Coronavirus Multiplication Strategy. I. Identification and Characterization of Virus-specified RNA

We examined the synthesis of intracellular RNA in primary chicken embryo kidney cells infected with the avian coronavirus infectious bronchitis virus. Infected cells were labeled with (32)P(i) in the presence of actinomycin D for the duration of the viral multiplication cycle, and nucleic acids were extracted, denatured, and analyzed on agarose slab gels. Six major RNA species were found. None of these RNAs was found in extracts of mock-infected cells. All six of the virus-specified RNAs (designated species A through F) were single stranded, and RNA species F had the same electrophoretic mobility as purified viral genome RNA. The molecular weights of the five subgenomic RNAs were estimated to be 0.8 x 10(6), 0.9 x 10(6), 1.3 x 10(6), 1.5 x 10(6), and 2.6 x 10(6) for species A through E, respectively. All of the RNAs were polyadenylated and are therefore likely to be viral mRNA's. The RNAs were synthesized in approximately constant proportions throughout the viral multiplication cycle. Intracellular RNA species A, B, C, D, and F and the purified viral genome were analyzed by RNase T(1) fingerprinting. The results confirmed the identification of RNA species F as the intracellular genome and the derivation of the four smaller RNAs from the genome. Fingerprinting also showed that the intracellular RNAs constitute a nested set such that the nucleotide sequence of each RNA is contained within all larger RNAs and each larger RNA contains an additional sequence congruent with its greater size. Finally, the possible modes of transcription and translation of the infectious bronchitis virus RNAs are discussed.