Dataset Information

A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

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.

SUBMITTER: Ishibashi K

PROVIDER: S-EPMC5627289 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

Ishibashi Kazuhiro K Matsumoto-Yokoyama Eiko E Ishikawa Masayuki M

Scientific reports 20171004 1

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 Sacchar ...[more]

PMID: 28978935

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Project description:BackgroundThe intergenic region (IR) of ambisense RNA segments from animal- and plant-infecting (-)RNA viruses functions as a bidirectional transcription terminator. The IR sequence of the Tomato spotted wilt virus (TSWV) ambisense S RNA contains stretches that are highly rich in A-residues and U-residues and is predicted to fold into a stable hairpin structure. The presence of this hairpin structure sequence in the 3' untranslated region (UTR) of TSWV mRNAs implies a possible role in translation.Methodology/principal findingsTo analyse the role of the predicted hairpin structure in translation, various Renilla luciferase constructs containing modified 3' and/or 5' UTR sequences of the TSWV S RNA encoded nucleocapsid (N) gene were analyzed for expression. While good luciferase expression levels were obtained from constructs containing the 5' UTR and the 3' UTR, luciferase expression was lost when the hairpin structure sequence was removed from the 3' UTR. Constructs that only lacked the 5' UTR, still rendered good expression levels. When in addition the entire 3' UTR was exchanged for that of the S RNA encoded non-structural (NSs) gene transcript, containing the complementary hairpin folding sequence, the loss of luciferase expression could only be recovered by providing the 5' UTR sequence of the NSs transcript. Luciferase activity remained unaltered when the hairpin structure sequence was swapped for the analogous one from Tomato yellow ring virus, another distinct tospovirus. The addition of N and NSs proteins further increased luciferase expression levels from hairpin structure containing constructs.Conclusions/significanceThe results suggest a role for the predicted hairpin structure in translation in concert with the viral N and NSs proteins. The presence of stretches highly rich in A-residues does not rule out a concerted action with a poly(A)-tail-binding protein. A common transcription termination and translation strategy for plant- and animal-infecting ambisense RNA viruses is being discussed.

Dataset Information

A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

Publications

A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

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