The Open Virology Journal

2011, 5 : 103-108
Published online 2011 September 6. DOI: 10.2174/1874357901105010103
Publisher ID: TOVJ-5-103

RESEARCH ARTICLE
 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 and Jean-François Eléouët, *
INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France

* 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..