Huang Cheng, Mantlo Emily, Paessler Slobodan
Department of Pathology, Galveston National Laboratory and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA.
Current address: Department of Microbiology & Immunology, Upstate Medical University, Syracuse, NY, USA.
bioRxiv. 2023 Dec 30:2023.04.12.536665. doi: 10.1101/2023.04.12.536665.
Lassa virus (LASV), a mammarenavirus from , is the causative agent of Lassa fever (LF) endemic in West Africa. Currently, there are no vaccines or antivirals approved for LF. The RNA-dependent RNA polymerases (RdRp) of RNA viruses are error-prone. As a negative-sense RNA virus, how LASV copes with errors in RNA synthesis and ensures optimal RNA replication are not well elucidated. LASV nucleoprotein (NP) contains a DEDDH 3'-to-5' exoribonuclease motif (ExoN), which is known to be essential for LASV evasion of the interferon response via its ability to degrade virus-derived double-stranded RNA. Herein, we present evidence that LASV NP ExoN has an additional function important for viral RNA replication. We rescued an ExoN-deficient LASV mutant (ExoN- rLASV) by using a reverse genetics system. Our data indicated that abrogation of NP ExoN led to impaired LASV growth and RNA replication in interferon-deficient cells as compared with wild-type rLASV. By utilizing PacBio Single Molecule, Real-Time (SMRT) long-read sequencing technology, we found that rLASV lacking ExoN activity was prone to producing aberrant viral genomic RNA with structural variations. In addition, NP ExoN deficiency enhanced LASV sensitivity to mutagenic nucleoside analogues in virus titration assay. Next-generation deep sequencing analysis showed increased single nucleotide substitution in ExoN- LASV RNA following mutagenic 5-flurouracil treatment. In conclusion, our study revealed that LASV NP ExoN is required for efficient viral RNA replication and mutation control. Among negative-sense RNA viruses, LASV NP is the first example that a viral protein, other than the RdRp, contributes to reduce errors in RNA replication and maintain genomic RNA integrity. These new findings promote our understanding of the basics of LASV infection and inform antiviral and vaccine development.
拉沙病毒（LASV）是一种来自沙粒病毒科的哺乳动物病毒，是西非地方性拉沙热（LF）的病原体。目前，尚无获批用于拉沙热的疫苗或抗病毒药物。RNA病毒的RNA依赖性RNA聚合酶（RdRp）容易出错。作为一种负链RNA病毒，拉沙病毒如何应对RNA合成中的错误并确保最佳RNA复制尚不清楚。拉沙病毒核蛋白（NP）含有一个DEDDH 3'至5'外切核糖核酸酶基序（ExoN），已知该基序对于拉沙病毒通过降解病毒衍生的双链RNA逃避干扰素反应至关重要。在此，我们提供证据表明拉沙病毒NP ExoN对病毒RNA复制具有重要的额外功能。我们使用反向遗传学系统拯救了一种ExoN缺陷型拉沙病毒突变体（ExoN - rLASV）。我们的数据表明，与野生型rLASV相比，NP ExoN的缺失导致拉沙病毒在干扰素缺陷细胞中的生长和RNA复制受损。通过利用PacBio单分子实时（SMRT）长读长测序技术，我们发现缺乏ExoN活性的rLASV易于产生具有结构变异的异常病毒基因组RNA。此外，在病毒滴定试验中，NP ExoN缺陷增强了拉沙病毒对诱变核苷类似物的敏感性。下一代深度测序分析显示，诱变5 - 氟尿嘧啶处理后，ExoN - 拉沙病毒RNA中的单核苷酸替换增加。总之，我们的研究表明拉沙病毒NP ExoN是高效病毒RNA复制和突变控制所必需的。在负链RNA病毒中，拉沙病毒NP是第一个除RdRp外，有助于减少RNA复制错误并维持基因组RNA完整性的病毒蛋白实例。这些新发现促进了我们对拉沙病毒感染基础的理解，并为抗病毒和疫苗开发提供了信息。
