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 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.
Sethna
P B
PB
Department of Microbiology, University of Tennessee, Knoxville 37996-0845, USA.
Brian
D A
DA
D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D016679:Genome, Viral; D008297:Male; D012367:RNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D013737:Testis; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000502:physiology*; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014779:Virus Replication
D012367:RNA, Viral; D015678:Viral Structural Proteins; D012194:RNA-Directed DNA Polymerase