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.
Funder
National Natural Science Foundation of China
Publisher
Proceedings of the National Academy of Sciences
Reference59 articles.
1. M. K. David , M. H. Peter , Fields Virology, (Lippincott Williams & Wilkins, ed. 6, 2013), pp. 1–2264.
2. A. Plyusnin , R. M. Elliott , The Bunyaviridae: Molecular and Cellular Biology (Caister Academic Press, 2011), pp. 1–222.
3. Tospoviruses: Diagnosis, Molecular Biology, Phylogeny, and Vector Relationships
4. Interaction between Rice stripe virus Disease-Specific Protein and Host PsbP Enhances Virus Symptoms
5. Tenuivirus utilizes its glycoprotein as a helper component to overcome insect midgut barriers for its circulative and propagative transmission;Lu;PLoS Pathog.,2019
Cited by
60 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献