Characterization of a nonstructural phosphoprotein of two orbiviruses

A phosphorylated nonstructural protein, NS2, was detected in bluetongue and African horsesickness virus (BTV and AHSV) infected-radiolabeled-cell lysates by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE). The NS2 proteins of both viruses have similar migration on one-dimensional (1D) 10% SDS-PAGE. Examination of infected cell lysates on two-dimensional (2D) gels (isoelectric focusing followed by SDS-PAGE) separated two phosphorylated isoelectric forms of BTV NS2 and four phosphorylated forms of AHSV NS2. The isoelectric points of both species of BTV NS2 were acidic relative to all forms of AHSV NS2. Nonphosphorylated NS2 polypeptides were not detected by 2D gels. A nonphosphorylated host protein, which comigrated with NS2 on 1D gels, could be distinguished from viral proteins by isoelectric focusing on 2D gels. High performance liquid chromatography (HPLC) elution profiles of NS2 tryptic peptides from the two orbiviruses were compared. Three 32P-labeled tryptic peptides were generated from both AHSV and BTV NS2 proteins, which had been isolated and eluted from SDS-polyacrylamide gels. The elution profile from reverse phase HPLC was very similar for the tryptic phosphopeptides; in contrast, 35S-labeled tryptic peptides displayed considerable differences in elution profiles for the two NS2 proteins. Phosphoamino acid analysis revealed only phosphoserine in hydrolysates of BTV and AHSV NS2.