Mutagenesis of the genome of mouse hepatitis virus by targeted RNA recombination.
Our laboratory has described a method for introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA. By using a thermolabile N protein mutant of MHV as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, engineered recombinant viruses were selected as heat-stable progeny resulting from cotransfection. We have recently reported an optimization of the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that recombinants can often be directly identified without employing a thermal selection. We present here a progress report on our use of this system to map MHV mutants and to construct N gene mutants which include (1) a mutant in which the internal open reading frame within the N gene (the I gene) has been disrupted, and (2) a series of recombinants in which portions of the MHV N gene have been replaced by the homologous regions from the N gene of bovine coronavirus. We also report on some mutants we have not been able to construct.
Masters
P S
PS
David Axelrod Institute, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-2002, USA.
Peng
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D
Fischer
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F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics*; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000235:genetics*; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics
D021382:Protein Sorting Signals; D012333:RNA, Messenger; D012367:RNA, Viral; D014758:Viral Core Proteins