluate the intracellular accessibility of the ribozymes by comparing the trans-splicing activ­ities in cells of several ribozymes targeting different sites of the HCV IRES RNA. We assessed the intra­cellular activities of the ribozymes by monitoring their target-specific induction degree of both reporter gene activity and cytotoxin expression. The ribozyme capable of targeting the most accessible site iden­tified by the mapping studies then harbored the most active trans-splicing activity in cells. These results suggest that the target sites predicted to be accessible are truly the most accessible in the cells, and thus, could be applied to the development of various RNA-based anti-HCV therapies.

• BMB Reports
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• 제36권6호
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• pp.538-544
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• 2003

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.

• 생명과학회지
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• 제7권4호
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• pp.392-401
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• 1997

폴리오바이러스는 바이러스들 중에서도 특히 커기가 작은 바이러스로서 피막(coat)을 둘러싸는 막(envelop) 이 없다. 폴리오바이러스는 (+) 가닥의 단일 RNA 게놈을 갖는데 이는 한 개의 해독판 (open reading frame)을 이용하여 다단백전구체를 만든 후 바이러스 자체의 단백질분해효소에 의해 스스로 잘라져서 궁극적으로느 특이한 기능을 갖는 여러개의 단백질이 된다. P1 다단백질전구체로부터 만들어지는 단백질들은 바이러스의 피막을 구성하는 성분이다. 단백질분해효소인 2A에 의한 최초의 절단은 구조단백질 P1 전구체와 구조단백질이 아닌 P2-P3간을 분리시켜준다. 단백질분해효소 2A는 진핵세포 판독개시인자(translation initiation factor) 4F의 한 subunit인 숙주단백질 p220의 절단에 간접으로 참여한다. 이 단백질의 절단은 캡(cap)에 의존하는 숙주세포의 대부분의 판독을 차단하게 되며 이는 판독에 사용되는 숙주세포의 모든 기구들을 캡에 의존하지 않는 폴리오바이러스 NA 특유의 판독을 위해 전적으로 사용할 수 있게 해준다. 2B, 2C, 2BC 단백질의 기능에 대해서는 많이 알려져 있지 않다. 2B, 2C, 2BC와 3CD 단백질들은 바이러스로 인해 만들어지는 소낭(vesicle)의 복제복합체에 함유되어 있으므로 바이러스의 RNA 복제시 중요한 역할을 함을 암시해준다. 새로이 만들어진 모든 바이러스 RNA는 VPg와 공유결합으로 연결되어 있다. VPg는 3AB로부터 만들어진 아미노산 22개 짜리의 폴리펩타이드이다. 3C와 3CD는 단백질분해소로 다단백질 전구체의 대부분의 절단부위를 잘라준다. 3C단백질은 숙주의 전사인자를 불활성화 시킴으로써 RNA polymer II와 III에 의한 전사를 저해한다. 3D는 RNA의존선RNA 중합효소이다. 폴리오바이러스는 (+)가닥 RNA 바이러스의 일반적인 복제양식을 따른다. 즉 (+) 가닥 RNA는 이와 상보적인 (-)가닥 RNA로 전사되고 이는 다시 (+)가닥 RNA의 합성을 위한 주형으로 사용된다. 폴리오바이러스의 RNA 합성은 세포내막에서 일어나는 데 RNA 복제에 요구되는 주형 RNA와 이때 필요한 단백질들이 어떤 방법으로 세포내막에서 모일 수 있는지는 아직 밝혀진 것이 적다. 바이러스입자의 형성은 세포막의 RNA 복제가 들어가는 데 피막단백질이 (+)가닥 RNA을 인식하는 표지 즉 packaging singal에 대해서는 거의 알려져 있지 않다. 폴리오바이러스 감염 후 첫 바이러스입자가 만들어지기 까는 약 6시간이 소요된다.