Manning Aidan C, Bashir Mahmoud M, Jimenez Ariana R, Upton Heather E, Collins Kathleen, Lowe Todd M, Tucker Jessica M
Department of Biomolecular Engineering, Baskin School of Engineering, University of California, Santa Cruz, Santa Cruz, CA, 95064, USA.
Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA.
bioRxiv. 2024 May 2:2024.05.01.592122. doi: 10.1101/2024.05.01.592122.
Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports biological function for tRNA cleavage products, including in the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to enhanced tRNA transcription. However, whether this has any influence on tRNA transcript processing, viral replication, or the host response is not known. Here, we combined two new approaches, sequencing library preparation by Ordered Two Template Relay (OTTR) and tRNA bioinformatic analysis by tRAX, to quantitatively profile full-length tRNAs and tRNA fragment (tRF) identities during MHV68 infection. We find that MHV68 infection triggers both pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tRFs. OTTR-tRAX revealed not only host tRNAome changes, but also the expression patterns of virally-encoded tRNAs (virtRNAs) and virtRFs made from the MHV68 genome, including their base modification signatures. Because the transcript ends of several host tRFs matched tRNA splice junctions, we tested and confirmed the role of tRNA splicing factors TSEN2 and CLP1 in MHV68-induced tRF biogenesis. Further, we show that CLP1 kinase, and by extension tRNA splicing, is required for productive MHV68 infection. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.
转运RNA（tRNA）在病毒感染期间对细胞和病毒基因表达都至关重要。此外，越来越多的证据支持tRNA切割产物的生物学功能，包括在应激和感染条件下对基因表达的控制。我们之前报道，感染模型小鼠γ疱疹病毒MHV68会导致tRNA转录增强。然而，这是否对tRNA转录本加工、病毒复制或宿主反应有任何影响尚不清楚。在这里，我们结合了两种新方法，即通过有序双模板中继（OTTR）制备测序文库和通过tRAX进行tRNA生物信息学分析，以定量分析MHV68感染期间全长tRNA和tRNA片段（tRF）的特征。我们发现，MHV68感染会触发前体tRNA和成熟tRNA的切割，导致特定tRF的积累。OTTR-tRAX不仅揭示了宿主tRNA组的变化，还揭示了由MHV68基因组产生的病毒编码tRNA（virtRNA）和virtRF的表达模式，包括它们的碱基修饰特征。由于几个宿主tRF的转录本末端与tRNA剪接接头匹配，我们测试并证实了tRNA剪接因子TSEN2和CLP1在MHV68诱导的tRF生物合成中的作用。此外，我们表明，有 productive MHV68感染需要CLP1激酶，进而需要tRNA剪接。我们的研究结果为γ疱疹病毒感染如何影响并依赖tRNA转录和加工提供了新的见解。
