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

Abstract
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.
AuthorsP B Sethna, D A Brian
JournalJournal of virology (J Virol) Vol. 71 Issue 10 Pg. 7744-9 (Oct 1997) ISSN: 0022-538X [Print] United States
PMID9311859 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • RNA, Viral
  • Viral Structural Proteins
  • RNA-Directed DNA Polymerase
Topics
  • Animals
  • Cell Fractionation
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Genome, Viral
  • Male
  • RNA, Viral (biosynthesis, isolation & purification)
  • RNA-Directed DNA Polymerase (isolation & purification, metabolism)
  • Swine
  • Testis
  • Transmissible gastroenteritis virus (genetics, physiology)
  • Viral Structural Proteins (biosynthesis, isolation & purification)
  • Virus Replication

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