Manning Aidan C, Bashir Mahmoud M, Rapchak Kyle, Huff Calyssa J, Jimenez Ariana R, Gonzalez Sheila, Woodruff Courtney L, 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, California, USA.
Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
mBio. 2025 May 30:e0087525. doi: 10.1128/mbio.00875-25.
Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports the biological function for tRNA cleavage products, including the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus 68, 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. 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, which regulates tRNA splicing among other RNA processing events, is required for efficient MHV68 replication. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.IMPORTANCEDiverse conditions of infection and cellular stress incite the cleavage of transfer RNAs (tRNAs), leading to the formation of tRNA fragments (tRFs) that can directly regulate gene expression. In our study of gammaherpesviruses, such as murine herpesvirus 68 and human oncogenic Kaposi sarcoma-associated herpesvirus, we discovered that tRNA regulation and cleavage are key components of gene reprogramming during infection. We present the first in-depth profile of tRF generation in response to DNA virus infection, using state-of-the-art sequencing techniques that overcome several challenges with tRNA sequencing. We present several lines of evidence that tRFs are made from newly transcribed premature tRNAs and propose that this may be a defining characteristic of tRNA cleavage during infection. Finally, we show that tRNA splicing machinery is involved with the formation of some MHV68-induced tRFs, with a key regulator of splicing, CLP1, required for maximal viral titer. Taken together, we posit that tRNA processing may be integral to the elegant shift in gene expression that occurs during viral takeover of the host cell.
转运RNA（tRNA）在病毒感染期间对于细胞和病毒基因表达均至关重要。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调控。我们之前报道过，用模式鼠γ疱疹病毒68（MHV68）感染会导致tRNA转录增强。然而，这是否会对tRNA转录本加工、病毒复制或宿主反应产生任何影响尚不清楚。在这里，我们结合了两种新方法，即通过有序双模板中继（OTTR）制备测序文库以及通过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。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，调节tRNA剪接以及其他RNA加工事件的CLP1激酶是高效MHV68复制所必需的。我们的研究结果为γ疱疹病毒感染如何影响并依赖于tRNA转录和加工提供了新的见解。
重要性
多种感染条件和细胞应激会引发转运RNA（tRNA）的切割，导致形成可直接调节基因表达的tRNA片段（tRF）。在我们对γ疱疹病毒（如鼠疱疹病毒68和人类致癌性卡波西肉瘤相关疱疹病毒）的研究中，我们发现tRNA调控和切割是感染期间基因重编程的关键组成部分。我们使用克服了tRNA测序中若干挑战的先进测序技术，首次深入分析了DNA病毒感染后tRF的产生情况。我们提供了多条证据表明tRF是由新转录的前体tRNA产生的，并提出这可能是感染期间tRNA切割的一个决定性特征。最后，我们表明tRNA剪接机制参与了一些MHV68诱导的tRF的形成，剪接的关键调节因子CLP1是达到最大病毒滴度所必需的。综上所述，我们认为tRNA加工可能是宿主细胞被病毒接管期间发生的基因表达巧妙转变所不可或缺的。
