Coronavirus genomic and subgenomic minus-strand RNAs copartition in membrane-protected replication complexes

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 net 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 completes, By contrast, the majority of minus-strand RN;is (genome length and subgenomic mRNA length) were found to be resistant to micrococcal nuclease (69%) or to remain suspended in association with membrane-protected completes following isopyenic 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 tract amounts of proteins hi and N, In no instance during fractionation were genome-length minus strands Pound segregated from sub-genome-length minus strands, These results indicate that all minus-strand species are components of similarly structured membrane-associated replication completes and support the concept that all are active in the synthesis of plus-strand RNAs.