The Insertion of Fluorescent Proteins in a Variable Region of Respiratory Syncytial Virus L Polymerase Results in Fluorescent and Functional Enzymes But with Reduced Activities



Jenna Fix, Marie Galloux, Marie-Lise Blondot, Jean-François Eléouët*
INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France


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© Fix et al.; Licensee Bentham Open

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http: //creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France; Tel: (33) 1 34 65 26 04; Fax: (33) 1 34 65 26 21; E-mail: jean-francois.eleouet@jouy.inra.fr


Abstract

The respiratory syncytial virus (RSV) Large protein L is the catalytic subunit of the RNA-dependent RNA polymerase complex. Currently, no structural information is available for RSV L. Sequence alignments of L protein from human and bovine strains of RSV revealed the existence of two variable regions, VR1 and VR2. Following comparison with morbillivirus and rhabdovirus L genes, VR2, which is located between domains V and VI, was chosen as an insertion site for sequences encoding the epitope tag HA or the fluorescent proteins eGFP and mCherry. Recombinant tagged-L proteins co-localized with RSV N and P proteins in transfected cells. These recombinant polymerases were shown to be functional using a viral minigenome system assay, their activities being reduced by ~70% compared to the unmodified L polymerase. We have also shown by site-directed mutagenesis that the GDNQ motif (residues 810-813 for the Long strain of HRSV) is essential for L activity.

Keywords: Respiratory syncytial virus, L protein, RNA-dependent RNA polymerase, HA tag, eGFP, mCherry..