Isolation of a soluble and template-dependent foot-and-mouth disease virus RNA polymerase

A large-scale purification method for the soluble RNA-dependent RNA polymerase from foot-and-mouth disease virus (FMDV)-infected BHK 21 cells is described. A cytoplasmic extract made 2.75 hr postinfection (fraction 1b) was chromatographed on DEAE-cellulose (fraction 2), poly(U)-Sepharose (fraction 3a, b) and sized on Ultrogel AcA 44 (fraction 4). Fraction 3a, b contained the viral protein P56 (the VIA antigen) plus small amounts of its precursor P72. The enzyme is capable of transcribing the synthetic template poly(A) primed with (Up) 5 U by a poly(U) polymerase activity. Furthermore, fraction 3a, b transcribed FMDV RNA with an RNA polymerase activity which did not require oligoribouridylate priming. These chromatographic procedures separated P72 from P56. P72 alone appeared to be transcriptionally inactive in vitro. Gel filtration of fraction 3a resulted in a high yield of pure P56. This fractionation step led to a considerable loss of transcribing activity.