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*
Identifiers and Pagination:Year: 2011
First Page: 103
Last Page: 108
Publisher Id: TOVJ-5-103
Article History:Received Date: 16/5/2011
Revision Received Date: 29/6/2011
Acceptance Date: 13/7/2011
Electronic publication date: 6/9/2011
Collection year: 2011
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.
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.