Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis, we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL), from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleoli of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleoli of transfected cells. Multiple-point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed; several NoLS-mutated LEF5 proteins were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production, and occlusion body production.IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleoli of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

中文翻译：

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加利福尼亚州卷柏多核多角体病毒LEF5的核仁定位信号的鉴定和表征。

加利福尼亚州产的Autographa californica多核多角体病毒（AcMNPV）晚期表达因子5（LEF5）在所有测序的杆状病毒基因组中均高度保守，并在感染性病毒后代的产生中发挥重要作用。在这项研究中，AcMNPV LEF5的核仁定位被表征。通过转录组分析，我们从Sf9细胞中鉴定出两种推定的核仁蛋白，即贪夜蛾（Spodoptera frugiperda nucleostemin）（SfNS）和原纤维蛋白（SfFBL）。免疫荧光分析表明，SfNS和SfFBL位于核仁中。AcMNPV感染导致受感染细胞的核仁重组。LEF5和SfNS的共定位显示AcMNPV LEF5定位于Sf9细胞中的核仁。生物信息学分析表明，LEF5的碱性氨基酸在第184至213位残基富集，可能含有核仁定位信号（NoLS）。绿色荧光蛋白（GFP）融合到AcMNPV LEF5的NoLS上，位于转染细胞的核仁中。多点突变分析表明，氨基酸残基197至204对于LEF5的核仁定位很重要。为了鉴定AcMNPV LEF5中的NoLS是否对病毒后代的产生很重要，构建了一个lef5空的AcMNPV杆粒。使用GFP报告基因，将几种NoLS突变的LEF5蛋白重新插入到lef5-null AcMNPV杆粒中。在AcMNPV LEF5的185至189或197至204残基处含有点突变的构建体导致感染杆状病毒的细胞中感染性病毒后代的产生减少和闭塞体产量降低。一起，这些数据表明AcMNPV LEF5含有NoLS，这对于LEF5的核仁定位，子代产生和闭塞体产生很重要。重要信息许多病毒，包括人和植物病毒，都将核仁功能作为其感染策略的一部分。然而，杆状病毒蛋白的核仁定位尚未被表征。在这项研究中，在Sf9细胞中鉴定了两个核仁蛋白SfNS和SfFBL。我们的研究结果表明，加州致癌的Autographa californica多核多角体病毒（AcMNPV）感染导致受感染细胞核仁的重新分布。我们证明了AcMNPV晚期表达因子5（LEF5）可以定位于核仁并包含核仁定位信号（NoLS），