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Open AccessArticle

The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion

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Division of Virology, Medical University of Innsbruck, 6020 Innsbruck, Austria
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Christian Doppler Laboratory for Viral Immunotherapy of Cancer, Medical University of Innsbruck, 6020 Innsbruck, Austria
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Division of Molecular Pathophysiology, Medical University of Innsbruck, 6020 Innsbruck, Austria
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Division of Genetic Epidemiology, Medical University of Innsbruck, 6020 Innsbruck, Austria
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ViraTherapeutics GmbH, 6020 Innsbruck, Austria
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Authors to whom correspondence should be addressed.
Viruses 2019, 11(11), 989; https://doi.org/10.3390/v11110989
Received: 13 September 2019 / Revised: 20 October 2019 / Accepted: 23 October 2019 / Published: 26 October 2019
(This article belongs to the Section Animal Viruses)
The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies. View Full-Text
Keywords: vesicular stomatitis virus; VSV; polymerase; insertion; methyltransferase vesicular stomatitis virus; VSV; polymerase; insertion; methyltransferase
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Heilmann, E.; Kimpel, J.; Geley, S.; Naschberger, A.; Urbiola, C.; Nolden, T.; von Laer, D.; Wollmann, G. The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion. Viruses 2019, 11, 989.

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