Cargando…

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

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 de...

Descripción completa

Detalles Bibliográficos
Autores principales: Heilmann, Emmanuel, Kimpel, Janine, Geley, Stephan, Naschberger, Andreas, Urbiola, Carles, Nolden, Tobias, von Laer, Dorotheé, Wollmann, Guido
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893670/
https://www.ncbi.nlm.nih.gov/pubmed/31717818
http://dx.doi.org/10.3390/v11110989
_version_ 1783476253313990656
author Heilmann, Emmanuel
Kimpel, Janine
Geley, Stephan
Naschberger, Andreas
Urbiola, Carles
Nolden, Tobias
von Laer, Dorotheé
Wollmann, Guido
author_facet Heilmann, Emmanuel
Kimpel, Janine
Geley, Stephan
Naschberger, Andreas
Urbiola, Carles
Nolden, Tobias
von Laer, Dorotheé
Wollmann, Guido
author_sort Heilmann, Emmanuel
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6893670
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-68936702019-12-23 The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion Heilmann, Emmanuel Kimpel, Janine Geley, Stephan Naschberger, Andreas Urbiola, Carles Nolden, Tobias von Laer, Dorotheé Wollmann, Guido Viruses Article 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. MDPI 2019-10-26 /pmc/articles/PMC6893670/ /pubmed/31717818 http://dx.doi.org/10.3390/v11110989 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Heilmann, Emmanuel
Kimpel, Janine
Geley, Stephan
Naschberger, Andreas
Urbiola, Carles
Nolden, Tobias
von Laer, Dorotheé
Wollmann, Guido
The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion
title The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion
title_full The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion
title_fullStr The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion
title_full_unstemmed The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion
title_short The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion
title_sort methyltransferase region of vesicular stomatitis virus l polymerase is a target site for functional intramolecular insertion
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893670/
https://www.ncbi.nlm.nih.gov/pubmed/31717818
http://dx.doi.org/10.3390/v11110989
work_keys_str_mv AT heilmannemmanuel themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT kimpeljanine themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT geleystephan themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT naschbergerandreas themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT urbiolacarles themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT noldentobias themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT vonlaerdorothee themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT wollmannguido themethyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT heilmannemmanuel methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT kimpeljanine methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT geleystephan methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT naschbergerandreas methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT urbiolacarles methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT noldentobias methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT vonlaerdorothee methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion
AT wollmannguido methyltransferaseregionofvesicularstomatitisviruslpolymeraseisatargetsiteforfunctionalintramolecularinsertion