Rescue of tomato spotted wilt virus entirely fromcomplementary DNA clones

Mingfeng Feng, Ruixiang Cheng, Minglong Chen, Rong Guo, Luyao Li, Zhike Feng, Jianyan Wu, Li Xie, Jian Hong, Zhongkai Zhang, R.J.M. Kormelink, Xiaorong Tao*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

4 Citations (Scopus)

Abstract

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 S 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 γb, but TSWV NSs 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.
Original languageEnglish
Pages (from-to)1181-1190
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number2
Early online date26 Dec 2019
DOIs
Publication statusPublished - 14 Jan 2020

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