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

Abstract

The synthesis of intracellular RNA in primary chicken embryo kidney cells infected with the avian coronavirus infectious bronchitis virus was examined. Infected cells were labeled with 32Pi 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 RNA was found in extracts of mock-infected cells. All 6 virus-specified RNA (designated species A through F) were single stranded; RNA species F had the same electrophoretic mobility as purified viral genome RNA. The MW of the 5 subgenomic RNA were estimated to be 0.8 .times. 106, 0.9 .times. 106, 1.3 .times. 106, 1.5 .times. 106 and 2.6 .times. 106 for species A through E, respectively. All RNA were polyadenylated and are probably viral mRNA. The RNA 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 T1 fingerprinting. The identification of RNA species F as the intracellular genome and the derivation of the 4 smaller RNA from the genome were confirmed. Fingerprinting also showed that the intracellular RNA constitute a nested set such that the nucleotide sequence of each RNA is contained within all larger RNA and each larger RNA contains an additional sequence congruent with its greater size. The possible modes of transcription and translation of the infectious bronchitis virus RNA are discussed.