IN-VITRO TRANSCRIPTION OF THE DOUBLE-STRANDED-RNA BACTERIOPHAGE-PHI-6IS INFLUENCED BY PURINE NTPS AND CALCIUM

The double-stranded RNA bacteriophage phi 6 contains a virion-associat ed RNA-dependent RNA polymerase complex. Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase comp lex, capable of synthesizing RNA strands of positive polarity. The in vitro plus strand synthesis (transcription) reaction of the particle o btained from the mature virion was optimized and its activation and in activation were investigated. Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, acti vated plus strand synthesis. GTP was the preferred NTP, but dGTP, ddGT P, and the noncleavable analog GMP-PCP could also switch on transcript ion. This NTP-binding site is probably different from that of the unsp ecific viral NTPase found in protein P4 and also from that of the rNTP -specific RNA polymerase active site. Binding of purine NTPs was suffi cient for the switch-on; hydrolysis of the NTP was not required. Besid es nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis. Magnesium ions are required for the activity but cal cium ions inhibit the reaction. Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exc eptionally high level plus strand synthesis. (C) 1995 Aacademic Press, Inc.