A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways.

IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here, we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle; thus, targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention.

登革热病毒（DENV）生物学的一个主要问题是募集宿主细胞蛋白质合成机制的分子策略。在这里，我们结合细胞分级分离，核糖体谱分析和转录组测序（RNA-seq），研究病毒基因组翻译和复制以及宿主细胞翻译及其对DENV感染的反应的亚细胞组织。我们报告说，在整个病毒生命周期中，DENV正链和负链RNA高度分配到内质网（ER），从而将ER确定为DENV翻译的主要位点。DENV感染伴有ER区域特异性翻译的重塑，其中ER的翻译能力已从宿主转录物转化为DENV加链RNA，特别是在感染后期。值得注意的是 在感染的整个实验过程中，胞浆区室中的翻译水平和模式仅受到中等程度的影响。DENV正链RNA和宿主mRNA的核糖体足迹密度的比较表明，DENV正链RNA仅稀疏地装载有核糖体。结合起来，这些观察结果提出了一种机制，其中可能是ER本地化的翻译和翻译控制机制顺式编码，用于将ER重新用于DENV病毒粒子生产。与此观点一致，我们发现通常与病毒感染有关的内质网相关的细胞应激反应途径，即干扰素反应和未折叠的蛋白质反应，在DENV感染期间仅被适度激活。这些数据支持一个模型，其中DENV重新编程ER蛋白的合成和加工环境，以促进病毒的生存和复制，同时最大程度地减少抗病毒和蛋白水解应激反应途径的激活。

重要性DENV是一种严重的人类健康威胁，没有得到广泛有效的治疗或特异性治疗，它依赖宿主细胞翻译机制进行病毒复制，免疫逃逸和病毒体生物发生。DENV指挥宿主细胞蛋白质合成机制以及DENV复制和病毒蛋白质合成的亚细胞组织的分子机制了解甚少。在这里，我们报道DENV具有几乎完全位于ER的生命周期，病毒复制和翻译在很大程度上局限于ER。出人意料的是，DENV感染在很大程度上只影响与ER相关的翻译，而对溶质中宿主细胞翻译的影响相对较小。DENV RNA的翻译效率很低，可能代表了一种最小化ER蛋白稳态破坏的策略。总的来说，这些发现表明，DENV已经进化出了一个ER隔间的生命周期。因此，以分子标记和DENV-ER相互作用格局的调控为目标可能会揭示治疗干预的策略。