Chen Jui-Kai, Hsiao Chiaolong, Wu Jian-Shin, Lin Shin-Yi, Wang Chi-Young
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, 40227, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan.
Arch Virol. 2019 Aug;164(8):2091-2106. doi: 10.1007/s00705-019-04292-z. Epub 2019 May 29.
Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg and Mn. Rep proteins containing mutation in the ATPase/GTPase motifs and the FTLNN, KKRLS, YCSK, and GKS motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.
喙羽病病毒（BFDV）属于圆环病毒科。有人提出BFDV采用基于复制相关蛋白（Rep）的滚环复制策略。BFDV的Rep基因被表达并纯化，结果显示它能切割含有圆环病毒复制起点中保守九核苷酸序列的短寡核苷酸。这种内切酶活性在二价金属离子镁和锰存在时最为高效。在ATP酶/GTP酶基序以及FTLNN、KKRLS、YCSK和GKS基序中含有突变的Rep蛋白缺乏内切酶活性。除N - 乙基马来酰亚胺（NEM）外，ATP酶抑制剂和GTP酶抑制剂均不影响内切酶活性，但用内切酶抑制剂L - 742001处理会使其活性降低。通过定点诱变以及缺失ATP酶/GTP酶和内切酶基序，ATP酶和GTP酶活性均降低。Rep蛋白能够结合包含BFDV复制起点茎环结构的P36双链DNA片段（dsP36）。所有Rep突变蛋白与该片段的结合能力均降低，这表明所有的ATP酶/GTP酶和内切酶基序都参与了结合过程。除NEM外，所有ATP酶、GTP酶和内切酶抑制剂均抑制Rep蛋白与dsP36片段的结合。这是首篇描述BFDV的Rep蛋白内切酶活性的报告。
