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Characterization of leader related small rna species in coronavirus infected cells further evidence for leader primed mechanism of transcription



Characterization of leader related small rna species in coronavirus infected cells further evidence for leader primed mechanism of transcription



Virus Research 3(1): 19-34



Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNA species containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNA species by a unique mechanism. Utilizing a leader-specific complementary DNA probe, several free leader RNA species were detected ranging from 70-82 nucleotides in length. The predominant leader RNA was .apprx. 75 nucleotides. In addition, larger distinct leader-containing RNA species were also detected ranging from 130-250 nucleotides in length. The 70-82-nucleotide leader-related RNA species were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNA species were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. A temperature-sensitive mutant was identified which synthesizes only leader RNA but not mRNA species at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNA species. These data support the leader-primed mechanism for coronavirus transcription and suggest that .gtoreq. 1 free leader RNA species are used as primers of mRNA synthesis.

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