Murine Norovirus (MNV) infection results in anti-inflammatory response downstream of amino acids depletion in macrophages.

Purpose: Characterisation of MNV induced host genetic reprogramming and translational control exerted over the host response by comparison of available cytoplasmic transcripts and their association with polysomes. Results: RNaseq data showed an inadequate response to MNV infection downstream of MDA5 activation coupled with a cellular response to a metabolic stress. In particular, all 296 significantly regulated transcripts showed a strong correlation between a transcript availability in the cytoplasm and its recrutment to the polysomal fraction as addressed using the R package 'Anota2seq'. Genome annotation analyses using Cytoscape highlighted the host response to the viral infection but showed a cluster of genes involved in response to stress and the related intrinsic apoptotic pathways suggesting a prevalence of a metabolic stress response over the innate immune response to the viral replication. Conclusions: Our study provides the first understanding analysis of the host response to MNV at the level of available cytoplasmic mRNA and their recruitment to the actively translating fractions. It described an absence of translational control over the host innate immune response but highlight and inadequate NF-kB response correlating with an ectopic anti-inflammatory response to amino acid starvation. Overall design: Methods: Cytoplasmic RNA were extracted from cells infected with MNV or UV-irradiated MNV at different time post-infection (6 and 10hpi). For each sample, half was purified directly (Total samples) and half loaded onto polysome gradient to obtain the RNA associated with the translation machinery (Polysomal samples) in triplicate. Samples were enriched for polyA-tailed mRNA and sequenced on Illumina HiSeq4000, outputs quality checked via FastQC and mapped onto mouse genome (Gencode release M12 GRCm38.p5) using Hisat2 and to the Murine Norovirus genome MNV1-CW1 (DQ285629) coding sequences (ORF1: ABB90153.1, ORF2: ABB90154.1, ORF3: ABB90155.1) using Bowtie. Genomic features of the host were defined using 'Rsubread' and in-built gene annotations for the mouse genome (NCBI RefSeq gene annotations Build 38.1). Host genomic features were then annotated using the R package 'org.Mm.eg.db', Ensembl and GenBank. The feature count matrix consisted of the set of Host and MNV feature count and genes annotated as rRNA, miRNA, snoRNA, snoRNA or scaRNA were filtered out of the feature count matrix. Filtering of lowly expressed genes was performed after library size normalisation by keeping genes with at least 0.25 CPM in at least 25% of the samples in both Total and Polysomal fractions and differential expression addressed using the GLM implementation of EdgeR. A final filtering criterion was applied to each individual significant gene using the genome visualisation tool Integrative Genome Viewer IGV hosted by the Broad Institute. qRT–PCR validation was performed using SYBR Green assays.

研究目的：通过比对可获取的胞质转录本及其与多聚核糖体（polysomes）的结合情况，解析鼠诺如病毒（MNV）诱导的宿主遗传重编程，以及宿主应答过程中所受的翻译调控机制。
研究结果：RNA测序（RNaseq）数据显示，黑色素瘤分化相关蛋白5（MDA5）激活下游通路对MNV感染的应答存在缺陷，同时伴随细胞对代谢应激的应答。具体而言，经R包'Anota2seq'分析的全部296个差异调控转录本，其胞质丰度与被招募至多聚核糖体组分的程度之间呈现极强的相关性。基于Cytoscape的基因组注释分析结果显示了宿主对病毒感染的应答特征，同时发现一组参与应激应答及相关内源性凋亡通路的基因簇，这提示相较于针对病毒复制的天然免疫应答，代谢应激应答占据主导地位。
研究结论：本研究首次从胞质可获取mRNA及其被招募至活跃翻译组分的层面，解析了宿主对MNV的应答机制。研究未发现宿主天然免疫应答存在翻译调控现象，但揭示了存在缺陷的核因子κB（NF-κB）应答，该缺陷应答与氨基酸饥饿引发的异位抗炎应答存在关联。
实验设计与方法：分别在感染后6小时和10小时（6hpi、10hpi），从感染MNV或紫外线灭活MNV的细胞中提取胞质RNA。每份样本取一半直接纯化（总RNA样本组），另一半经多聚核糖体梯度离心获取与翻译机器结合的RNA（多聚核糖体RNA样本组），每组设置3次生物学重复。对样本中的polyA尾mRNA进行富集后，使用Illumina HiSeq4000平台进行测序；测序数据通过FastQC进行质量质控，随后使用Hisat2将读段比对至小鼠基因组（Gencode release M12 GRCm38.p5），使用Bowtie将读段比对至鼠诺如病毒MNV1-CW1（DQ285629）的编码序列（ORF1: ABB90153.1, ORF2: ABB90154.1, ORF3: ABB90155.1）。宿主基因组特征通过'Rsubread'工具及小鼠基因组内置基因注释（NCBI RefSeq基因注释Build 38.1）进行定义；随后使用R包'org.Mm.eg.db'、Ensembl及GenBank对宿主基因组特征进行注释。特征计数矩阵包含宿主及MNV的特征计数，将注释为rRNA、miRNA、snoRNA、snoRNA或scaRNA的基因从特征计数矩阵中移除。在文库大小标准化后进行低表达基因过滤：保留总RNA样本组与多聚核糖体RNA样本组中至少25%的样本里CPM值不低于0.25的基因；随后使用EdgeR的广义线性模型（GLM）流程进行差异表达分析。最后，使用布罗德研究所（Broad Institute）提供的基因组可视化工具整合基因组浏览器（Integrative Genome Viewer, IGV）对每个显著差异基因进行最终过滤验证。采用SYBR Green荧光染料法进行qRT-PCR验证。