The majority of
porcine transmissible gastroenteritis coronavirus plus-strand RNAs (genome and subgenomic mRNAs), at the time of peak
RNA synthesis (5 h postinfection), were not found in membrane-protected complexes in lysates of cells prepared by Dounce homogenization but were found to be susceptible to
micrococcal nuclease (85%) or to sediment to a pellet in a
cesium chloride gradient (61%). They therefore are probably free molecules in
solution or components of easily dissociable complexes. By contrast, the majority of minus-strand RNAs (genome length and
subgenomic mRNA length) were found to be resistant to
micrococcal nuclease (69%) or to remain suspended in association with membrane-protected complexes following isopycnic sedimentation in a
cesium chloride gradient (85%). Furthermore, 35% of the suspended minus strands were in a dense complex (1.20 to 1.24 g/ml) that contained an
RNA plus-to-minus-strand molar ratio of approximately 8:1 and
viral structural proteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/ml) that contained nearly equimolar amounts of plus- and minus-strand RNAs and only trace amounts of
proteins M and N. In no instance during fractionation were genome-length minus strands found segregated from sub-genome-length minus strands. These results indicate that all minus-strand species are components of similarly structured membrane-associated replication complexes and support the concept that all are active in the synthesis of plus-strand RNAs.