Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

We have previously shown that avian reovirus (ARV) sigmaA and sigmaNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on sigmaA (I and II) and three epitopes (A, B, and C) on sigmaNS. To further define the location of epitopes on sigmaA and sigmaNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of sigmaA and sigmaNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on sigmaA was located at amino acid residues 340QWVMAGLVSAA350 and epitope B on sigmaNS at amino acid residues 180MLDMVDGRP188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of sigmaA and sigmaNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete sigmaNS but not with truncated sigmaNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on sigmaNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on sigmaA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the sigmaNS with MAb 1F9. The sigmaNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on sigmaNS is required for ssRNA binding because its deletion fully abolished the ssRNA binding activity of sigmaNS.