DOUBLE-STRANDED-RNA BACTERIOPHAGE-PHI-6 PROTEIN P4 IS AN UNSPECIFIC NUCLEOSIDE TRIPHOSPHATASE ACTIVATED BY CALCIUM-IONS

Double-stranded RNA bacteriophage phi 6 has an envelope surrounding th e nucleocapsid (NC). The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase comp lex enclosing the segmented viral genome. Empty polymerase complex par ticles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphospha tes (NTPs). Photoaffinity labelling of both the NC and the procapsid h as earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacki ng surface protein P8) and purified protein P4, we demonstrate here th at multimeric P4 is the active NTPase. Isolation of multimeric P4 is s uccessful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NT Ps to the corresponding nucleoside diphosphates. The K-m of the reacti on for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis b y P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition expe riments indicate that Mg2+ and Ca2+ ions have approximately equal bind ing affinities for P4. They might compete for a common binding site. T he nucleotide specificity and enzymatic properties of the P4 NTPase ar e similar to the NTP hydrolysis reaction conditions needed to transloc ate and condense the viral positive-sense RNAs to the procapsid partic le.