Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth
Characterization of Localization and Export Signals of Bovine Torovirus Nucleocapsid Protein Responsible for Extensive Nuclear and Nucleolar Accumulation and Their Importance for Virus Growth
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DOI：
10.1128/jvi.02111-20
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发表时间：
2021-02-01
影响因子：
5.4
通讯作者：
Taguchi，Fumihiro
中科院分区：
文献类型：
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作者：
Ujike，Makoto;Kawachi，Yukako;Taguchi，Fumihiro
虽然在历史上属于冠状病毒科，但最近将Torovirus（ToV）归类为Tobaniviridae新科。CoV的核衣壳（N）蛋白主要位于病毒复制的细胞质中，但在某些情况下，蛋白质部分位于核仁中。许多研究已经研究了CoV N蛋白的亚细胞定位和核质运输信号，但对ToV N蛋白知之甚少。在这里，我们研究了牛ToV（BToV）N蛋白（BToN）的亚细胞定位，并表征其核质运输信号。与其他CoV不同，在感染细胞中的BToN在感染早期主要被运输到核仁，但在感染晚期主要分布在核质中而不是核仁中。有趣的是，在感染期间，在细胞质中检测到少量的BToN。对与增强型绿色荧光蛋白（EGFP）融合的BToN的一组全面的取代或缺失突变体的检查揭示，精氨酸（R）残基簇包含核/核仁定位信号（NLS/NoLS），并且C-末端区域充当染色体维持1（CRM 1）-独立的核输出信号（内斯）。此外，在NLS/NoLS中具有突变但保留核积累的重组病毒被成功拯救并显示出略微降低的生长能力，而失去NLS/NoLS介导的BToN核积累的病毒没有被拯救。这些结果表明，BToN独特的积累主要是在核隔室在感染过程中，由一个富含R的NLS/NoLS和CRM 1独立的内斯，和NLS/NoLS驱动的核隔室中的BToN的积累是重要的病毒growth. IMPORTANCEToV是在许多物种，包括人类中检测到的病原体引起的脑炎。BToV已经在全球范围内传播，导致经济损失，目前没有可用的治疗方法或疫苗。包括ToV在内的正链RNA病毒在细胞质中复制，其结构蛋白通常在细胞质中积累。有趣的是，BToN在所有感染过程中主要积累在细胞核/核仁中，尽管是主要的结构蛋白，但只有一小部分积累在细胞质中。此外，我们确定了独特的核质运输信号，并证明了NLS/NoLS对病毒生长的重要性。本研究首次对BToN的亚细胞定位和细胞内运输信号进行了深入研究。我们的研究结果还表明，NLS/NoLS介导的BToN核积累对病毒复制很重要。了解BToV的独特功能，不仅可以为ToV的组装机制提供新的见解，还可以为其他正链RNA病毒提供新的见解。
Torovirus(ToV) has recently been classified into the new familyTobaniviridae, although historically, it belonged to theCoronavirus(CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with enhanced green fluorescent protein (EGFP) revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth.IMPORTANCEToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.