Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.

乙型肝炎病毒 (HBV) 是一种小型双链 DNA 病毒，可长期感染 2.96 亿人。超过一半的紧凑基因组以两个重叠的阅读框编码蛋白质，在进化过程中，多种选择压力可以作用于共享的核苷酸。这项研究将基于 RNA 的 HBV 细胞培养系统与深度突变扫描 (DMS) 相结合，以解开 HBV 基因组中的顺式和反式作用序列要求。结果支持聚合酶翻译的渗漏核糖体扫描模型，提供了单核苷酸分辨率的 HBV 聚合酶的适应性图，并鉴定了与 HBV 聚合酶终止密码子相邻的保守脯氨酸，这些脯氨酸使核糖体停滞。进一步的实验表明，停滞的核糖体将新生聚合酶束缚在其模板 RNA 上，确保 HBV 基因组的顺式优先 RNA 包装和逆转录。