Studies of coronavirus DI RNA replication using in vitro constructed DI cDNA clones.
Sequence analysis of an intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) revealed that it is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5'-end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3'-end of the parental MHV genome. DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. A system was developed for generating DI RNAs to study the mechanism of MHV RNA replication. A cDNA copy of DIssE RNA was placed downstream of T7 RNA polymerase promoter to generate DI RNAs capable of extremely efficient replication in the presence of a helper virus. We demonstrated that, in the DI RNA-transfected cells, the leader sequence of these DI RNAs was switched to that of the helper virus during one round of replication. This high-frequency leader sequence exchange was not observed if a nine-nucleotide stretch at the junction between the leader and the remaining DI sequence was deleted. This observation suggests that a free leader RNA is utilized for the replication of MHV RNA.
Makino
S
S
Department of Microbiology, University of Southern California, School of Medicine, Los Angeles 90033.
Lai
M M
MM
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D006378:Helper Viruses / Q000235:genetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral