Biochemical mapping of the simian rotavirus SA11 genome

Abstract

Biochemical mapping experiments of the simian rotavirus SA11 genome were performed to determine which double-stranded RNA segment coded for each of the viral polypeptides. Viral RNA transcripts were synthesized in vitro by using the endogenous viral RNA polymerase and fractionated by electrophoresis in acid-urea agarose gels. The fractionated transcripts were translated in 2 cell-free systems: micrococcal nuclease-treated rabbit reticulocyte lysates and wheat germ extracts. The polypeptide products were identified by polyacrylamide gel electrophoresis and partial peptide analysis and compared with polypeptides synthesized in infected rhesus monkey kidney MA-104 cells or found in purified virus. The RNA segment that coded for each transcript was determined by hybridization of the fractionated transcripts to the double-stranded RNA genome and analysis of the hybrids by electrophoresis in polyacrylamide gels. Primary gene products were assigned for 10 of the rotavirus transcripts and 10 of the double-stranded RNA segments. The coding assignments are as follows: the inner-capsid polypeptides, VP1, VP2 and VP6, were assigned to segments 1, 2 and 6, respectively; the major outer-capsid polypeptides, VP3 and VP7, were assigned to segments 4 and 9, respectively; segments 5, 7 and 8 coded for nonstructural polypeptides with MW of 53,000, 34,000 and 35,000, respectively; segment 10 coded for the 20,000 MW precursor to the 29,000 MW glycosylated nonstructural polypeptide; and segment 11 coded for a 26,000 MW polypeptide that may be the precursor to the minor outer-capsid polypeptide VP9. Several methods were used to determine the product of gene segment 3, and the problems associated with the identification of this gene product are discussed.