PROTEIN P7 OF PHAGE PHI-6 RNA-POLYMERASE COMPLEX, ACQUIRING OF RNA PACKAGING ACTIVITY BY IN-VITRO ASSEMBLY OF THE PURIFIED PROTEIN ONTO DEFICIENT PARTICLES

The RNA polymerase complex of double-stranded RNA bacteriophage phi 6 is composed of four proteins, P1, P2, P4 and P7. These four proteins a re capable of performing all the functions required for the replicatio n of the double-stranded RNAs of the phi 6 genome. The polymerase comp lex containing the three genomic dsRNA segments is the core particle o f the phi 6 virion. In this study purified protein P7 was found to for m highly asymmetric dimers. Using polyclonal anti-P7 antibody, P7 was shown to be accessible on the surface of the nucleocapsid. Treatment o f nucleocapsids with polyclonal anti-P7 antibody released coat protein P8 with ensuing activation of the plus strand RNA synthesis from the resulting core particles. Purified P7 could be assembled onto particle s lacking P7 and particles lacking both P2 (RNA polymerase) and P7. In both cases RNA packaging activity was acquired. Assembly of P7 onto d eficient particles took place also in the absence of host proteins. Pr otein P7 is known to be necessary for stable packaging of the three ge nomic phi 6 plus strand RNAs into preformed polymerase complex particl es. Additionally, protein P7 seems to be involved in the regulation of plus strand synthesis (i.e. transcription) as a fidelity factor. Part icles lacking protein P7 produce anomalous size transcripts. Analysis of the polymerase complex stability revealed that proteins P2, P4 and P7 are independently associated with the major structural protein P1. The number of P7 molecules in one virion was estimated to be 60 and a location at the 5-fold symmetry position is proposed. (C) 1997 Academi c Press Limited.