Screening and Characterization of Anti-enterovirus 71 Compounds that target viral translation initiation

• Main Authors: Kuan-Yin Wu, 吳冠瑩
• Other Authors: Szu-Hao Kung
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/36197396716995001317

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 103 === Human Enterovirus 71 (EV71), a member of genus enterovirus of the family Picornaviridae, commonly causes Hand, Food and Mouth Disease and occasionally neurological complications in young children. To date, there is no specific antiviral agent known to be effective in clinically treating EV71 infections. Previous studies from a drug screen identified an anthracycline compound idarubicin (IDR) that showed anti-EV71 activities by selectively targeting the viral translation initiation. The translation initiation of the enterovirus occurs via the highly structured internal ribosomal entry site (IRES) located at the 5’ untranslated region (5’UTR) of the virus RNA genome, and it is regulated by certain cellular factors called IRES trans-acting factors (ITAFs). As anthracycline compounds are known as DNA intercalators, we hypothesized that IDR may inhibit the viral IRES activity by preferentially binding with the viral IRES RNA. By the fluorescence quenching assay, we showed that IDR binds strongly with viral IRESs from EV71, echovirus 9 and coxsackievirus A16 but only weakly with cellular p53 IRES. To investigate whether IDR exhibited a specific binding or multiple bindings with the viral IRES RNA, three EV71 IRES subfragments covering domain I-V, IV and IV-linker were generated. All these EV71 IRES subfragments displayed strong binding affinities with IDR, indicating that IDR bind at multiple sites within the viral IRES RNA. Moreover, pull-down assay revealed that IDR intervened binding between EV71 IRES RNA and heterogenous nuclear ribonucleoprotein A1 (hnRNPA1), a representative ITAF, in a dose-dependent manner. The hnRNPA1 is known to bind the domains II and VI of the EV71 IRES RNA. We further showed that IDR intervened with the hnRNPA1 bindings with the IRES subfragments containing the domains I-V, and IV-linker, but not the domain IV. These data support a working model that IDR may exhibit an inhibitory effect on enterovirus IRES activity by selectively binding to enteroviral IRES at multiple sites, thereby blocking the binding of ITAFs and the consequent IRES activity. Moreover, because of the secondary structure conservation yet sequence diversity among the enteroviral IRESs, IDR likely acts in a structure-dependent rather than a sequence-dependent manner. These findings are consistent with the previous failure to isolate drug-resistant virus. To find other anthracycline compounds with anti-EV71 activity, Aclarubicin (ACL), Pirarubicin (PIR), and Valrubicin (VAL) have been evaluated. The results showed that ACL displayed stronger EV71 inhibition in viral protein synthesis and infection rates among the anthracyclines tested. However, ACL showed higher cytotoxicity (CC50: 107.9μM) than IDR (CC50: 192.1μM). In sum, we have come with a molecular mechanism that elaborates the IDR action in selective inhibition of enterovirus IRES activity. The finding may point to development of a broad-spectrum anti-EV drug based on the anthracycline analogs.