RNA transcriptome analysis during HSV-1 infection. Homo sapiens

For Samples 1-8 and 11-18: The innate immune sensor retinoic acid-inducible gene-I (RIG-I) detects double-stranded RNA derived from RNA viruses, and recent studies have demonstrated that RIG-I also plays a role in the antiviral response to DNA viruses. To identify the physiological RNA species that are recognized by RIG-I during HSV-1 infection, we purified the RNAs that co-immunoprecipitated with FLAG-tagged RIG-I in transfected human embryonic kidney (HEK) 293T cells that had been infected with a recombinant HSV-1 (hereafter referred to as HSV-1 mut) containing a mutation (K220A) in the viral serine/threonine protein kinase US3 that abolishes its catalytic activity, as the viral kinase is known to antagonize type-I IFN responses. As controls, RNA species bound to FLAG-RIG-I in uninfected cells and RNA bound to FLAG-GFP from both HSV-1 mut-infected and uninfected cells were also purified. RIG-I-bound RNA and total RNA extracted from uninfected and HSV-1 mut-infected cells were analyzed by RNAseq, and the resulting sequences were mapped to both the HSV-1F-strain and human genome (hg38). This analysis revealed that several human transcripts were highly enriched in the RIG-I-bound fraction from infected cells; in contrast, the enrichment of viral sequences was low. The cellular transcripts that were most abundant in the RIG-I fraction were predominantly non-coding RNAs from different subclasses, as well as some coding RNAs. For Samples 9 and 10: HSV-1 infection is known to induces changes in the transcriptional profile of the infected cell. To analyze global changes in RNA transcript levels in infected cells, total RNA was extracted from HEK 293T cells that were infected with wild-type (WT) HSV-1. For comparison, total RNA was extracted from HEK 293T cells that remained uninfected. Next, RNAseq analysis was performed. The resulting sequences were mapped to the human genome, and gene inductions were calculated and normalized to uninfected samples to determine changes in gene expression upon infection. Overall design: Cells, which were not infected or infected with either wildtype HSV-1 or mutated HSV-1 were either subjected to a pulldown isolating RLR/GFP associated RNA (8 samples) or the corresponding total RNA (8 samples) was extracted from the infected cells and sequenced. Additionally, non-transfected cells were infected and total RNA extracted and sequenced (2 samples)

针对样本1-8及11-18：固有免疫感受器视黄酸诱导基因I（retinoic acid-inducible gene-I, RIG-I）可识别RNA病毒来源的双链RNA，近期研究表明RIG-I在针对DNA病毒的抗病毒应答中同样发挥作用。为鉴定单纯疱疹病毒1型（herpes simplex virus type 1, HSV-1）感染期间被RIG-I识别的生理状态RNA物种，我们在感染了携带病毒丝氨酸/苏氨酸蛋白激酶US3位点K220A突变（该突变可使其催化活性丧失）的重组HSV-1（以下简称HSV-1 mut）的转染人胚肾（human embryonic kidney, HEK）293T细胞中，纯化了与FLAG标签标记的RIG-I共免疫沉淀的RNA；已知该病毒激酶可拮抗I型干扰素（type I interferon, IFN）应答。作为对照，我们同时纯化了未感染细胞中与FLAG-RIG-I结合的RNA，以及HSV-1 mut感染和未感染细胞中与FLAG-GFP结合的RNA。对RIG-I结合的RNA以及从未感染和HSV-1 mut感染细胞中提取的总RNA进行RNA测序（RNAseq）分析，将得到的序列比对至HSV-1 F毒株和人类基因组（hg38）。分析结果显示，感染细胞的RIG-I结合组分中存在多种人类转录本显著富集；与之相反，病毒序列的富集程度较低。RIG-I结合组分中丰度最高的细胞转录本主要来自不同子类的非编码RNA（non-coding RNAs），以及部分编码RNA（coding RNAs）。

针对样本9和10：已知HSV-1感染可改变受感染细胞的转录组谱。为分析受感染细胞中RNA转录本水平的整体变化，我们从感染野生型（wild-type, WT）HSV-1的HEK 293T细胞中提取总RNA；作为对照，从未感染的HEK 293T细胞中提取总RNA。随后进行RNAseq分析，将得到的序列比对至人类基因组，计算基因诱导倍数并以未感染样本为参照进行标准化，以确定感染后基因表达的变化。

整体实验设计：分别设置未感染、感染野生型HSV-1或突变型HSV-1的细胞组，其中8个样本通过pull-down实验分离与RIG-I样受体（RIG-I-like receptor, RLR）/GFP结合的RNA，另外8个样本从受感染细胞中提取总RNA并测序；此外设置未转染细胞感染后提取总RNA并测序的组别（共2个样本）。