Genome-wide Viral Nascent RNA Sequencing Unveils Polymerase Pausing Landscape at Single-nucleotide Precision

Understanding viral replication and transcription mechanisms is critical for developing effective antiviral strategies. The study of viral gene regulation in host cellular environment is an important bridge for translating mechanistic discoveries from in vitro studies to in vivo but it remains stagnated due to the absence of technological advancement. Current methods for studying viral transcription and replication have been limited to capturing only the mature viral RNAs, obscuring the dynamic intermediates and mechanistic details of these crucial processes. Here, we introduce an original technology we called "Total Elongating Nascent VIral Polymerase single-molecule Sequencing (TenVIP-seq)", which isolates and analyses newly synthesized RNA within the viral RNA-dependent RNA polymerase (RdRp) complex, enabling the discovery of mechanisms critical for both viral replication and transcription. Our first characterization of nascent RNA species for an RNA virus showed that RdRP exhibited non-random pausing with profile signatures along the eight influenza A virus (IAV) gene segments. We also revealed genome-wide pausing at known regulatory sites, such as the poly(U) tract polyadenylation site, and new putative regulatory sites at single-nucleotide resolution. Distinct pausing features between the viral genomic (vRNA) and anti-genomic (cRNA/mRNA) templates were observed, suggesting that RdRp processes transcription and replication differentially on positive and negative sense RNA. The NTP analog drug T-705 (favipiravir) intensified RdRp pauses during genome replication and transcription without introducing new pausing sites, while TRIM25 knockout in host cells infected with virus reduced RdRp pausing globally across the viral genome. Strikingly, we observed that terminal nucleotide misincorporations of nascent RNA sequencing of paused RdRP, which were previously undetectable, were as high as an average of 51.6% (vRNA) and 44.0% (cRNA/mRNA), suggesting that the mutational rate of viruses is much higher than previously thought. We demonstrated that TenVIP-seq holds potential in providing insights into the molecular mechanisms that control viral genome replication, gene expression and regulation, mutation, antiviral drug treatment, and virus-host interaction.