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In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements.

ABSTRACT: Requirements for capped leader sequences for use during transcription initiation by tomato spotted wilt virus (TSWV) were tested using mutant alfalfa mosaic virus (AMV) RNAs as specific cap donors in transgenic Nicotiana tabacum plants expressing the AMV replicase proteins. Using a series of AMV RNA3 mutants modified in either the 5'-non-translated region or in the subgenomic RNA4 leader, sequence analysis revealed that cleaved leader lengths could vary between 13 and 18 nucleotides. Cleavage occurred preferentially at an A residue, suggesting a requirement for a single base complementarity with the TSWV RNA template, which could be confirmed by analyses of host mRNAs used in vivo as cap donors.

SUBMITTER: Duijsings D

PROVIDER: S-EPMC125463 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements.

Duijsings D D Kormelink R R Goldbach R R

The EMBO journal 20010501 10

Requirements for capped leader sequences for use during transcription initiation by tomato spotted wilt virus (TSWV) were tested using mutant alfalfa mosaic virus (AMV) RNAs as specific cap donors in transgenic Nicotiana tabacum plants expressing the AMV replicase proteins. Using a series of AMV RNA3 mutants modified in either the 5'-non-translated region or in the subgenomic RNA4 leader, sequence analysis revealed that cleaved leader lengths could vary between 13 and 18 nucleotides. Cleavage oc ...[more]

PMID: 11350944

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Cap-snatching in TSWV

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Cap-snatching mechanism in yeast L-A double-stranded RNA virus.

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Cap snatching in yeast L-BC double-stranded RNA totivirus.

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Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation

Project description:The influenza polymerase cleaves host RNAs ~10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs.

2015-04-23 | GSE65101 | GEO

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