Characterization of replicative intermediate RNA of mouse hepatitis virus: presence of leader RNA sequences on nascent chains.
Mouse hepatitis virus A59 codes for seven mRNAs in infected cells. These mRNAs are transcribed from a minus (-) strand template of genome length and contain a leader RNA at their 5' ends. To further elucidate the mechanism of coronavirus transcription, we examined the structure of mouse hepatitis virus replicative intermediates (RIs) isolated by 2 M NaCl precipitation and Sepharose 2-B column chromatography. Purified RIs migrated as a single species on agarose gels and sedimented between 12 and 38S on 10 to 25% sucrose gradients. The complexes were readily heat denatured into a heterogeneous population of smaller RNA molecules which probably represent nascent plus (+) strands. RNase A digestion of RIs produced a single replicative form which sedimented between 30 and 32S. These data suggest that the RI is composed of a single genome-sized (-) strand hydrogen bonded to an average of 4 to 6.5 nascent (+) strands. In contrast, a column-purified replicative form was extremely resistant to RNase A digestion and heat denaturation and migrated as a single RNA species on agarose gels and sucrose gradients. Oligonucleotide fingerprinting of an RI revealed the presence of the 5' leader RNA on the nascent (+) strands. In addition, an average of 6.2 cap structures were present in each RI, which agrees with the average number of nascent (+) strands per RI. These data suggest that the leader RNA is utilized as a primer for mouse hepatitis virus RNA transcription and is not added to mRNA post-transcriptionally.
D000818:Animals; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002847:Chromatography, Agarose; D004587:Electrophoresis, Agar Gel; D051379:Mice; D006517:Murine hepatitis virus / Q000235:genetics*; D009843:Oligoribonucleotides / Q000032:analysis; D012315:RNA Caps / Q000032:analysis; D012367:RNA, Viral / Q000302:isolation & purification*
D009843:Oligoribonucleotides; D012315:RNA Caps; D012367:RNA, Viral