Studies on rotavirus replication

Sammanfattning: Rotavirus is the leading cause of dehydrating diarrhea in infants and young children globally. In cells infected with rotavirus SA11 the synthesis of viral RNA and protein predominated already after a few hours. Cellular RNA remained intact, but viral mRNA was preferentially utilised for protein synthesis. As a reaction to the infection, the cellular double-stranded RNA-activated protein kinase (PKR) was induced and activated. Its major substrate, the translation initiation factor 2α became phosphorylated. However, rotavirus protein synthesis was resistant to this antiviral response, even when it was induced before infection by treatment of the cells with interferon alpha. The porcine rotavirus strain CC86, having partial duplication in RNA segment 11, was used for an investigation of genetic stability. In serial cloning of CC86 by plaque to plaque transfer the virus lost fitness, probably due to mutations in all RNA segments. Mutations in segment 11 were identified by nucleotide sequence determination of cDNA. The overall mutation rate of this segment was 1x10-5 per replicted base at two nucleotide positions mutations and reversions occurred at much higher frequencies.The properties of the nonstructural protein NSP5, encoded by gene segment 11, were analysed. NSP5 isolated from infected cells was phosphorylated at multiple serine residues. Similar phosphorylated forms were formed when the polypeptide was expressed in the absence of the other viral proteins. This result and the properties of NSP5 expressed in bacteria show that NSP5 is a protein kinase of unusual type capable of autophosphorylation.