title: 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.
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.
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France.
authors.forename: Jenna
authors.initials: J
authors.lastname: Fix
authors.forename: Marie
authors.initials: M
authors.lastname: Galloux
authors.forename: Marie-Lise
authors.initials: ML
authors.lastname: Blondot
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eléouët