Defective Interfering Particle RNAs Research Articles

Studies on the generation of vesicular stomatitis virus (Indiana serotype) defective interfering particles

Using freshly cloned vesicular stomatis virus (Indiana serotype) (VSV IND) serial undiluted high-multiplicity passage in four different cell lines has produced different size classes of defective interfering (DI) particles at different passage numbers. The same clonal isolates of VSV in a given cell type appear to produce identical patterns of particles, although different clonal isolates produce different size classes of DI particles. Thus, the classes of DI particles generated during serial passages appear to relate in some way to the clone of virus and to the type of host cell used. All the DI particles generated in different cell types are capable of homologous interference in any cell type. Oligonucleotide sequence analyses of five DI particle RNAs indicate that they are all generated from a specific region of the standard B virion genome and contain only negative-stranded genomic RNA.

Inverted Complementary Terminal Sequences in Single-Stranded RNAs and Snap-Back RNAs from Vesicular Stomatitis Defective Interfering Particles

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.

Expression of genetic information contained in the rna of a defective interfering particle of vesicular stomatitis virus

Extracellular HR Indiana defective interfering (DI) particles were generated by cells coinfected with these particles and vesicular stomatitis virus (VSV) New Jersey. Proteins of purified isolates of these heterotypically generated DI particles were analyzed by electrophoresis in stacked polyacrylamide gels. Under these conditions, it was possible to demonstrate that the M and G proteins of Indiana and New Jersey serotypes of VSV were present in the isolates. It was shown that the Indiana proteins could not have originated from a low level of Indiana virion contamination in the original HR DI particle inoculum. Proteins of both serotypes were also found in HR DI particle isolates which were repeatedly passed with New Jersey virions. It is concluded that the genetic information of the HR DI particle RNA was transcribed and translated in vivo. Moreover, the results indicated that New Jersey L protein could be utilized in the transcription of HR DI particle RNA and in the formation of hybrid particles. During the course of these studies, electrophoretic differences between the M proteins of two Indiana virions (HR and Gla) were observed.

Use of defective interfering particle RNA probes in the determination of the order of in vitro transcription of vesicular stomatitis virus genes

Annealing of various defective interfering particle RNAs of vesicular stomatitis virus to early in vitro viral transcription products has shown that transcription is sequential, with 13S to 18S mRNA's being produced first. Based on this finding, the 30S mRNA cistron is located at the 5' end of the viral genome.

Cyclic production of vesicular stomatitis virus caused by defective interfering particles.

Purified standard and defective interfering (DI) particles of vesicular stomatitis virus (VSV) were reconstituted, and the mixture was continuously passaged for six 24-hr growth periods in Chinese hamster ovary cells in suspension cultures. The cultures were monitored for cellular viability, for intracellular virus-specific RNA, for total extracellular viral particles, and for production of plaque-forming standard VSV. During the six passages, there was a cyclic, overlapping pattern of synthesis of both DI and standard VSV particles; decline in the production of standard virus was always preceded by large amounts of DI particles. Intracellular virus-specific RNA synthesis reflected the pattern of production of extracellular particles. Viral antigens represented by nucleocapsids accumulated in cells when DI particles were being produced in large quantities. These nucleocapsids contained RNA of DI particles. Under other conditions, infected cells were able to survive longer than usual and were resistant to superinfection by standard VSV. Preliminary evidence indicates that this resistance was due to surface interference. Therefore, a wide variety of different interactions between virus and host occurs simply by varying the total concentration of viral particles and the relative concentrations of standard and OI particles in these preparations.