Abstract
Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus of the order Bunyavirales, family Tospoviridae, genus Orthotospovirus. TSWV infects a broad range of plant species, causing serious economic losses. Despite its agronomic importance, molecular biological understanding of TSWV has been limited, partly due to the lack of a reverse genetics system, which would enable genetic manipulation of the virus. Here, we report that RNA synthesis by TSWV RNA polymerase occurs in the yeast Saccharomyces cerevisiae using a segment of the TSWV genome, S RNA expressed from cloned cDNA, as a template. Viral nucleocapsid protein was required for RNA synthesis. Replacement of the protein-coding and intergenic regions of TSWV S RNA by a yellow fluorescent protein (YFP)-coding sequence drastically increased the accumulation of both sense and antisense strands of the RNA, showing that this RNA was replicated. Using this system, we revealed that efficient RNA synthesis by TSWV RNA polymerase in yeast requires the 5′-terminal 17-nt and 3′-terminal ~50-nt regions of the TSWV S cRNA (complementary RNA to the genomic RNA) template.
Highlights
One of the most important achievements in RNA virus research is the development of reverse genetics systems which make possible the genetic manipulation of viruses[1]
Tomato spotted wilt virus (TSWV) S cDNA was sandwiched between a hammerhead ribozyme and a hepatitis delta virus ribozyme and inserted downstream of the CUP1 promoter such that TSWV S cRNA [i.e., complementary RNA to the TSWV S virion RNA] with precise terminal sequences was presumably generated after transcript self-cleavage
These results indicate that S virion RNA (vRNA) synthesis using the S cRNA template by TSWV RNA polymerase occurred in yeast
Summary
One of the most important achievements in RNA virus research is the development of reverse genetics systems which make possible the genetic manipulation of viruses[1]. Schnell et al (1994) described the recovery of recombinant rabies virus, a non-segmented negative-strand RNA virus, from host cells transformed with plasmids expressing viral RNA and proteins[2]. This was the first report of a reverse genetics system for a negative-strand RNA virus. M and S RNAs are ambisense and encode protein NSm and glycoproteins G1 and G2, and proteins NSs and N (nucleocapsid protein), respectively Functional analyses of these proteins using heterologous expression systems have revealed that NSs is an RNA silencing suppressor[9] and NSm is a cell-to-cell movement protein[10]. We aimed to develop a TSWV replicon system in yeast, and to determine the sequence requirements for TSWV RNA replication
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