The isolation of Mengo virus stable non-capsid polypeptides from infected L cells and preliminary characterization of an RNA polymerase activity associated with polypeptide E.
Isolation of the Mengo virus stable non-capsid virus polypeptides E, F, G and I from infected L cells has been achieved. Unstable precursors were eliminated by incubation in the presence of pactamycin and capsid polypeptides were removed by ultracentrifugation and affinity chromatography. Subsequent sodium dodecyl sulphate (SDS)-hydroxylapatite chromatography resolved the non-capsid proteins into two major peaks which comprised F plus G and E plus I, respectively. The individual polypeptide species were separated by gel filtration on Sephadex G-100 in the presence of SDS. Polypeptide E was isolated in an undenatured form by gel filtration of infected cell extracts (from which precursor and capsid polypeptides had been removed) on Bio-Gel A-5m agarose beads. Purified polypeptide E was found to co-sediment with Mengo virion RNA during centrifugation in a sucrose density gradient and it was also capable of binding to poly(A)-Sepharose. Assay mixtures containing polypeptide E exhibited an RNA polymerase activity which was dependent upon exogenous virus RNA template and oligo(U) primer and which was not affected by the addition of virus capsid polypeptides or extracts from uninfected cells.
D002845:Chromatography; D002850:Chromatography, Gel; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D007739:L Cells; D008574:Mengovirus / Q000032:analysis* / Q000201:enzymology; D009843:Oligoribonucleotides / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification; D012367:RNA, Viral / Q000378:metabolism; D014764:Viral Proteins / Q000302:isolation & purification*
D009843:Oligoribonucleotides; D010455:Peptides; D012367:RNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases