Foot-and-mouth Disease Virus Ribonucleic Acid

-The foot-and-mouth disease virus-RNA polymerase complex was released from membrane particulates present in the cytoplasm of infected baby hamster kidney cells. The soluble polymerase complex was fractionated by zonal centrifugation in sucrose gradients. Two polymerase complexes (RNA and protein complex) active in the cell-free system were isolated and had S-rate ranges of 20-70S and 100-300S, respectively. The light polymerase complex contained 20S double-stranded RNA; and the heavy polymerase complex contained a polydisperse, partially RNase-resistant RNA. The cell-free product of these two polymerase complexes was analyzed by zonal centrifugation in sucrose gradients. The light polymerase complex synthesized only 20S doublestranded RNA. The product of the heavy polymerase complex contained no detectable 20S double-stranded RNA and only a peak of single-stranded RNA with S-rate corresponding to 37S viral RNA. A third polymerase complex was isolated with S-rate greater than 300S, and it contained a polydisperse, partially RNase-resistant RNA. This third polymerase complex synthesized both 37S viral RNA and 20S double-stranded RNA in the cell-free system, and it is probably the native polymerase complex still bound to cellular particulates. Introduction.-Studies by Eikhom et al.1 with coliphage Q13 RNA-dependent RNA polymerase have shown that it contains two components, both of which are required for replication of the parent, single-stranded RNA. However, the role of these two components in the RNA replication mechanism has not been elucidated. Work with temperature-sensitive mutants by Lodish and Zinder2 provides strong evidence that two enzymes are required to replicate F2 bacteriophage RNA. Progress in animal virus-RNA replication has been hampered because of membrane components and excessive levels of nucleases. We have partially overcome these difficulties3'5 and have approached the study of the mechanism of foot-and-mouth disease virus (FMDV) RNA synthesis by analysis and purification of the active RNA-enzyme complex. Analysis of the FMDV-polymerase complex by centrifugation gave three RNAenzyme components: a light component which synthesized 20S doublestranded RNA; a heavy component which synthesized 37S single-stranded viral RNA; and a third component, designated aggregate polymerase component, which synthesized both single-stranded and double-stranded RNA. The latter may represent the native FMDV-RNA replication unit. Materials and Methods.-Nucleoside triphosphates and H3-NTP were purchased from Schwarz BioResearch, Inc.; Tris, phosphoenolpyruvic acid (PEP), and PEP kinase from