The fitness of defective interfering murine coronavirus DI-a and its derivatives is decreased by nonsense and frameshift mutations.
The genome of the defective interfering (DI) mouse hepatitis virus DI-a carries a large open reading frame (ORF) consisting of ORF1a, ORF1b, and nucleocapsid sequences. To test whether this fusion ORF is important for DI virus replication, we constructed derivatives of the DI-a genome in which the reading frame was truncated by a nonsense codon or a frameshift mutation. In vitro-transcribed DI RNAs were transfected into mouse hepatitis virus-infected cells followed by undiluted passage of the resulting virus-DI virus stocks. The following observations were made. (i) Truncation of the fusion ORF was not lethal but led to reduced accumulation of DI RNA. (ii) When pairs of nearly identical in-frame and out-of-frame DI RNAs were directly compared by cotransfection, DI viruses containing in-frame genomic RNAs prevailed within three successive passage even when the out-of-frame RNAs were transfected in 10-fold molar excess. (iii) When DI viruses containing out-of-frame genomic RNAs were passaged, mutants emerged and were selected for that had restored the reading frame. We conclude that translation of the fusion ORF is indeed required for efficient propagation of DI-a and its derivatives.
de Groot
R J
RJ
Department of Virology, Institute of Medical Microbiology, Faculty of Medicine, Leiden University, The Netherlands.
van der Most
R G
RG
Spaan
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003673:Defective Viruses / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D016368:Frameshift Mutation; D005814:Genes, Viral; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics*; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012367:RNA, Viral; D011995:Recombination, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D012367:RNA, Viral