VAMP8 contributes to TRIM6-mediated type-I interferon antiviral response upon West Nile virus (WNV) infection

Purpose: Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Therefore, we aimed to investigate the mechanism of IFN-I regulation upon West Nile virus infection using TRIM6 knockout (TRIM6-KO) human airway epithelial cells (A549). Methods: RNAs were extracted from wild-type (WT) and TRIM6KO A549 cells infected with WNV or mock-treated. RNA quality was assessed by visualization of 18S and 28S RNA bands using an Agilent BioAnalyzer 2100 (Agilent Technologies, CA); the electropherograms were used to calculate the 28S/18S ratio and the RNA Integrity Number. Poly-A+ RNA was enriched from total RNA (1 µg) using oligo dT-attached magnetic beads. First and second strand synthesis, adapter ligation, and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit as recommended by the manufacturer (Illumina, Inc). Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1500 using protocols defined by the manufacturer. The alignment of NGS sequence reads was performed using the Spliced Transcript Alignment to a Reference (STAR) program, version 2.5.1b, using default parameters. We used the human hg38 assembly as a reference with the UCSC gene annotation file. The –quantMode GeneCounts option of STAR provided read counts per gene, which were input into the DESeq2 (version 1.12.1) differential expression analysis program to determine expression levels and differentially expressed genes. Processed data are presented as reads per kilobase transcript per million (RPKM). Results: Using next generation sequencing, we identified VAMP8 as a factor involved in this TRIM6-mediated antiviral response. In support, VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNß treatment. Conclusions: Our results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system Overall design: A549 cells (WT and TRIM6KO) cells were infected with WNV at MOI = 5 or mock-treated with similar volumes of PBS. RNA was extracted at 24h post-infection and subjected to deep-sequencing analysis in duplicate using NSG on an Illumina HiSeq 1500 sequencing system.

研究目的：三结构域蛋白（tripartite motif, TRIM）家族的E3泛素连接酶多个成员可调控包括抗病毒I型干扰素（IFN-I）系统在内的免疫通路。本研究旨在利用TRIM6敲除（TRIM6-KO）人气道上皮细胞（A549），探究西尼罗病毒（West Nile virus, WNV）感染后IFN-I系统的调控机制。
研究方法：分别从感染WNV或模拟处理的野生型（wild-type, WT）及TRIM6-KO A549细胞中提取总RNA。采用安捷伦生物分析仪2100（Agilent BioAnalyzer 2100，安捷伦科技公司，美国加利福尼亚州）通过可视化18S和28S RNA条带评估RNA质量，利用电泳图谱计算28S/18S比值与RNA完整性指数。从1μg总RNA中使用偶联寡聚dT的磁珠富集Poly-A+ RNA。按照制造商推荐的操作流程，使用Illumina TruSeq RNA样本制备试剂盒完成第一链、第二链cDNA合成、接头连接及文库扩增（Illumina公司）。采用安捷伦DNA-1000芯片在安捷伦2100生物分析仪上评估文库质量。通过qPCR结合已知大小的参考标准品对文库DNA模板进行定量。使用TruSeq PE Cluster Kit v3（Illumina）及Illumina cBot工作站，按照制造商推荐的条件完成文库DNA模板的簇生成。采用TruSeq SBS Kit v3（Illumina），在Illumina HiSeq 1500平台上按照制造商规定的流程完成50碱基双端合成测序。使用剪接转录本比对到参考基因组（Spliced Transcript Alignment to a Reference, STAR）程序（版本2.5.1b），以默认参数完成下一代测序（NGS）序列读数的比对。以人类hg38组装版本作为参考基因组，结合UCSC基因注释文件进行分析。通过STAR程序的--quantMode GeneCounts选项获取每个基因的测序读数计数，将其输入DESeq2软件（版本1.12.1）进行差异表达分析，以确定基因表达水平及差异表达基因。处理后的数据以每百万读取每千碱基转录本（RPKM）进行呈现。
研究结果：通过下一代测序技术，我们鉴定出VAMP8蛋白是参与TRIM6介导的抗病毒应答的关键因子。进一步验证显示，敲低VAMP8可在WNV感染或β干扰素（IFNβ）处理后，降低Janus激酶1（Jak1）与信号转导与转录激活因子1（STAT1）的磷酸化水平，并削弱多种干扰素刺激基因（ISGs）的诱导表达。
研究结论：本研究结果证实TRIM6可参与针对WNV的抗病毒应答，并鉴定VAMP8为IFN-I系统的新型调控因子。
整体实验设计：将A549细胞（WT及TRIM6-KO）以感染复数（MOI）=5的剂量感染WNV，或以等量PBS进行模拟处理。于感染后24小时提取总RNA，采用Illumina HiSeq 1500测序系统进行双重复深度测序分析。