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

Abstract

We examined the synthesis of intracellular RNA in primary chicken embryo kidney celis infected with the avian coronavirus infectious bronchitis virus. Infected cells were labeled with 'Pi 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 106, 0.9 X 106, 1.3 x 106, 1.5 x 106, and 2.6 x 106 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, fingerprinting.

Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot

“…Recently, however, we described the first non-retroviral, higher eukaryotic example of the phenomenon, in the avian coronavirus infectious bronchitis virus (IBV) (Brierley et al, 1987). IBV, the type species of the Coronaviridae (Siddell et al, 1983), has a large, single-stranded, continuous RNA genome of positive polarity (Schochetman et al, 1977;Stern and Kennedy, 1980). Three-quarters of the genomic coding capacity is contained in two large ORFs, Fl and F2, which potentially encode 440 kd and 300 kd polypeptides respectively (Boursnell et al, 1987).…”

Sequence of the membrane protein gene from avian coronavirus IBV

“…Work on the membrane protein of the murine coronavirus MHV-A59 suggests that part of the molecule may also project from the inner surface of the membrane (Sturman et al, 1980). Six major species of RNA have been observed in IBV-infected cells (Stern and Kennedy, 1980a). These include RNA F. which is the same length as the viral genome, and five smaller species, RNAs A-E, RNA A being the smallest.…”

The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM

“…Acrylamide, xylene cyan01 FF, and bromphenol blue were obtained from Eastman Chemical. Hydroxylapatite (HA; DNA grade) was obtained from Bio-Rad and prepared as described by Stern and Kennedy (1980a).…”

“…The RNA was eluted from HA columns by two washes of 1.0 ml of 0.4 M sodium phosphate, 1 mM EDTA (pH 7.0) which were similarly forced from the columns by centrifugation at 1OOOg for 10 min. The phosphate eluates were pooled and the RNA precipitated with CTAB as described by Stern and Kennedy (1980a). The CTAB precipitates were dissolved in 0.2 ml of 50 mM Tris, pH 7.4, containing 1 M NaCl and 1 mM EDTA and the RNA precipitated with 1.0 ml of ethanol.…”

“…Ten microliters of a solution containing 5 M urea, 50% sucrose, 0.1% bromphenol blue, and 0.1% xylene cyan01 FF was added to the digestion products. The RNase T1-resistent oligonucleoties were separated by two-dimensional polyacrylamide gel electrophoresis as described by Stern and Kennedy (1980a) with the following modification. After electrophoresis in the first dimension the gel strips were washed for 25 min with two changes of 100 mM Tris borate buffer containing 2.5 mM EDTA, pH 8.3, prior to pouring the second dimension gel (Lee et al, 1979).…”