Persistence of ambigrammatic narnaviruses requires translation of the reverse open reading frame

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods and nematodes. Though initially described as simple single-gene non-segmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as “ambigrammatic” harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2 kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8 kb “Robin” RNA. We developed a system to launch CxNV1 in a naïve mosqu...诺达病毒（Narnaviruses）是一类在多种真菌、植物、原生生物、节肢动物及线虫中检出的RNA病毒。最初人们认为其为仅编码RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRp）的简单单基因非分段病毒。部分被称为"双义型（ambigrammatic）"的诺达病毒拥有独特的基因组结构：由两条互补链上编码的重叠开放阅读框（open reading frames, ORFs）组成。系统发育分析显示存在维持这种特殊基因组组织形式的选择压力，但相关功能研究仍较为匮乏。本研究以可感染蚊子的库蚊诺达病毒1型（Culex narnavirus 1, CxNV1）为模型，旨在探究重叠ORFs在诺达病毒复制过程中的功能作用。在CxNV1中，一条与已知蛋白无同源性的反向ORF几乎覆盖了整条编码RdRp的3.2kb基因组片段。此外，在第二个推测的CxNV1基因组片段——0.8kb的"Robin"RNA上，还存在两条方向相反且几乎完全重叠的新型ORFs。我们开发了一套可在未感染的蚊子中启动CxNV1感染的系统……