ABSTRACT

Entry of circovirus into the host cell nucleus is essential for viral replication during the early stage of infection. However, the mechanisms by which nucleolar shuttle proteins are used during viral replication is still not well understood. Here, we report a previously unidentified nucleolar localization signal in circovirus capsid protein (Cap), and that circovirus hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate its replication. Colocalization analysis showed that NPM1 translocates from the nucleolus to the nucleoplasm and cytoplasm during viral infection. Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that Cap interacts directly with NPM1. Binding domain mapping showed that the arginine-rich N-terminal motif ¹MTYPRRRYRRRRHRPRSHLG²⁰ of Cap, and residue serine-48 of the N-terminal oligomerization domain of NPM1, are essential for the interaction. Virus rescue experiments showed that all arginine to alanine substitution in the N-terminal arginine-rich motif of Cap resulted in diminished viral replication. Knockdown of NPM1 and substitution of serine-48 in NPM1 to glutamic acid also decreased viral replication. In addition, binding assays showed that the arginine-rich motif of Cap is a nucleolar localization signal. Taken together, our findings demonstrate that circovirus protein Cap is a nucleolus-located, and regulates viral replication by directly binding to NPM1.

摘要

在感染的早期阶段，圆环病毒进入宿主细胞核对于病毒复制至关重要。然而，在病毒复制过程中使用核仁穿梭蛋白的机制仍然不很清楚。在这里，我们报告圆环病毒衣壳蛋白（Cap）中以前未鉴定的核仁定位信号，并且该圆环病毒劫持了核仁磷蛋白nucleophosmin-1（NPM1）以促进其复制。共定位分析表明，NPM1在病毒感染过程中从核仁转移到核质和细胞质。免疫共沉淀和谷胱甘肽S-转移酶下拉实验表明Cap与NPM1直接相互作用。绑定域映射显示，富含精氨酸的N端基序¹ MTYPRRR ÿ RRRR ħ - [R P - [R SHLG ²⁰帽的和残基的丝氨酸-48 NPM1的N-末端寡聚化结构域的，是用于相互作用所必需。病毒抢救实验显示，Cap的N端富含精氨酸的所有精氨酸替换为丙氨酸，导致病毒复制减少。击倒NPM1和将NPM1中的丝氨酸48替换为谷氨酸也降低了病毒复制。另外，结合测定显示Cap的富含精氨酸的基序是核仁定位信号。两者合计，我们的发现表明，圆环病毒蛋白Cap位于核仁，并通过直接结合NPM1调节病毒复制。