REPLICATION AND AMPLIFICATION OF NOVEL VESICULAR STOMATITIS-VIRUS MINIGENOMES ENCODING VIRAL STRUCTURAL PROTEINS

We have developed a system in which vesicular stomatitis virus (VSV) m inigenomes encoding viral structural proteins can be expressed from pl asmids. These RNAs can be replicated, transcribed, and packaged into i nfectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [sta nds for G minigenome]) or both the G and matrix (M) protein genes (GMM G [stands for G/M minigenome]) from the Indiana serotype of VSV flanke d by the trailer and leader regions from the wild-type VSV genome. Nor thern (RNA) blot analysis showed that the minigenome RNAs were replica ted and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV po lymerase 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 expres sed. Expression of the G and M proteins from GMMG resulted in the asse mbly and release of infectious particles that could be passaged on cel ls expressing the N, P, and L proteins only. Amplification occurred du ring successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs p roduced in the GMMG-infected cells also showed that the minigenomes ac curately 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 cri tical to its own propagation.