Stillman E A, Rose J K, Whitt M A
Department of Microbiology and Immunology, University of Tennessee at Memphis 38163, USA.
J Virol. 1995 May;69(5):2946-53. doi: 10.1128/JVI.69.5.2946-2953.1995.
We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.
我们开发了一种系统，其中编码病毒结构蛋白的水疱性口炎病毒（VSV）微型基因组可从质粒中表达。当与其他VSV蛋白共表达时，这些RNA可以被复制、转录并包装成感染性颗粒。微型基因组包含来自VSV印第安纳血清型的糖蛋白（G蛋白）基因（GMG [代表G微型基因组]）或G和基质（M）蛋白基因（GMMG [代表G/M微型基因组]），两侧是野生型VSV基因组的拖尾和前导区域。Northern（RNA）印迹分析表明，微型基因组RNA被复制，并且当在体内与核衣壳（N）蛋白以及两种VSV聚合酶蛋白（磷蛋白[P]和大催化亚基[L]）共表达时，合成了正义复制中间体。此外，功能性mRNA从微型基因组模板转录而来，并表达了相应编码的蛋白质。GMMG中G和M蛋白的表达导致感染性颗粒的组装和释放，这些颗粒可以仅在表达N、P和L蛋白的细胞上进行传代。在连续传代过程中发生了扩增，经过四次传代后，约30%的细胞同时表达了G和M蛋白。对GMMG感染细胞中产生的RNA的分析还表明，微型基因组准确地再现了VSV感染细胞中正常发生的所有复制和转录事件。因此，GMMG是一种新型的缺陷型颗粒，它编码对自身传播至关重要的功能性病毒蛋白。
