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Rescue of tomato spotted wilt virus entirely from complementary DNA clones

文献类型： 外文期刊

作者： Feng, Mingfeng 1 ; Cheng, Ruixiang 1 ; Chen, Minglong 1 ; Guo, Rong 1 ; Li, Luyao 1 ; Feng, Zhike 1 ; Wu, Jianyan 1 ; Xie 1 ;

作者机构： 1.Nanjing Agr Univ, Dept Plant Pathol, Nanjing 210095, Peoples R China

2.Zhejiang Univ, Anal Ctr Agrobiol & Environm Sci, Hangzhou 317502, Peoples R China

3.Yunnan Acad Agr Sci, Inst Biotechnol & Genet Resources, Yunnan Prov Key Lab Agri Biotechnol, Kunming 650223, Yunnan, Peoples R China

4.Wageningen Univ, Dept Plant Sci, Lab Virol, NL-6708 PB Wageningen, Netherlands

关键词： reverse genetics system; tomato spotted wilt virus; negative-strand RNA virus; minireplicon; genome-length infectious cDNA clones

期刊名称：PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA （ 影响因子：11.205； 五年影响因子：12.291 ）

ISSN： 0027-8424

年卷期： 2020 年 117 卷 2 期

页码：

收录情况： SCI

摘要： 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.

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