Modulating the function of the measles virus RNA-dependent RNA polymerase by insertion of green fluorescent protein into the open reading frame.
Measles virus (MV) is the type species of the Morbillivirus genus and its RNA-dependent RNA polymerase complex is comprised of two viral polypeptides, the large (L) and the phospho- (P) proteins. Sequence alignments of morbillivirus L polymerases have demonstrated the existence of three well-conserved domains (D1, D2, and D3) which are linked by two variable hinges (H1 and H2). Epitope tags (c-Myc) were introduced into H1 and H2 to investigate the tolerance of the variable regions to insertions and to probe the flexibility of the proposed domain structures to spatial reorientation. Insertion into H1 abolished polymerase activity whereas introduction into H2 had no effect. The open reading frame of enhanced green fluorescent protein was also inserted into the H2 region of the MV L gene to extend these observations. This resulted in a recombinant protein that was both functional and autofluorescent, although the overall polymerase activity was reduced by over 40%. Two recombinant viruses which contained the chimeric L genes EdtagL(MMc-mycM) and EdtagL(MMEGFPM) were generated. Tagged L proteins were detectable, by indirect immunofluorescence in the case of EdtagL(MMc-mycM) and by autofluorescence in the case of EdtagL(MMEGFPM). We suggest that D3 enjoys a limited conformational independence from the other domains, indicating that the L polymerases of the Mononegavirales may function as multidomain proteins.
Duprex
W Paul
WP
School of Biology and Biochemistry, The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom. p.duprex@qub.ac.uk
Collins
Fergal M
FM
Rima
Bert K
BK
D000818:Animals; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008459:Measles virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D014709:Vero Cells
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012324:RNA-Dependent RNA Polymerase