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J Virol. 1980 September; 35(3): 732-740

Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.

T A Van Dyke and J B Flanegan

ABSTRACT

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.

J Virol. 1980 September; 35(3): 732-740




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