Molecular biological characterization of defective interfering particles of vesicular stomatitis virus.
Creators: name: Choi, Woo-Young.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1997
Subjects: subject: Biology, Microbiology.

description: Defective interfering particles (DIPs) of vesicular stomatitis virus generated and/or amplified during serial high multiplicity passage in BHK-21 cells. Two IND-DIPs (IND-ST and IND-LT) were derived from the Indiana (Ind) serotype and two NJ-DIPs (Ogd-DI and Haz-DI) were generated from Ogden and Hazelhurst strains, respectively, of the New Jersey (NJ) serotype of VSV. The biological activity of each DIP was examined by the interference assay. Two IND-DIPs interfered homotypically and heterotypically. In contrast, two NJ-DIPs interfered homotypically but could not interfere heterotypically. Intracellular RNA synthesis in standard virus infected cells and in cells infected with both standard virus and DIPs revealed that the level of RNA synthesis of the standard virus was markedly reduced if DIP interfered with the production of the standard virus. In contrast, the level of mRNA transcription was not affected if DIPs did not interfere with replication of the standard virus. Furthermore, DIP genomes were replicated only if the DIP interfered with the replication of the standard virus. These data strongly suggest that the first step of DIP-mediated viral interference is at the level of viral RNA synthesis. In an effort to understand how these DIPs are generated, which genetic elements are involved in viral interference and how the DIPs interfere with the replication of standard virus, the complete sequence analyses of cloned cDNAs from the four different DIPs were carried out by the dideoxy chain termination method. The size of each DIP genome was 2,660 bases (IND-ST), 5,313 bases (IND-LT), 2,984 bases (Ogd-DI), and 6,110 bases (Haz-DI). The DIP genomes of IND-ST, Ogd-DI, and Haz-DI were derived from the 5$\sp\prime$ end of the standard virus genome and retained different lengths of inverted complementary termini (54 bp, 18 bp, and 71 bp, respectively), to form panhandle stem structures. In contrast, IND-LT retained the 3$\sp\prime$ half of the standard virus genome (4,974 bases) and 530 bases of the 5$\sp\prime$ end of the standard virus genome. This DIP genome represented a single deletion of a part of the L gene and thus contained the same 3$\sp\prime$ and 5$\sp\prime$ termini as the standard virus genome. Examination of the DIP genomes revealed that they all retained the 5$\sp\prime$ end of the standard virus. These findings suggest that the 5$\sp\prime$ terminus may be essential for control of DIP replication and interference activity. To understand the mechanism(s) of viral interference mediated by DIPs, a reverse genetic system for VSV was employed. All five viral genes from each of the three different strains of VSV were cloned and placed under the control of the T7 RNA polymerase promoter. Furthermore, cDNAs of four different DIPs were subcloned into a specialized transcription vector containing the T7 RNA polymerase promoter, hepatitis delta virus ribozyme, and the T7 terminator to generate DIP RNAs with the exact 3$\sp\prime$ and 5$\sp\prime$ termini identical to those of the authentic DIP genomic RNAs. The T7 RNA polymerase expressed by a recombinant vaccinia virus successfully rescued the cloned DIP genomes, which were amplified by subsequent passages. Furthermore, the rescued recombinant DIPs had the same biological activities as the original DIPs. Future experiments will help to identify cis-acting sequences in the DIP genomes and the roles of individual viral proteins involved in viral interference.