Beak and feather disease virus (BFDV) is a single-stranded circular DNA icosahedral virus that belongs to the Circoviridae family. This virus is the causative pathogen of beak and feather disease, which leads to feather loss, malformed claws, and immunosuppression of psittacine birds. Our study produced BFDV virus-like particles (VLPs) including capsid proteins, mutant Cap proteins (Cap ΔNLS₅₄, Cap ΔNLS₆₂, Cap C₂₂₈S, and Cap ΔNES) and chimeric Cap proteins carrying the epitope (amino acid residues 64–70) of the replication-associated protein (R-Cap, Cap-R, R-Cap ΔNLS₅₄, and Cap ΔNLS₅₄-R). All of the aforementioned VLPs were observed via transmission electron microscopy and verified through immunogold labeling. The nuclear localization sequence (NLS) of the Cap protein was identified between amino acid residues 55–62. Nuclear export of the Cap protein depended on the nuclear export sequence (NES). All VLPs except Cap ΔNLS₆₂ and Cap ΔNES entered the cells 2 h post-infection (hpi) and were shuttled into the nucleus at 8 hpi. Wheat germ agglutinin (WGA) blocked the nuclear entry of Cap proteins at 8 hpi and the nuclear export of Cap proteins at 16 hpi was inhibited by leptomycin B. The nuclear entry of Cap protein was inhibited by importin α and importin β inhibitors, as well as NLS peptides. Moreover, the interactions of Cap proteins and Cap VLPs with both importin α and importin β were characterized via the GST pull-down and immunofluorescence assays. These interactions were blocked by the presence of importin α and importin β inhibitors, as well as NLS peptides. Therefore, our study is the first to describe the precise position of the NLS of the BFDV Cap protein and the interaction of Cap protein with importin α and importin β in vitro.

喙羽病病毒 (BFDV) 是一种单链环状 DNA 二十面体病毒，属于圆环病毒科。这种病毒是喙和羽毛病的病原体，可导致鹦鹉羽毛脱落、爪子畸形和免疫抑制。我们的研究产生了 BFDV 病毒样颗粒 (VLP)，包括衣壳蛋白、突变 Cap 蛋白（Cap ΔNLS ₅₄、Cap ΔNLS ₆₂、Cap C ₂₂₈ S 和 Cap ΔNES）和带有表位的嵌合 Cap 蛋白（氨基酸残基 64-70 ) 的复制相关蛋白（R-Cap、Cap-R、R-Cap ΔNLS ₅₄和 Cap ΔNLS ₅₄-R)。所有上述 VLP 均通过透射电子显微镜观察并通过免疫金标记进行验证。Cap 蛋白的核定位序列 (NLS) 在氨基酸残基 55-62 之间被鉴定。Cap 蛋白的核输出取决于核输出序列 (NES)。除 Cap ΔNLS ₆₂外的所有 VLPCap ΔNES 在感染后 2 小时 (hpi) 进入细胞，并在 8 hpi 穿梭到细胞核中。小麦胚芽凝集素 (WGA) 在 8 hpi 阻止 Cap 蛋白的核进入，并且在 16 hpi 时 Cap 蛋白的核输出被细霉素 B 抑制。 Cap 蛋白的核进入被 importin α 和 importin β 抑制剂抑制作为 NLS 肽。此外，通过 GST 下拉和免疫荧光分析表征了 Cap 蛋白和 Cap VLP 与输入蛋白 α 和输入蛋白 β 的相互作用。这些相互作用被 importin α 和 importin β 抑制剂以及 NLS 肽的存在阻断。因此，我们的研究首次在体外描述了BFDV Cap蛋白NLS的精确位置以及Cap蛋白与importin α和importin β的相互作用。.