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 sy nthesis (5 h postinfection). were net found in membrane-protected comp lexes in lysates of cells prepared by Dounce homogenization but were f ound to be susceptible to micrococcal nuclease (85%) or to sediment to a pellet in a cesium chloride gradient (61%), They therefore are prob ably free molecules in solution or components of easily dissociable co mpletes, By contrast, the majority of minus-strand RN;is (genome lengt h and subgenomic mRNA length) were found to be resistant to micrococca l nuclease (69%) or to remain suspended in association with membrane-p rotected completes following isopyenic sedimentation in a cesium chlor ide gradient (85%), Furthermore, 35% of the suspended minus strands we re in a dense complex (1.20 to 1.24 g/ml) that contained an RNA plus-t o-minus-strand molar ratio of approximately 8:1 and viral structural p roteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/m l) that contained nearly equimolar amounts of plus-and minus-strand RN As and only tract amounts of proteins hi and N, In no instance during fractionation were genome-length minus strands Pound segregated from s ub-genome-length minus strands, These results indicate that all minus- strand species are components of similarly structured membrane-associa ted replication completes and support the concept that all are active in the synthesis of plus-strand RNAs.