目的：1型单纯疱疹病毒（HSV-1）在全球有着较高的感染率，感染后可潜伏在宿主感觉神经节，裂解性复制和再激活时能够引发多种疾病，且在单细胞水平感染表现具有异质性。HSV-1能够改变细胞RNA代谢与合成并影响细胞信号通路。本研究旨在借助多供体混合感染模型结合单细胞转录组测序（scRNA-seq），深入探究HSV-1感染早期宿主免疫应答机制，明确差异表达基因及关键信号通路激活模式等早期免疫反应，为阐明发病原理提供单细胞证据，发现新治疗靶点。并基于继续结果，筛选差异表达基因，在体外应用细胞感染模型进行基因表达调控，从转录和复制层面评估这些基因对HSV-1增殖的影响，研究其在HSV-1感染中的功能，为阐明病毒劫持宿主细胞并利用宿主细胞的转录翻译系统完成自身复制周期的分子机制提供新思路。

方法：本研究通过构建多供体混合感染模型，收集来自19名健康供体的永生化B淋巴细胞（B-LCL），等比例混合后接种HSV-1，以模拟个体感染情况。首先通过梯度MOI（1、10、20、50）的HSV-1感染B-LCL，以检测B-LCL对HSV-1的易感性，并通过RT-qPCR对HSV-1在B-LCL内的转录活性进行检测，以确定进行scRNA-seq的最佳实验条件。设立病毒感染组与正常对照组，制备单细胞悬液，利用10x Genomics平台进行scRNA-seq检测，全面获取感染前后宿主细胞的基因表达数据。对所得数据进行生物信息学分析，筛选出差异表达基因，进行GO/KEGG富集分析，阐明差异表达基因参与的生物学过程、分子功能及关键信号通路，探究细胞早期免疫反应的共同机制。基于scRNA-seq数据分析结果，筛选与代谢重编程和免疫调控密切相关的差异表达基因，包括HMGCS1、PSAT1、MSMO1、ITM2A、ARHGAP6、STPG4、PKLR、TNFRSF10D。以人支气管上皮细胞系Beas-2B为工具细胞，利用siRNA介导的基因沉默技术和重组质粒转染的过表达技术进行基因表达调控，分别构建基因表达下调和上调的细胞模型，随后接种病毒。采用实时荧光定量 PCR技术，定量检测病毒基因的转录活性以及病毒基因组复制水平，综合评估这些宿主基因对HSV-1增殖的影响。

结果：梯度MOI感染实验结果显示，在MOI=1和MOI=10时病毒滴度呈逐渐上升趋势，MOI=20和MOI=50时呈先上升后下降趋势，证实了B-LCL对HSV-1具有易感性。对RT-qPCR对病毒的转录活性的测定显示，在MOI=50时病毒的转录活性最高。基于scRNA-seq的数据分析显示，正常对照组与HSV-1感染组分别捕获了14000和10286个细胞，且两组样本UMI有效率均达到99.9%，表明测序质量高度可靠。HSV-1感染组与正常对照组相对比，以P＜0.05为阈值，共鉴定出3856个差异表达基因，包括1287个上调基因和2569个下调基因。GO富集和KEGG富集分析显示，这些差异基因显著富集于代谢相关通路与过程。选取HMGCS1、PSAT1、MSMO1、ITM2A、ARHGAP6、STPG4、PKLR、TNFRSF10D进行功能验证。基因沉默实验结果显示，敲低MSMO1和HMGCS1可显著抑制HSV-1复制，病毒基因转录活性和病毒DNA拷贝数均显著降低。构建过表达质粒并转染后，MSMO1、PKLR和TNFRSF10D的过表达均能使病毒DNA拷贝数显著增加；HMGCS1、MSMO1、TNFRSF10D、PKLR和STPG4的过表达能显著提升病毒基因转录活性。其中HMGCS1、MSMO1结果与RNAi结果相对应，因此认为这两个基因具有促进病毒复制的作用。

结论：我们的研究证明了B淋巴母细胞系对HSV-1具有易感性，为建立    HSV-1感染模型提供了理想的细胞系统。通过scRNA-seq，发现了HSV-1能够影响宿主的代谢网络。其中MSMO1和HMGCS1能够促进HSV-1的复制。

Objective: Herpes simplex virus type 1 (HSV-1) exhibits a high global infection rate and can establish latent infections in sensory ganglia, with lytic replication and reactivation causing diverse clinical manifestations. Notably, HSV-1 infection displays significant heterogeneity at the single-cell level. HSV-1 alters cellular RNA metabolism and synthesis and modulates host signaling pathways. This study aims to employ a multi-donor mixed infection model combined with single-cell RNA sequencing (scRNA-seq) to investigate early host immune responses during HSV-1 infection, identify differentially expressed genes (DEGs), and elucidate key signaling pathway activation patterns. These findings will provide single-cell-level insights into HSV-1 pathogenesis and reveal novel therapeutic targets. Furthermore, based on scRNA-seq data, we screened host genes with significant expression changes post-infection and evaluated their impact on HSV-1 proliferation through in vitro infection models and gene expression modulation, thereby systematically characterizing viral hijacking of host cellular mechanisms.

Methods: A multi-donor mixed infection model was established using immortalized B lymphoblastoid cells (B-LCLs) from 19 healthy donors to simulate HSV-1 infection. scRNA-seq was performed to obtain comprehensive gene expression profiles before and after infection. Bioinformatics analyses identified DEGs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to uncover key signaling pathways and early immune response mechanisms. Based on scRNA-seq results, host genes with significant expression changes post-HSV-1 infection were selected for functional validation. The human bronchial epithelial cell line Beas-2B was used to construct gene knockdown (via siRNA) and overexpression (via recombinant plasmids) models for HMGCS1, PSAT1, MSMO1, ITM2A, ARHGAP6, STPG4, PKLR, and TNFRSF10D. Viral gene transcription levels were quantified by RT-qPCR, and viral DNA copy numbers were measured to assess genome replication, collectively evaluating the role of these host genes in HSV-1 proliferation.

Results: Gradient MOI (1, 10, 20, 50) infection assays combined with viral titer measurements confirmed the susceptibility of B-LCLs to HSV-1. Viral titers increased gradually at MOI=1 and 10 but peaked and declined at MOI=20 and 50. Maximal intracellular viral transcripts were observed at MOI=50 after 24 hours. scRNA-seq data analysis captured 14,000 and 10,286 cells in the control and HSV-1-infected groups, respectively, with a UMI efficiency of 99.9%, indicating high sequencing reliability. The infected group exhibited elevated viral loads, further confirming B-LCL susceptibility. Comparative analysis identified 3,856 DEGs (1,287 upregulated, 2,569 downregulated), significantly enriched in metabolic pathways (P < 0.05). Functional validation revealed that silencing MSMO1 and HMGCS1 significantly suppressed HSV-1 replication (reduced viral transcription and DNA copies). Conversely, overexpression of MSMO1, PKLR, and TNFRSF10D increased viral DNA copies, while HMGCS1, MSMO1, TNFRSF10D, PKLR, and STPG4 overexpression enhanced viral gene transcription.

Conclusion: Our study demonstrated that B-lymphoblastoid cell lines (B-LCLs) exhibit susceptibility to HSV-1 infection, establishing an ideal cellular system for modeling HSV-1 infection. Through single-cell RNA sequencing (scRNA-seq) analysis, we revealed that HSV-1 can reprogram the host metabolic network. Specifically, we identified MSMO1 and HMGCS1 as host factors that significantly promote HSV-1 replication.