Phylogenetic Studies of the United States Bluetongue Viruses and Characterization of the Viral VP4 Protein

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4. The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region of the M1 gene, which encodes the VP4 protein, possesses an open reading frame with an initiation codon (ATG) at nucleotides #9-11 and a stop codon (TAA) at nucleotides #1941-1943. This open reading frame encodes a protein of 644 amino acid residues with a predicted molecular weight of about 75 kDa. A potential leucine zipper motif was detected near the carboxyl terminus of the deduced VP4 amino acid sequence. The phylogenetic analysis of bluetongue viruses using the sequences of these five cognate M1 genes is consistent with the results of previous phylogenetic studies. Serotypes-10, -11, -13, and -17 are closely related and serotype-2 is the most distantly related among the five US BTV serotypes. Heterologously expressed bluetongue virus VP4 protein was purified to near homogeneity. Six linear epitopes of VP4 were mapped at both termini and in the middle of the protein. By using enzyme-linked immunosorbent assay and peptide competition assay, six linear epitopes were found to be surface accessible. The VP4 protein was shown to be an oligomer by chemical cross-linking. VP4 protein was identified as a ssRNA-binding protein. The VP4 protein has binding activity towards both capped and non-capped ssRNA. RNA-binding activity was not specific to BTV ssRNA. A leucine-zipper motif of VP4 is not required for RNA-binding activity. One RNA-binding domain was mapped between amino acid residues #112-158 by a Northwestern assay and by deletion mutant analysis. Using sequence-specific synthetic peptides corresponding to VP4 in the arginine-and lysine-rich regions, four potential ssRNA-binding domains of VP4 protein were mapped