From the Key Laboratory of Animal Vaccine and Protein Engineering and.
Laboratory of Functional Proteomics (LFP) and Research Center of Reverse Vaccinology (RCRV), College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China.
J Biol Chem. 2018 Sep 28;293(39):15221-15232. doi: 10.1074/jbc.RA118.004823. Epub 2018 Aug 14.
Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 μm Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 μm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions.
猪圆环病毒 2 型（PCV2）是最小的、无包膜的单链 DNA 病毒之一。PCV2 衣壳蛋白（Cap）是唯一的病毒结构蛋白和主要抗原决定簇。先前的序列分析表明，PCV2 Cap 的 N 端含有富含正电荷残基的核定位信号（NLS）。在这里，我们报告 PCV2 的 NLS 可以作为细胞穿透肽（CPP）发挥作用。我们观察到，当该 NLS 与 NLS 融合时，可以携带大分子、增强型 GFP（EGFP）进入细胞，表明它可以像源自 HIV-1 转录激活蛋白（HIV TAT）的经典 CPP 一样发挥作用。我们还发现 NLS 的前 17 个残基（NLS-A）在细胞摄取中起关键作用。除了通过多种内吞作用进入细胞外，NLS-A 还可以通过增加的膜通透性进行快速的直接转位而被内化，并且当其在细胞培养物中的浓度≥10μm 时，均匀分布在细胞中。值得注意的是，细胞内的 NLS-A 摄取效率比 HIV TAT 高约 10 倍。我们推断，PCV2 Cap 的外翻 NLS 可能在局部膜区域积聚到高浓度（≥10μm），增加膜通透性以促进病毒进入细胞，从而将其基因组释放到病毒 DNA 复制中心。我们得出结论，NLS-A 具有作为将外源分子穿梭进入细胞的通用载体的潜力，包括用于治疗干预的药物。
