Veterinary and Biomedical Sciences, Department of


Date of this Version



JOURNAL OF VIROLOGY, June 1990, p. 2948-2957


Copyright © 1990, American Society for Microbiology


Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have untD now been attained only in ceUs coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When ceUs expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) ofVSV were superiofected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of anyone of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wDd-type helper VSV by 8- to IO-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.