Properties of poliovirus associated protein kinase

Abstract

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.

Poliovirus-associated protein kinase: destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+

“…We showed previously that preincubation of poliovirus at this temperature in the Mg2+-dependent phosphorylation reaction enhances the phosphorylation of viral and exogenous proteins significantly (18).…”

“…In all experiments, Mahoney type I poliovirus was used. HeLa cells were grown and virus was prepared as previously described (25) with modifications (18). Mahoney type I poliovirus was also obtained from green monkey kidney (GMK) cells.…”

“…Infected cells were incubated for 6 h at 37°C under 5% CO, and the cultures were harvested by scraping the cells of the dishes with a rubber policeman. Poliovirus was purified as previously described (18,25).…”

“…It was shown in previous studies that protein kinase activity is associated with highly purified poliovirus particles (18,25). Viral capsid proteins are the preferred substrates for virus-associated kinase.…”

“…These findings indicate a destabilization of the virus particle during the phosphorylation reaction. For this reason, it was suggested that phosphorylation of viral capsid proteins plays a role in the uncoating of poliovirus (18).…”