Schmitz A, Tobler K, Suter M, Ackermann M
Institute of Virology, University of Zurich, Switzerland.
Adv Exp Med Biol. 1998;440:775-80. doi: 10.1007/978-1-4615-5331-1_100.
Wild type (wt) and cell culture adapted (ca) strains of the coronavirus PEDV differ in their ability to cause diarrhea in neonate piglets: the wt strains are virulent; the ca strains are attenuated. Comparison of the available nucleotide sequences obtained from the different viral isolates revealed almost complete sequence identity with the exception of variations and truncations in open reading frame 3 (ORF3) observed exclusively in ca-PEDV isolates. In order to study the biological function(s) of the putative ORF3 product, the molecule was expressed as a heterodimeric fusion protein in E. coli. ORF3 was fused in frame to the alkaline phosphatase gene. Simultaneously, the construct was designed to form specific heterodimers by inclusion of the well known leucine zipper motiv of Jun and Fos. The heterodimerization partner contained the E. coli heat-labile enterotoxin subunit B (LTB) to allow specific binding to the eukaryotic cell receptor GM1. Our results indicate that heterodimeric fusion protein containing a truncated form of ORF3 was produced in high amounts, carried the expected ORF3 epitope, showed phosphatase activity, and was able to bind to the GM1 receptor. In contrast, a fusion protein containing the entire sequence of the ORF3 product was produced in minute amounts, indicating that it may have biological activity in prokaryotes, which led to the reduction of the amounts of proteins expressed.
冠状病毒猪流行性腹泻病毒（PEDV）的野生型（wt）毒株和细胞培养适应型（ca）毒株在引起新生仔猪腹泻的能力上存在差异：wt毒株具有毒性；ca毒株则是减毒株。对从不同病毒分离株获得的现有核苷酸序列进行比较后发现，除了在ca - PEDV分离株中仅观察到的开放阅读框3（ORF3）中的变异和截短外，它们几乎具有完全的序列同一性。为了研究假定的ORF3产物的生物学功能，该分子在大肠杆菌中作为异源二聚体融合蛋白表达。ORF3与碱性磷酸酶基因读框融合。同时，通过包含众所周知的Jun和Fos的亮氨酸拉链基序，将构建体设计成形成特异性异源二聚体。异源二聚化伴侣包含大肠杆菌不耐热肠毒素亚基B（LTB），以允许与真核细胞受体GM1特异性结合。我们的结果表明，含有截短形式ORF3的异源二聚体融合蛋白大量产生，携带预期的ORF3表位，具有磷酸酶活性，并且能够与GM1受体结合。相比之下，含有ORF3产物完整序列的融合蛋白产生量极少，这表明它在原核生物中可能具有生物学活性，从而导致表达的蛋白量减少。
