University of Nevada, Reno School of Medicine, Department of Microbiology & Immunology, Reno, NV 89557, USA.
University of Nevada, Reno, Nevada Bioinformatics Center (RRID: SCR_017802), Reno, NV 89557, USA.
Virus Res. 2024 Nov;349:199456. doi: 10.1016/j.virusres.2024.199456. Epub 2024 Sep 7.
Kaposi's sarcoma-associated herpesvirus (KSHV) relies on many cellular proteins to complete replication and generate new virions. Paraspeckle nuclear bodies consisting of core ribonucleoproteins splicing factor proline/glutamine-rich (SFPQ), Non-POU domain-containing octamer-binding protein (NONO), and paraspeckle protein component 1 (PSPC1) along with the long non-coding RNA NEAT1, form a complex that has been speculated to play an important role in viral replication. Paraspeckle bodies are multifunctional and involved in various processes including gene expression, mRNA splicing, and anti-viral defenses. To better understand the role of SFPQ during KSHV replication, we performed SFPQ immunoprecipitation followed by mass spectrometry from KSHV-infected cells. Proteomic analysis showed that during lytic reactivation, SFPQ associates with viral proteins, including ORF10, ORF59, and ORF61. These results are consistent with a previously reported ORF59 proteomics assay identifying SFPQ. To test if the association between ORF59 and SFPQ is important for replication, we first identified the region of ORF59 that associates with SFPQ using a series of 50 amino acid deletion mutants of ORF59 in the KSHV BACmid system. By performing co-immunoprecipitations, we identified the region spanning amino acids 101-150 of ORF59 as the association domain with SFPQ. Using this information, we generated a dominant negative polypeptide of ORF59 encompassing amino acids 101-150, that disrupted the association between SFPQ and full-length ORF59, and decreased virus production. Interestingly, when we tested other human herpesvirus processivity factors (EBV BMRF1, HSV-1 UL42, and HCMV UL44) by transfection of each expression plasmid followed by co-immunoprecipitation, we found a conserved association with SFPQ. These are limited studies that remain to be done in the context of infection but suggest a potential association of SFPQ with processivity factors across multiple herpesviruses.
卡波氏肉瘤相关疱疹病毒（KSHV）依赖许多细胞蛋白来完成复制并产生新的病毒粒子。由核心核糖核蛋白剪接因子脯氨酸/谷氨酰胺富含（SFPQ）、非 POUM 结构域结合八聚体结合蛋白（NONO）和核小体蛋白成分 1（PSPC1）以及长非编码 RNA NEAT1 组成的核小体，形成了一种复合物，该复合物被推测在病毒复制中发挥重要作用。核小体是多功能的，参与包括基因表达、mRNA 剪接和抗病毒防御在内的各种过程。为了更好地了解 SFPQ 在 KSHV 复制过程中的作用，我们从 KSHV 感染的细胞中进行了 SFPQ 免疫沉淀，然后进行了质谱分析。蛋白质组学分析表明，在裂解性再激活过程中，SFPQ 与病毒蛋白，包括 ORF10、ORF59 和 ORF61 结合。这些结果与先前报道的 ORF59 蛋白质组学测定鉴定 SFPQ 的结果一致。为了测试 ORF59 与 SFPQ 之间的关联是否对复制很重要，我们首先使用 KSHV BACmid 系统中的一系列 50 个氨基酸缺失突变体鉴定了与 SFPQ 结合的 ORF59 区域。通过进行共免疫沉淀，我们确定了 ORF59 跨越氨基酸 101-150 的区域作为与 SFPQ 结合的区域。利用这一信息，我们生成了一个包含氨基酸 101-150 的 ORF59 显性负性多肽，该多肽破坏了 SFPQ 与全长 ORF59 之间的关联，并减少了病毒的产生。有趣的是，当我们通过转染每种表达质粒并进行共免疫沉淀来测试其他人类疱疹病毒的连续性因子（EBV BMRF1、HSV-1 UL42 和 HCMV UL44）时，我们发现了与 SFPQ 的保守关联。这些研究有限，仍有待在感染背景下进行，但表明 SFPQ 可能与多种疱疹病毒的连续性因子有关。
