RNA-dependent RNA polymerase sequence specificities of capsnatching viruses are tailored to aid viral replication. Homo sapiens

Influenza A virus (IAV) is a major respiratory pathogen that causes severe illness and mortality worldwide. IAV critically depends on host factors for multiple functions that are not encoded by the viral genome. Notably, the IAV RNA-dependent RNA polymerase (RdRP) complex lacks intrinsic 7-methylguanosine (m7G) capping activity and instead “snatches” the 5’m7G caps and the adjacent 10-15nts of host transcripts to prime viral mRNA production, allowing viral transcripts to be recognized by the host translation machinery. We developed a new approach to simultaneously assay the 5’ ends of host and viral transcripts in infected cells and identify the host capsnatch origins for viral mRNAs. We used this technique to study the progression of IAV infection in nuclear and cytoplasmic compartiments and determined that the RdRP complex has specific sequence preferences that results in preferential capsnatching and avoidance of sub-populations of host RNAs, which can be reverted by the addition of a cap-snatch inhibitor. Notably, genes avoided by CapSnatch Seq have biological functions enriched for mRNA processing, translation, and viral replication. This selectivity and functional enrichment of avoided genes is conserved among capsnatching viruses including Influenza B Virus and Lymphocytic Choriomeningitis Virus, despite differences in RdRP domains and subcellular locations of RNA substrates. This suggests that this preference convergently arose as a viral strategy to allow cap-snatching viruses to avoid targeting genes necessary for its own replication. Overall design: This study contains 33 samples comprising capsnatch sequencing and directional RNA sequencing. Experimental setup includes extraction of RNA at various timepoints after viral infection, separation of cytosolic and nucleic extacts, treatment with capsnatch inhibitor drug or DMSO, and comparison of different viral strains.

甲型流感病毒（Influenza A virus, IAV）是一种在全球范围内引发重症疾病与死亡的主要呼吸道病原体。IAV的多项非病毒基因组编码的功能，严格依赖宿主因子完成。值得注意的是，IAV的RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRP）复合物本身不具备内在的7-甲基鸟苷（7-methylguanosine, m7G）加帽活性，而是通过“抢帽”方式获取宿主转录本的5’端m7G帽结构及相邻的10-15个核苷酸序列，以此作为病毒mRNA合成的引物，使病毒转录本能够被宿主翻译系统识别。
我们开发了一种全新方法，可同时检测感染细胞内宿主与病毒转录本的5’端序列，并鉴定病毒mRNA的宿主帽抢起源位点。我们利用该技术研究了IAV感染在细胞核与细胞质区室中的进展过程，发现RdRP复合物具有特定的序列偏好性，这会导致其优先进行帽抢并避开特定亚群的宿主RNA；而加入帽抢抑制剂可逆转这一现象。
值得注意的是，被帽抢测序（CapSnatch Seq）所避开的基因，其生物学功能显著富集于mRNA加工、翻译及病毒复制相关通路。这种选择性及避靶基因的功能富集特征，在包括乙型流感病毒（Influenza B Virus）与淋巴细胞性脉络丛脑膜炎病毒（Lymphocytic Choriomeningitis Virus）在内的多种帽抢病毒中均保守存在，尽管这些病毒的RdRP结构域与RNA底物的亚细胞定位存在差异。这表明该序列偏好性作为一种病毒策略趋同演化而来，使帽抢病毒能够避开靶向自身复制所必需的宿主基因。
实验整体设计：本研究共包含33份样本，涵盖帽抢测序与定向RNA测序（directional RNA sequencing）实验。实验设置包括：在病毒感染后的不同时间点提取RNA，分离细胞质与细胞核提取物，使用帽抢抑制剂或二甲基亚砜（DMSO）进行处理，并开展不同病毒毒株的对比分析。