Sandoval-Jaime Carlos, Green Kim Y, Sosnovtsev Stanislav V
Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD, USA.
Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD, USA.
J Virol Methods. 2015 Jun 1;217:1-7. doi: 10.1016/j.jviromet.2015.02.003. Epub 2015 Feb 16.
Reverse genetics systems constitute one of the most important and powerful tools to study the molecular biology of viruses. We developed a new strategy for the recovery of murine norovirus from a single plasmid in which a bacteriophage T7 RNA polymerase (T7pol) promoter for transcription and an EMCV IRES for efficient translation were engineered immediately upstream of the viral genome. Infectious noroviruses were recovered following transfection of the newly designed plasmid into nonpermissive BHK-21 and HEK293T cell lines that were engineered to express T7pol constitutively. Recovery of the virus did not require the presence of a ribozyme at the 3'-end of the virus genome. The strategy worked also for the efficient recovery of feline calicivirus in these normally nonpermissive cell types. This simplified reverse genetics approach may be broadly applicable to other caliciviruses.
反向遗传学系统是研究病毒分子生物学最重要、最强大的工具之一。我们开发了一种从单个质粒中拯救小鼠诺如病毒的新策略，其中用于转录的噬菌体T7 RNA聚合酶（T7pol）启动子和用于高效翻译的EMCV IRES被设计在病毒基因组的紧邻上游。将新设计的质粒转染到经基因工程改造以组成型表达T7pol的非允许性BHK-21和HEK293T细胞系后，可拯救出感染性诺如病毒。病毒拯救不需要在病毒基因组的3'端存在核酶。该策略在这些通常为非允许性的细胞类型中对猫杯状病毒的高效拯救也有效。这种简化的反向遗传学方法可能广泛适用于其他杯状病毒。
