The characterization of inner core protein VP6 of African horsesickness virus

Abstract

VP6 is one of the minor structural core proteins of African horsesickness virus. The minor core proteins VP1, VP4 and VP6 are presumed to constitute the dsRNA dependent RNA polymerase transcription complex of the virus. In the Orbivirus prototype bluetongue virus (BTV), VP6 has a helicase activity. The aim of this investigation was to characterize the primary structure and nucleic acid binding function of the inner core protein VP6 of African horsesickness virus (AHSV). To characterize the primary structure of AHSV VP6, VP6 genes of serotypes 3 and 6 were cloned and sequenced. Both genes encode a 369 amino acid polypeptide. A comparison to the VP6 proteins of other Orbiviruses indicated that in all cases the proteins are rich in basic residues and in glycine. The proteins are highly conserved within serogroups but the conservation between serogroups is low. VP6 of AHSV-3 and AHSV-6 have 93.5% identity and 96% similarity in amino acid residues. AHSV-6 VP6 has 27% identical and 46% similar amino acid residues to BTV-10 VP6. Phylogenetic analysis of four orbivirus VP6 genes indicated that AHSV and BTV are most closely related to each other. Motifs characteristic of known helicases were identified by sequence analysis. Glycine rich protein motifs and a N-glycosylation signal were present. No nucleic acid binding motifs identified in other proteins were found in AHSV VP6. To characterize the VP6 protein of AHSV VP6, the genes were expressed using both a baculovirus and a bacterial expression system. Proteins were found to be soluble and the VP6 expressed in insect cells was found to be N-glycosylated. The nucleic acid binding function of AHSV VP6 was investigated. Bacterially expressed VP6 was demonstrated to bind nucleic acids by electrophoretic mobility shift assays. Baculovirus expressed VP6 bound double and single-stranded RNA and DNA in nucleic acid overlay protein blot assays. Competition assays indicated that VP6 may have a preference for binding to RNA rather than DNA. Glycosylation was found to play no direct role in nucleic acid binding but the binding is strongly dependent on the NaCl concentration. A series of truncated VP6 peptides were produced to investigate the importance of localized regions in nucleic acid binding. Two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. Binding appeared to be influenced by charge as reflected by the isoelectric points (pI) of the peptides and experiments indicating the effect of pH on the binding activity. However, only peptides containing amino acid residues 190 to 289 showed binding activity. This region corresponded to the region on BTV VP6 that contains two binding domains. It is proposed that the dsRNA binding domain in AHSV VP6 is a sequence of positively charged amino acids constituting a domain that determines the nucleic acid binding characteristics of the peptide. The mechanism of binding of baculovirus expressed VP6 in a nucleic acid overlay protein blot is proposed to be charge related.