Generation of infectious nucleocapsids by in vitro assembly of the shell protein on to the polymerase complex of the dsRNA bacteriophage phi 6.
A method for the in vitro uncoating of the phi 6 nucleocapsid (NC) was developed. The resulting particle, designated as the NC core, containing the genomic double-stranded (ds) RNA segments and the proteins P1, P2, P4 and P7, was not infectious but had a highly enhanced in vitro transcriptase activity compared to that of the intact NC. The NC shell protein P8 was purified by immunoaffinity chromatography, and it was shown to self-assemble to shell-like structures upon addition of calcium ions. The conditions for the self-assembly of the shell were optimized. Shell reassembly on to the NC cores restored the infectivity but resulted in a decrease of transcriptase activity. No reassembly of the shell on to RNA-less cores (procapsids) produced from a cDNA construction in Escherichia coli was observed. Our results suggest that the intracellular uncoating of the NC is the event activating the phi 6 dsRNA transcriptase and that the NC shell is necessary for infectivity, probably for the passage of the NC through the host cytoplasmic membrane. Packaging of the dsRNA segments into the procapsid appears to be a prerequisite for NC shell assembly.
Olkkonen
V M
VM
Department of Genetics, University of Helsinki, Finland.
Ojala
P M
PM
Bamford
D H
DH
D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D002122:Calcium Chloride / Q000494:pharmacology; D002213:Capsid / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014758:Viral Core Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014758:Viral Core Proteins; D014764:Viral Proteins; D011189:Potassium Chloride; D012321:DNA-Directed RNA Polymerases; D002122:Calcium Chloride