Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, Japan.
Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan.
J Virol. 2024 Mar 19;98(3):e0163823. doi: 10.1128/jvi.01638-23. Epub 2024 Feb 14.
Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology.
The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.
逆转录遗传系统在开发用于广泛病毒研究的重组病毒方面发挥了核心作用。环形聚合酶延伸反应（CPER）方法已应用于正链 RNA 病毒的研究，使研究人员能够绕过病毒 cDNA 克隆的分子克隆，从而快速产生重组病毒。然而，到目前为止，CPER 方案仅在使用依赖于帽结构的 RNA 病毒中建立。在这里，我们证明了 CPER 方法的一个修改版本可以成功应用于使用非依赖性于帽结构、内部核糖体进入位点（IRES）介导的翻译的正链 RNA 病毒。作为概念验证，我们采用了不同类型（I、II 和 III 类）IRES 的哺乳动物病毒来优化 CPER 方法。我们使用丙型肝炎病毒（HCV，III 类）发现，在 CPER 组装中包含 RNA 聚合酶 I 启动子和终止子，而不是聚合酶 II 的启动子和终止子，可提高病毒产量。该方法在转染 MDBK/293T 共培养物以克服低转染效率后，也成功地产生了重组牛病毒性腹泻病毒（III 类）。此外，我们还成功地从临床标本中生成了重组病毒。我们的改进 CPER 可用于生成甲型肝炎病毒（HAV，I 型）和生成脑炎心肌炎病毒（II 型）。最后，我们生成了复制与野生型亲本病毒相当的重组 HCV 和 HAV 报告病毒。重组 HAV 报告病毒有助于评估抗病毒药物。综上所述，本研究为病毒学提供了方法学上的进展。
