Comparison of the expression and phosphorylation of the non-structural protein NS2 of three different orbiviruses: evidence for the involvement of an ubiquitous cellular kinase.
The non-structural protein NS2 of epizootic haemorrhagic disease (EHD), bluetongue (BT) and African horsesickness (AHS) viruses has each been expressed to high levels using a baculovirus vector gene expression system. It was found that the recombinant baculovirus-expressed EHDV NS2 protein was resolved as a doublet following PAGE. Peptide mapping of these protein bands indicated that they were identical. The difference in the sizes of the NS2 protein bands could not be attributed to the phosphorylation of NS2 or other posttranslational modification such as N-glycosylation and remains obscure. The EHDV, BTV and AHSV baculovirus-expressed NS2 proteins were all phosphorylated in vitro without the addition of an exogenous kinase. An unphosphorylated form of EHDV NS2, obtained by expressing the NS2 gene as a fusion protein in Escherichia coli cells, could be phosphorylated in vitro by a protein kinase associated with the cytoplasm of insect cells. The phosphorylated version of this protein was found to be significantly less efficient in binding ssRNA, compared to the unphosphorylated version.
Theron
J
J
Department of Microbiology and Plant Pathology, University of Pretoria, South Africa.
Uitenweerde
J M
JM
Huismans
H
H
Nel
L H
LH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D018163:Orbivirus / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011072:Poly U / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D011072:Poly U; D012313:RNA; D011494:Protein Kinases