The Protein Covalently Linked to the 5' End of Poliovirus RNA

Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labelled with P) in 0.5% sodium dodecyl sulfate 32 or 6 H guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5 end of poliovirion RMA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5-terminal uridine. After acid hydrolysis of [PH) tyrosine-labelled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labelled with either 32p or [PH] tyrosine yields tyrosine-phosphate. There appears to be only 1 tyrosine residue per VPg molecule. VTg can be recovered from poliovirus RIMA chains varying in length from 7,500 nucleotides (full-sized RNA) to about 500 nucleotides. No other type of 5 terminus can be demonstrated on nascent RNA, and the yield of VPg is consistent with one molecule of the protein on each nascent chain. These results are consistent with the concept that the protein is added to the 5' end of the growing RNA chains at a very early stage, possibly a a primer of RNA synthesis.

The 5' terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of ++ HeLa cells and is activated by ig. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that removal of VPg from poliovirus RNA by a cellular enzyme (unlinking enzyme) is a normal event during polio infection. Specific hypotheses for the roles of VPg and unlinking enzyme in infected cells are discussed. The substrate specificity of this enzyme has been investigated in vitro. VPg-pU or VPg-pUp is resistant to cleavage under conditions where either VPg or the protease-K-resistant RNA-bound oligopeptide fragment of VPg is removed from full length poliovirus RNA. Possible roles for unlinking enzyme in uninfected cells are discussed.