Intracellular murine hepatitis virus-specific RNAs contain common sequences

Abstract

A major polyadenylated viral RNA of approximately 0.8 X lo6 daltons was isolated from murine hepatitis virus (A59)-infected cells by preparative polyacrylamide gel electrophoresis in formamide. This RNA was shown to encode the viral nucleocapsid protein by direct in vitro translation in a cell-free, reticulocyte-derived system. Single stranded q-labeled complementary DNA was prepared from this RNA and was demonstrated to be virus specific. Using this complementary DNA in a Northern blotting procedure, we were able to identify six major virus-specific intracellular RNA species with estimated molecular weights of 0.8, 1.1, 1.4, 1.6, 3, and 4 X l@ daltons. All of these RNA species were polyadenylated. Our results support the idea that coronavirus-infected cells contain multiple intracellular polyadenylated RNAs which share common sequences. et al., 1959) of mouse fibroblasts.

Quantitative sense-specific determination of murine coronavirus RNA by reverse transcription polymerase chain reaction

“…Using equimolar methodology, qssRT-PCR showed that at maximal infection 12 h after inoculation, FL5 cells contained 0.86 ng or 2.7× 10 9 molecules of RNA competing with N-del template per microgram of total cellular RNA and, further, that \99% of the MHV-4 RNA was of positive sense (data not shown). Assuming that the yield of total RNA extracted from FL5 cells was approximately 5.0×10 − 6 mg of RNA/cell (RNA STAT 60 instructions), on a molar basis these results are in close agreement with prior studies based on hybridization kinetics (Cheley et al, 1981) and Northern blots (Hof-mann et al, 1990) which indicate that at the peak of coronavirus infection in vitro in the order of 10 4 molecules of viral RNA are present in each infected cell. The consistency among different methods of estimating the level of coronavirus expression suggests that the qssRT-PCR assay yields reasonably accurate results when applied to complex biological samples, such as RNA derived from virally-infected cells.…”

Temperature-sensitive mutants of mouse hepatitis virus strain A59: Isolation, characterization and neuropathogenic properties

“…feature of the coronavirus r~~li~at~~~ strategy is that seven, or six in the avian system, virus-specific RNAs are synthesized in the infected cell. The largest ot?e is the intracellular form of ge~~rne~ the others are a nested set of sub~e~~~i~ mRNAs with common ~-proximal sequences (Spaan et al, 198 al., 1981;Lai et al, 1981;Cheley et al, 1981;bowitz, 1981;Stern and Kenne RNAs 3, 6, and '7 have be code the three major virion proteins (Slddell et al+, 1980;Rottier et aZ., 19818. et al, 1981;Leibowitz et al, 19 and 2 of MHV encode nonstruc teins Si 1981). ts mutants will be useful to identify the functions of these and other ~~~~tr~~ tural proteins and for the study of the transcription mechanism.…”

RNA-dependent RNA Polymerase Activity in Murine Coronavirus-infected Cells

“…In infected cells, up to seven RNA species with the characteristics of mRNAs have been identified in association with polysomes. The largest is of genome length; the other six are subgenomic RNAs which form a 'nested set' as originally found for the avian coronavirus, infectious bronchitis virus (IBV) (Stern & Kennedy, 1980) in which the sequence of each RNA is contained within the sequences of all larger RNAs, extending inward from the 3'-terminus of genome RNA (Cheley et al, 1981 ;Leibowitz & Weiss, 1981 ;Spaan et al, 1982). There is evidence that these RNAs are functionally monocistronic (Siddell et al, 1980 with only the Y-terminal portion available for translation by ribosomes.…”