Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient virion production
Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient virion production
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DOI：
10.1128/jvi.02006-23
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发表时间：
2024-02-09
影响因子：
5.4
通讯作者：
Barr，John N.
中科院分区：
文献类型：
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作者：
Byford，Owen;Shaw，Amelia B.;Barr，John N.
淋巴细胞性脉络丛脑膜炎病毒（LCMV）是布尼亚病毒目沙粒病毒科的一种双节段负义RNA病毒。LCMV与免疫功能低下人群中的致命性疾病相关，并且作为原型沙粒病毒，充当该组中许多严重人类病原体的模型。在这里，我们研究了依赖LCMV增殖细胞运输组件使用重组LCMV表达增强绿色荧光蛋白结合策展的siRNA文库。筛选揭示了对外壳蛋白1（COPI）外被体和衔接蛋白4（AP-4）复合物的亚基的需要。通过拯救携带FLAG标记的糖蛋白（GP-1）包膜刺突的重组LCMV（rLCMV-GP 1-FLAG），我们发现感染导致单个COPI和AP-4组分与LCMV核蛋白（NP）和GP-1的显著共定位，这与它们参与病毒过程一致。为了进一步研究COPI和AP-4复合物在LCMV感染期间的作用，我们利用了防止复合物形成的ARF-1抑制剂布雷菲德菌素A（BFA）。在一个单一的12小时的病毒增殖周期内，BFA预处理引起LCMV特异性RNA合成没有显着变化，同时LCMV NP表达没有显着变化，如通过BFA添加时间实验所测量的。相比之下，BFA的加入导致释放的病毒滴度显著下降，在相同的12小时内接近50倍，在24小时内上升到600倍以上。两者合计，这些研究结果表明，COPI和AP-4复合物是重要的宿主细胞因子所需的形成和释放的传染性LCMV.IMPORTANCEArenaviruses是啮齿动物传播的，分段的，负义RNA病毒，有几个成员负责致命的人类疾病，与原型成员淋巴细胞性脉络丛脑膜炎病毒（LCMV）被低估的病原体能够造成致命的结果神经系统感染。关于沙粒病毒如何破坏宿主细胞过程以完成其增殖周期的详细了解是不完整的。在这里，使用基因消融和药理学抑制技术的组合，我们表明，宿主细胞COPI和AP-4复合物，在细胞囊泡运输的天然作用，需要有效的LCMV生长。我们进一步表明，这些复合物作用于增殖周期的后期阶段，即基因表达后，对感染性病毒的逃逸有显著影响。总的来说，我们的研究结果提高了对沙粒病毒宿主-病原体相互作用的理解，并揭示了感染期间所需的关键细胞运输途径。
Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within theArenaviridaefamily of theBunyaviralesorder. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV.IMPORTANCEArenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member lymphocytic choriomeningitis virus (LCMV) being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. A detailed understanding of how arenaviruses subvert host cell processes to complete their multiplication cycle is incomplete. Here, using a combination of gene ablation and pharmacological inhibition techniques, we showed that host cellular COPI and AP-4 complexes, with native roles in cellular vesicular transport, were required for efficient LCMV growth. We further showed these complexes acted on late stages of the multiplication cycle, post-gene expression, with a significant impact on infectious virus egress. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal critical cellular trafficking pathways required during infection.