ABSTRACT

Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5′ and 3′ untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5′ and 3′ UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1–4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14^th nucleotide uridine (U14′) of the 5′ UTR and the 13^th nucleotide adenosine (A13) of the 3′ UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14′ and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13′ and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.

摘要

甲型流感病毒的八段负义单链基因组 RNA 以 5' 和 3' 非翻译区 (UTR) 终止。所有片段在 5' 和 3' UTR 处分别具有 13 和 12 个核苷酸的高度保守的末端，构建了病毒 RNA (vRNA) 启动子。在两条保守链之间的 3 个碱基对 (bps) 的双链体茎附近，额外的 1-4 bps 以片段特异性方式存在。我们研究了5' UTR 的^第14^个核苷酸尿苷 (U14') 和第 13^个核苷酸之间的基质 (M) 片段特异性碱基对的作用。通过制备可能的 vRNA 启动子，命名为 vXY，以及命名为 cYX 的 cRNA 启动子，来提取 3' UTR 的核苷酸腺苷 (A13)。我们使用小基因组复制子系统和体外酶分析系统分析了它们的 RNA 依赖性 RNA 复制效率，并使用合成 RNA 启动子。值得注意的是，与具有 A14' 和 C13 的合成 vRNA 启动子 vAC(s) 相比，具有 A13' 和 C14 的 cAC(s) 中互补 RNA (cRNA) 启动子的碱基对破坏不仅减少了病毒细胞中的 RNA 复制但也从头受损启动未引物的 vRNA 合成。反向遗传学实验证实，cRNA 启动子的这种断裂影响了传染性病毒的拯救。目前的研究表明，第一个片段特异性碱基对通过调节 cRNA 而不是 vRNA 的启动子活性，在产生感染性病毒方面发挥着重要作用。它可以深入了解片段特异性核苷酸在病毒基因组复制中的作用，以实现可持续感染。