Rescue of tomato spotted wilt virus entirely from complementary DNA clones

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of 5 RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and gamma b, but TSWV Ms interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

负链/双义RNA病毒（NSVs）不仅包括具有医学重要性的危险病原体，还包括具有农学重要性的严重植物病原体。番茄斑萎病毒（TSWV）是最重要的植物NSVs之一，可感染1000多种植物，对全球粮食安全构成重大威胁。然而，TSWV的分节段负链/双义RNA基因组一直是分子遗传操作的主要障碍。在本研究中，我们报道了完全从互补DNA（cDNA）克隆中恢复具有感染性的TSWV。首先，基于由35S驱动的S（-）基因组（g）或S（+）反基因组（ag）RNA模板构建体建立了具有复制和转录能力的微型基因组复制系统，其两侧为丁型肝炎病毒的5'锤头结构和3'核酶序列、一个核衣壳（N）蛋白基因以及密码子优化的病毒RNA依赖的RNA聚合酶（RdRp）基因。接下来，基于M（-） - gRNA开发了具有移动能力的微型基因组复制系统，该系统能够补充由5个RNA复制子重构的核糖核蛋白复合物（RNPs）的细胞间和系统移动。最后，通过同时表达编码S（+） - agRNA、M（-） - gRNA和L（+） - agRNA的全长cDNA构建体（其中M（-） - gRNA的糖蛋白基因序列经过优化），在植物中拯救出具有感染性的TSWV以及携带eGFP报告基因的衍生物。在添加包括P19、HcPro和γb在内的各种RNA干扰抑制因子的情况下发生了病毒拯救，但TSWV的M基因干扰了基因组RNA的拯救。这种针对TSWV的反向遗传学系统现在允许对病毒感染周期和致病性的各个方面进行详细的分子遗传学分析。