Reid David W, Campos Rafael K, Child Jessica R, Zheng Tianli, Chan Kitti Wing Ki, Bradrick Shelton S, Vasudevan Subhash G, Garcia-Blanco Mariano A, Nicchitta Christopher V
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.
J Virol. 2018 Mar 14;92(7). doi: 10.1128/JVI.01766-17. Print 2018 Apr 1.
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 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. 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）生物学中的一个主要问题是招募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、核糖体分析和转录组测序（RNA-seq），以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高度定位于内质网（ER），确定ER是DENV翻译的主要位点。DENV感染伴随着翻译的ER区室特异性重塑，其中ER翻译能力从宿主转录本转向DENV正链RNA，特别是在感染后期。值得注意的是，在整个感染实验时间过程中，胞质区室中的翻译水平和模式仅受到适度影响。DENV正链RNA和宿主mRNA的核糖体足迹密度比较表明，DENV正链RNA仅稀疏地加载有核糖体。综合这些观察结果表明，一种机制是ER定位的翻译和翻译控制机制（可能是编码的）被用于将ER重新用于DENV病毒粒子的产生。与此观点一致，我们发现通常与病毒感染相关的ER连接的细胞应激反应途径，即干扰素反应和未折叠蛋白反应，在DENV感染期间仅被适度激活。这些数据支持一个模型，即DENV重新编程ER蛋白质合成和加工环境，以促进病毒存活和复制，同时最小化抗病毒和蛋白质稳态应激反应途径的激活。DENV是对人类健康的一个重大威胁，没有广泛有效的或特异性的治疗方法，它依赖宿主细胞翻译机制进行病毒复制、免疫逃避和病毒粒子生物发生。DENV征用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尚不清楚。在这里，我们报告说，DENV几乎完全有一个ER定位的生命周期，病毒复制和翻译主要限于ER。令人惊讶的是，DENV感染主要仅影响与ER相关的翻译，对胞质中的宿主细胞翻译影响相对较小。DENV RNA翻译非常低效，可能代表一种将ER蛋白质稳态破坏最小化的策略。总体而言，这些发现表明DENV已经进化出一个ER区室化的生命周期；因此，针对DENV-ER相互作用景观的分子特征和调控可能揭示治疗干预策略。
