FIG. 6.

TNTase activity within φ6 nucleocapsids. Nucleocapsids were incubated with a [α-³²P]UTP donor under standard TNTase reaction conditions, and the reaction products (lanes 1 and 2 in panel A and lane 1 in panel B) were separated by either native agarose gel electrophoresis (A) or strand-separating gel electrophoresis (B). (A) The reaction products were treated with RNase III prior to gel analysis as indicated below the panel. Lane N is as defined in the legend to Fig. 1A (control for labeled positive strands). Lane R is a product of the replication reaction using purified P2 and positive strands (s⁺, m⁺, and l⁺) of the φ6 genome (control for labeled negative strands). The mobilities of the dsRNA segments (S, M, and L in panel A), the positive strands (s⁺, m⁺, and l⁺ in panels A and B), and the negative strands (s⁻, m⁻, and l⁻ in panel B) are indicated. (C) Schematic presentation of the φ6 replication cycle. (a) The viral particles partly uncoated upon entry initiate the production of positive-sense RNA segments within the cell cytoplasm. Transcription occurs via a semiconservative strand displacement mechanism (59); the newly produced positive strand stays connected with the negative strand in the dsRNA, while the positive strand produced within the previous host exits the viral particle. The viral mRNA molecules extruded from the polymerase complexes direct viral protein synthesis. (b) The newly produced proteins (including P2) assemble to form empty polymerase complexes which subsequently package one copy of each of the positive-sense RNA segments (s⁺, m⁺, and l⁺) (solid lines). (c) The packaged segments direct the synthesis of complementary negative strands (dotted lines). (d) To induce the production of additional viral proteins, the dsRNA-filled particles initiate a new cycle of transcription. Subsequently, the particles mature and the progeny virions exit the cell (44).