Huang Pi H, Li Ying J, Su Yu P, Lee Long H, Liu Hung J
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Virology. 2005 Feb 20;332(2):584-95. doi: 10.1016/j.virol.2004.12.005.
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.
我们之前已经表明，禽呼肠孤病毒（ARV）的σA和σNS蛋白具有双链RNA和单链RNA结合活性，并提出σA上有两个表位（I和II），σNS上有三个表位（A、B和C）。为了进一步确定σA和σNS蛋白上表位的位置，并通过使用针对ARV S1133株的单克隆抗体（MAb）62、1F9、H1E1和4A123进一步阐明这些表位的生物学功能，通过聚合酶链反应（PCR）产生的ARV S2和S4基因的全长和缺失片段被克隆到pET32表达载体中，融合蛋白在大肠杆菌BL21株中过表达。使用MAb以及ARV S1133株的σA和σNS的大肠杆菌表达缺失片段、合成肽进行表位作图，以及MAb与异源ARV株的交叉反应表明，σA上的表位II位于氨基酸残基340QWVMAGLVSAA350处，σNS上的表位B位于氨基酸残基180MLDMVDGRP188处。针对表位II和B的MAb（62、1F9和H1E1）不需要σA和σNS的天然构象，这表明它们的结合活性不依赖于构象。另一方面，MAb 4A123在蛋白质印迹中仅与完整的σNS反应，而不与截短的σNS融合蛋白反应，这表明MAb与σNS上表位A的结合活性依赖于构象。氨基酸序列分析以及MAb 62与异源ARV株的结合试验表明，σA上的表位II在ARV株中高度保守，并且该表位适合作为检测鸡自然感染后ARV抗体的血清学标志物。相反，ARV表位B中第183位（M到V）的氨基酸取代可能会阻碍σNS与MAb 1F9的反应性。具有单链RNA结合活性的ARV的σNS可被单克隆抗体1F9阻断。σNS上的表位B是单链RNA结合所必需的，因为其缺失完全消除了σNS的单链RNA结合活性。
