In vitro replication of cowpea mosaic virus RNA: I. Isolation and properties of the membrane-bound replicase.
A fraction which contained the membrane-bound cowpea mosaic virus RNA replicase was isolated from cowpea mosaic virus-infected cowpea leaves. The replicase activity appeared on day 1 after inoculation, then increased to reach a maximal on day 4. The increase in enzyme activity preceded the most-rapid virus multiplication. The membrane-bound replicase activity was almost completely insensitive to actinomycin D and DNase. The corresponding fraction from healthy leaves had no RNA-dependent RNA polymerase activity. The viral RNA synthesis in vitro proceeded linearly for 20 min and required all four ribonucleoside triphosphates and Mg(2+) ions. Mn(2+) was a poor substitute for Mg(2+). The reaction was optimal at pH 8.2. During the whole period of RNA synthesis the in vitro synthesized RNA was at least 70% resistant against RNase in 2 x SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digestable by RNase in 0.1 x SSC. Analysis of the products by sucrose gradient centrifugation followed by treatment of separate fractions with RNase demonstrated that both single-and double-stranded RNA were present. Double-stranded RNA sedimented at about 20S, with a shoulder at 16S to 17S. A minor part of the double-stranded RNA sedimented below 10S. Single-stranded RNA sedimented with the same rate as the two viral RNAs, 26S and 34S.
D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009029:Mosaic Viruses / Q000254:growth & development / Q000378:metabolism*; D010942:Plant Viruses / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D012367:RNA, Viral / Q000096:biosynthesis*; D012260:Ribonucleases; D012265:Ribonucleotides / Q000378:metabolism; D014316:Tritium; D014500:Uracil Nucleotides / Q000378:metabolism; D014779:Virus Replication
D012367:RNA, Viral; D012265:Ribonucleotides; D014500:Uracil Nucleotides; D014316:Tritium; D003609:Dactinomycin; D008345:Manganese; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases; D012260:Ribonucleases; D008274:Magnesium