Highly purified virulent poliovirus preparations harbour an endogenous protein kinase. Enzyme activity increases significantly upon purification of infectious virus particles from infected HeLa cells. Enzyme activity is stimulated by divalent cations. The substrate specificity and the degree of stimulation of the kinase are dependent on the nature of the divalent cations included in the assay. The preferred substrates for this kinase are the viral capsid proteins. Exogenously added proteins such as alpha-casein, phosvitin and protamine are also phosphorylated by the kinase. Moreover, these proteins enhance the phosphorylation of viral proteins. In the presence of Mg++ VP 2 and VP 0 are highly phosphorylated, while in the presence of Zn++ only VP 2 and VP 4, but not VP 0 or exogenous proteins are phosphorylated. Poliovirus associated protein kinase exhibits optimal activity at pH 7.9 in the presence of 10 mM Mg++. The Km for ATP is shown to be 40 microM. By testing different nucleotides as phosphate donors a specificity of the phosphorylation reaction for ATP is demonstrated. Phosphoamino acid analysis of hydrolysates of the substrates phosphorylated in the presence of Mg++ by thin layer electrochromatography and HPLC yielded phosphoserine and phosphothreonine from viral capsid proteins while hydrolysates of protamine yield only phosphoserine. Destabilization of the viral capsid, e.g. by preincubation at 42 degrees C for 20 minutes results in a stimulation of kinase activity. Moreover, phosphorylation of the poliovirus capsid proteins itself results in destabilization of the viral capsid. These findings suggest that phosphorylation of the viral coat proteins triggers or enhances the uncoating of poliovirus leading to the release of viral RNA.

The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VPO and VP2 as well as exogenous proteins in the presence of Mg2+. In this paper, the effect of Zn2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg2+. Phosphorylation patterns of viral and other proteins depend on the divalent cation present. In the presence of Zn2+, phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VPO and exogenous phosphate acceptor proteins is not detected. Our results indicate the activation of more than one virus-associated protein kinase by Zn2+. The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn2+. The destabilization leads to a substantially increased permeability of virus particles to ethidium bromide and RNase, concomitant with decreased infectivity of the sample. This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. High-performance liquid chromatography-purified viral protein VP2 is phosphorylated by the released enzymes on serine, threonine, and tyrosine in the presence of Zn2+. We suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus.

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