The mechanism of replication: a novel polarity reversal in the in vitro synthesis of Q-beta-RNA and its complement.

The synthesis of biologically competent viral RNA has been demonstrated1-3 with a purified Qf3-replicase4 (an RNA-dependent RNA polymerase induced by the RNA bacteriophage Qt3). A kinetic analysis of the reaction was carried out using sucrose gradients' and electrophoretic separation in acrylamide gels.6-8 Two classes of complexes containing both initiating templates and early product were shown9' 10 to materialize prior to the formation of new infectious strands. The first complexes (HS) to appear correspond to the structures found in vivo and Hofschneider, 1 and one minute later a second class (FS) is synthesized re-sembling the complexes found by Franklin12 in cells infected with R-17. The temporal order of their appearance with respect to each other and the final product is consistent with a mechanism of synthesis which involves the following sequence of events: 5 to 10 volumes of methanol. The precipitate was collected by centrifugation and resuspended in 1 ml of water. The sodium salt of the triphosphate was then obtained by Dowex 50 (H+) treatment and neutralization with NaOH. In order to further purify the triphosphate from resid- ual traces of diphosphate, tetraphosphate, and pyrophosphate the solution was concentrated to 0.2 ml and subjected to paper electrophoresis in 0.1 M ammonium acetate, pH 3.8, at 5 kv and 50 ma for 90 min on Whatman 52 paper. The position of the triphosphate was identified by radioautography and its ultraviolet (UV) absorption. The triphosphate was eluted with water at 40C, paper flock removed by centrifugation, and the triphosphate again purified by barium precipitation and finally converted to the sodium salt. Triphosphates prepared in this manner were free, as determined by paper electrophoresis, from nucleotide mono-, di-, and tetraphosphates as well as pyrophosphates, or P32-orthophosphate. lower concentra- tion of 6,,y-labeled triphosphate to conserve material. Unlabeled Qft RNA was used as a template at 2 psg to minimize reincorporation of the product viral RNA. Reaction mixtures were incubated at 380 and terminated by addition of sodium dodecyl sulfate (0.2% final concentra-tion). Contamination with radioactive substrates was reduced in some experiments by passage through Sephadex G-25 as described by Haruna and Spiegelman.Y0 Fractions containing RNA were pooled and concentrated by placing in a dialysis bag and immersing in dry Sephadex G-100 until a volume of 0.2 ml was attained. Each RNA sample was mixed with sucrose crystals and loaded on a polyacrylamide gel for electrophoresis and subsequent counting of gel slices as de- tailed previously.10