Characterization of the nucleic acid binding activity of inner core protein VP6 of African horse sickness virus.
Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.
Department of Genetics, University of Pretoria, Pretoria, South Africa.
P J
PJ
de Waal
H
H
Huismans
D000356:African Horse Sickness Virus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001820:Bluetongue virus / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D015678:Viral Structural Proteins