Center for Shockwave Medicine and Tissue Engineering, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 123 Tai-Pei Road, Niao Sung District, Kaohsiung, Taiwan, 833.
Graduate Institute of Biotechnology, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, 40402, Taiwan.
Virol J. 2019 Apr 5;16(1):45. doi: 10.1186/s12985-019-1153-5.
VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1.
Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection.
A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm.
Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.
鸡贫血病毒（CAV）的 VP1 是一种结构蛋白，是病毒包装所必需的。VP1 蛋白存在于细胞核和细胞质中；然而，VP1 的功能核定位信号（NLS）和核输出信号（NES）仍然未知。本研究旨在使用生物信息学方法和多位点片段缺失，对 VP1 的 NLS 和 NES 基序进行特征分析，并通过与 VP1 共转染来研究 VP2 从细胞核到细胞质的穿梭。
通过 WoLF PSORT 和 NLStradamus 程序从 VP1 的氨基酸序列预测了两个假定的 NLS 基序。通过 NetNES 1.1 Server 和 ELM 服务器程序预测了 VP1 的三个 NES 基序。所有突变体均通过多位点片段缺失诱变产生。通过质粒转染在细胞中共同表达 VP1 和 VP2。
鉴定出 VP1 氨基酸残基 3 到 10（RRARRPRG）的功能性 NLS 基序。细胞内核输入中关键氨基酸 3 到 10 显著参与，并通过系统缺失突变进行了评估。通过 NetNES 1.1 Server 和 ELM 服务器程序预测了 VP1 的三个 NES 基序。鉴定出 VP1 氨基酸残基 375 到 388（ELDTNFFTLYVAQ）的功能性 NES。莱普霉素 B（LMB）处理表明 VP1 从细胞核输出到细胞质是通过染色体区域维持 1（CRM1）依赖性途径发生的。在细胞中共表达 VP1 和 VP2 时，我们观察到 VP1 可能将 VP2 从细胞核转运到细胞质。
我们的数据表明，CAV 的 VP1 含有功能性 NLS 和 NES 基序，通过 CRM1 依赖性途径调节核输入和输出。此外，VP1 可能在 VP2 从细胞核到细胞质的转运中发挥作用。
