cortactin 蛋白与猪瘟病毒 core 蛋白互作对外泌体与病毒感染的影响

effects of interaction between cortactin protein and classical swine fever virus core protein on exosomes and viral infection

摘要

[目的]本文旨在筛选并验证与猪瘟病毒(classical swine fever virus, CSFV)核心(Core)蛋白互作的宿主调控因子，系统解析外泌体依赖性 CSFV 感染的分子机制，为基于宿主细胞工程化改造优化疫苗生产工艺提供理论依据。[方法]基于体外蛋白互作沉淀(pull-down)-质谱联用技术筛选 Core 互作宿主蛋白；采用免疫共沉淀(Co-IP)和激光共聚焦显微术验证皮层肌动蛋白(cortactin, CTTN)与 Core 的亚细胞共定位；构建 CTTN 过表达(over-expression, OE)及敲低(knock-down, KD)细胞株；运用纳米颗粒追踪分析(NTA)定量外泌体分泌动力学，联合蛋白免疫印记(Western blot, WB)和间接免疫荧光(IFA)技术解析病毒蛋白时空分布特征及感染效价。[结果]蛋白质互作组学鉴定 CTTN 为 Core 蛋白互作蛋白，Co-IP 与共聚焦成像证实二者在细胞质各处均存在特异性互作；WB 分析显示 CTTN 敲低使外泌体的 Core 载量提升并加速其分泌，而 CTTN 过表达则延迟 Core 外泌体装载；NTA 定量表明 CTTN-KD 显著提高外泌体分泌效率，CTTN-OE 则抑制分泌活性；IFA 检测显示 CTTN 敲低通过外泌体途径提升胞外病毒滴度，而 CTTN 过表达导致总病毒滴度下降。[结论]本研究证实宿主 CTTN 蛋白与 CSFV Core 蛋白存在互作，并对外泌体介导的 CSFV 病毒组分释放及感染起重要调控作用，为基于宿主细胞改造的猪瘟病毒增殖效率优化及疫苗生产模式改进提供了全新靶标。

Abstract

[Objectives] The paper aimed to screen and validate host regulatory factors interacting with classical swine fever virus (CSFV) Core protein, systematically elucidate the molecular mechanism of exosome-dependent CSFV infection, and provide a theoretical basis for optimizing vaccine production processes based on host cell engineering. [Methods] Core-interacting host proteins were screened using pull-down coupled with mass spectrometry (pull-down-MS). The subcellular colocalization of cortactin (CTTN) and Core was verified by co-immunoprecipitation (Co-IP) and confocal microscopy. Stable cell lines with CTTN over-expression (OE) or knock-down (KD) were constructed. Nanoparticle tracking analysis (NTA) was employed to quantify exosome secretion dynamics, while Western blot (WB) and indirect immunofluorescence assay (IFA) were used to analyze the spatiotemporal distribution of viral proteins and infection efficacy. [Results] Proteomic interaction analysis identified CTTN as a Core-interacting protein, and Co-IP and confocal imaging confirmed their specific interaction throughout the cytoplasm. WB analysis revealed that CTTN-KD increased Core loading into exosomes and accelerated its secretion, whereas CTTN-OE delayed Core exosomal packaging. NTA quantification demonstrated that CTTN-KD significantly enhanced exosome secretion efficiency, while CTTN-OE suppressed secretory activity. IFA detection showed that CTTN-KD elevated extracellular viral efficacy via the exosomal pathway, whereas CTTN-OE led to a reduction in total infection efficacy. [Conclusions] This study is the first to confirm the interaction between host CTTN protein and CSFV Core protein, highlighting its critical regulatory role in exosome-mediated CSFV component release and infection. These findings not only provided deeper insights into CSFV infection and virus-host interaction mechanisms but also offer a novel target for optimizing CSFV proliferation efficiency and improving vaccine production strategies through host cell engineering.

关键词

猪瘟病毒 / 核心蛋白(Core) / 皮层肌动蛋白(CTTN) / 外泌体 / 病毒-宿主互作

Key words

classical swine fever virus (CSFV) / core protein / cortactin (CTTN) / exosomes / virus-host interaction

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基金资助

江苏省农业科技自主创新项目(CX(24)2004)

江苏省重点研发计划(社会发展)项目(BE2022787)