Semiconservative strand-displacement transcription of the M2 dsRNA segment of Ustilago maydis virus.

The P1 strain of the Ustilago maydis virus (UmV) is a segmented dsRNA virus with segments designated H1, H2, M1, M2, and L. Incubation of purified virus with a mixture of nucleotides containing 32P-UTP resulted in labeled dsRNA which was retained in the capsid and labeled ssRNA which was released from the capsid. This in vitro transcription reaction was dependent on Mg2+ ion and the optimum concentration for maximum incorporation was 10 mM. The pH and temperature optima were 8.0 and 30 degrees C, respectively. The ssRNA transcripts were precipitated from the supernatant solution of the reaction mixture after ultracentrifugation to separate the virus. Transcription products from supernatant solution hybridized with all five virion dsRNAs. Further studies of the M2 segment indicated that it was labeled within 2 h and the label was completely chased out in 2 h. Analysis of the labeled M2 dsRNA segment by strand-separation gel showed that only one strand (slow moving) was labeled. When both strands were tested in an in vitro translation system, only the slow-moving strand was translated to produce a 24 kDa product. Thus the M2 dsRNA segment of UmV P1 transcribes by a semiconservative strand-displacement mechanism.