Institute of Pathogenic Microorganism, Jiangxi Agricultural University, Nanchang 330000, China.
Nanchang City Key Laboratory of Animal Virus and Genetic Engineering, Nanchang 330000, China.
Int J Mol Sci. 2024 Aug 8;25(16):8678. doi: 10.3390/ijms25168678.
Monkeypox virus (MPXV) is a cross-kingdom pathogen infecting both humans and wildlife, which poses a significant health risk to the public. Although MPXV attracts broad attention, there is a lack of adequate studies to elucidate pathogenic mechanisms associated with viral infections. In this study, a high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to explore the transcriptional and metabolic responses of MPXV A23 protein to HEK293T cells. The protein-protein interactions and signaling pathways were conducted by GO and KEGG analyses. The localization of A23 protein in HEK293T cells was detected by immunofluorescence. A total of 648 differentially expressed genes (DEGs) were identified in cells by RNA-Seq, including 314 upregulated genes and 334 downregulated genes. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected 115 cellular proteins that interact with the A23 proteins. Transcriptomic sequencing analysis revealed that transfection of MPXV A23 protein modulated genes primarily associated with cellular apoptosis and DNA damage repair. Proteomic analysis indicated that this protein primarily interacted with host ribosomal proteins and histones. Following the identification of the nuclear localization sequence RKKR within the A23 protein, a truncated mutant A23 was constructed to investigate the subcellular localization of A23 protein. The wild-type A23 protein exhibits a significantly higher nuclear-to-cytoplasmic ratio, exceeding 1.5, in contrast to the mutant A23, which has a ratio of approximately 1. Immunofluorescence assays showed that the A23 protein was mainly localized in the nucleus. The integration of transcriptomics and proteomics analysis provides a comprehensive understanding of the interaction between MPXV A23 protein and the host. Our findings highlight the potential role of this enzyme in suppressing host antiviral immune responses and modulating host gene expression.
猴痘病毒 (MPXV) 是一种跨物种病原体，既能感染人类，也能感染野生动物，对公众健康构成重大威胁。尽管 MPXV 引起了广泛关注，但缺乏足够的研究来阐明与病毒感染相关的发病机制。在这项研究中，我们采用高通量 RNA 测序（RNA-seq）和液相色谱-串联质谱（LC-MS/MS）方法，研究了 MPXV A23 蛋白对 HEK293T 细胞的转录和代谢反应。通过 GO 和 KEGG 分析进行蛋白质-蛋白质相互作用和信号通路分析。通过免疫荧光检测 A23 蛋白在 HEK293T 细胞中的定位。通过 RNA-Seq 在细胞中鉴定出 648 个差异表达基因（DEGs），包括 314 个上调基因和 334 个下调基因。此外，液相色谱-串联质谱（LC-MS/MS）检测到 115 种与 A23 蛋白相互作用的细胞蛋白。转录组测序分析表明，MPXV A23 蛋白转染主要调节与细胞凋亡和 DNA 损伤修复相关的基因。蛋白质组学分析表明，该蛋白主要与宿主核糖体蛋白和组蛋白相互作用。在鉴定出 A23 蛋白中的核定位序列 RKKR 后，构建了截短突变体 A23，以研究 A23 蛋白的亚细胞定位。野生型 A23 蛋白的核质比显著升高，超过 1.5，而突变型 A23 的核质比约为 1。免疫荧光实验表明 A23 蛋白主要定位于细胞核。转录组学和蛋白质组学分析的整合提供了对 MPXV A23 蛋白与宿主相互作用的全面理解。我们的研究结果表明，该酶可能在抑制宿主抗病毒免疫反应和调节宿主基因表达方面发挥作用。
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