Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription

• Published in: Cell Vol. 187; no. 11; pp. 2735 - 2745.e12
• Main Authors: Yu, Yingpu, Kass, Maximilian A., Zhang, Mengyin, Youssef, Noor, Freije, Catherine A., Brock, Kelly P., Aguado, Lauren C., Seifert, Leon L., Venkittu, Sanjana, Hong, Xupeng, Shlomai, Amir, de Jong, Ype P., Marks, Debora S., Rice, Charles M., Schneider, William M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.05.2024
• Subjects: Cell Line; cis preference; deep mutational scanning; diproline; Genome, Viral - genetics; hepatitis B virus; Hepatitis B virus - genetics; Humans; Mutation; Reverse Transcription; ribosome stalling; Ribosomes - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; ribosome stalling; deep mutational scanning; diproline; cis preference; hepatitis B virus; reverse transcription
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2024.04.008

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. [Display omitted] •Deep mutational scanning (DMS) provides a high-resolution fitness map of HBV polymerase•Initiating HBV replication with RNA uncouples cis- and trans-acting protein functions•Ribosome pausing tethers HBV Pol to RNA, enforcing cis-preferential reverse transcription•DMS of the HBV genome supports a leaky ribosome scanning model for polymerase translation Deep mutational scanning reveals that conserved proline codons at the 3′ end of the hepatitis B virus polymerase open reading frame stall ribosomes. As a result, the ribosome physically tethers the nascent polymerase to its encoding RNA, which enforces cis-preferential genome RNA packaging and reverse transcription.