Purification and Polypeptide Composition of Semliki Forest Virus RNA Polymerase

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Abstract
A purification method for Semliki Forest virus-specified RNA-dependent RNA polymerase from BHK [baby hamster kidney] cells is described. The procedure entails the preparation of a crude cell lysate by Dounce homogenization of cells 3.5 h post-infection, differential centrifugation to give a 15,000 g ''mitochondrial'' pellet, equilibrium centrifugation on discontinuous sucrose gradients (Friedman et al. 1972) to give a membranous band of density 1.16 g/ml, solubilization with Triton N-101 and velocity centrifugation to give a 25 S solubilized polymerase complex, and affinity chromatography through an oligo (dT)-cellulose matrix bearing immobilized 42 S virus particle RNA. The overall purification was approximately 360-fold with a 5% recovery of activity. Of the various intermediate fractions in the purification procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNA found in infected cells. Other fractions incorporated precursor only into replicative intermediate (RI) or replicative form (RF). Analysis of the product RF showed that it was of the same size and could bind to the same extent to oligo (dT)-cellulose as the RF isolated directly from lysates of infected cells. Displacement hybridization and RNase digestion suggested that the purified polymerase initiated progeny positive strands using negative strands as template and, even in its most highly purified form, was still tightly bound to its template. Analysis on polyacrylamide slab gels revealed the presence of 3 35S-labeled polypeptides in the purified polymerase preparation, but a polypeptide which had identical electrophoretic mobility to the lowest MW polypeptide of the purified polymerase was also present in material from mock-infected cells which were taken through the purification procedure. Only 2 virus-specified polypeptides are apparently present in the polymerase.

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