In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification

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Abstract
A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated from tobacco mosaic virus-infected tobacco [Nicotiana tabacum] callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. Viral RNA synthesis in vitro proceeded linearly for 30 min and required the 4 nucleotide triphosphates and Mg2+. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate) but completely digested by RNase in 0.1X SSC. Analysis of the product by polyacrylamide gel electrophoresis revealed a double-stranded RNA (4.0 .times. 106 daltons) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized the enzyme. The solubilized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.