Cytoplasmic polyhedrosis viruses (CPVs), like other members of the order Reovirales, produce viroplasms, hubs of viral assembly that shield them from host immunity. Our study investigates the potential role of NSP9, a nucleic acid-binding non-structural protein encoded by CPVs, in viroplasm biogenesis. We determined the crystal structure of the NSP9 core (NSP9ΔC), which shows a dimeric organization topologically similar to the P9-1 homodimers of plant reoviruses. The disordered C-terminal region of NSP9 facilitates oligomerization but is dispensable for nucleic acid binding. NSP9 robustly binds to single- and double-stranded nucleic acids, regardless of RNA or DNA origin. Mutagenesis studies further confirmed that the dimeric form of NSP9 is critical for nucleic acid binding due to positively charged residues that form a tunnel during homodimerization. Gel migration assays reveal a unique nucleic acid binding pattern, with the sequential appearance of two distinct complexes dependent on protein concentration. The similar gel migration pattern shared by NSP9 and rotavirus NSP3, coupled with its structural resemblance to P9-1, hints at a potential role in translational regulation or viral genome packaging, which may be linked to viroplasm. This study advances our understanding of viroplasm biogenesis and Reovirales replication, providing insights into potential antiviral drug targets.

中文翻译：

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CPV NSP9 的晶体结构和核酸结合模式：对 Reovirales 病毒质的影响


细胞质多角体病毒 （CPV） 与 Reovirales 目的其他成员一样，产生病毒质，即病毒组装的中心，保护它们免受宿主免疫。我们的研究调查了 NSP9 的潜在作用，NSP9 是一种由 CPV 编码的核酸结合非结构蛋白，在病毒质生物发生中的作用。我们确定了 NSP9 核心 （NSP9ΔC） 的晶体结构，其显示拓扑结构与植物呼肠孤病毒的 P9-1 同源二聚体拓扑相似。NSP9 无序的 C 端区域促进了寡聚化，但对于核酸结合来说是必不可少的。NSP9 与单链和双链核酸稳健结合，与 RNA 或 DNA 来源无关。诱变研究进一步证实，NSP9 的二聚体形式对核酸结合至关重要，因为在同源二聚化过程中会形成带正电荷的残基形成隧道。凝胶迁移分析揭示了一种独特的核酸结合模式，两种不同的复合物的连续出现取决于蛋白质浓度。NSP9 和轮状病毒 NSP3 共享的凝胶迁移模式，加上其与 P9-1 的结构相似，暗示在翻译调控或病毒基因组包装中可能发挥作用，这可能与病毒质有关。这项研究促进了我们对病毒质生物发生和 Reovirales 复制的理解，为潜在的抗病毒药物靶点提供了见解。