Two murine coronavirus genes suffice for viral RNA synthesis.
We identified two mouse hepatitis virus (MHV) genes that suffice for MHV RNA synthesis by using an MHV-JHM-derived defective interfering (DI) RNA, DIssA. DIssA is a naturally occurring self-replicating DI RNA with nearly intact genes 1 and 7. DIssA interferes with most MHV-JHM-specific RNA synthesis, except for synthesis of mRNA 7, which encodes N protein; mRNA 7 synthesis is not inhibited by DIssA. Coinfection of MHV-JHM containing DIssA DI particles and an MHV-A59 RNA- temperature-sensitive mutant followed by subsequent passage of virus at the permissive temperature resulted in elimination of most of the MHV-JHM helper virus. Analysis of intracellular RNAs at the nonpermissive temperature demonstrated efficient synthesis of DIssA and mRNA 7 but not of the helper virus mRNAs. Oligonucleotide fingerprinting analysis demonstrated that the structure of mRNA 7 was MHV-JHM specific and therefore must have been synthesized from the DIssA template RNA. Sequence analysis revealed that DIssA lacks a slightly heterogeneous sequence, which is found in wild-type MHV from the 3' one-third of gene 2-1 to the 3' end of gene 6. Northern (RNA) blot analysis of intracellular RNA species and virus-specific protein analysis confirmed the sequence data. Replication and transcription of another MHV DI RNA were supported in DIssA-replicating cells. Because the products of genes 2 and 2-1 are not essential for MHV replication, we concluded that expression of gene 1 proteins and N protein was sufficient for MHV RNA replication and transcription.
Kim
K H
KH
Department of Microbiology, University of Texas at Austin 78712-1095.
Makino
S
S
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D018076:DNA, Complementary; D005814:Genes, Viral; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000502:physiology; D009363:Neoplasm Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014779:Virus Replication
D018076:DNA, Complementary; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012367:RNA, Viral