Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy.
The 2B protein of enteroviruses is the viral membrane-active protein that is responsible for the modifications in host cell membrane permeability that take place in enterovirus-infected cells. The 2B protein shows structural similarities to the group of lytic polypeptides, polypeptides that permeate membranes either by forming multimeric membrane-integral pores or, alternatively, by lying parallel to the lipid bilayer and disturbing the curvature and symmetry of the membrane. Our aim is to gain more insight into the molecular architecture of the 2B protein in vivo. In this study, the possible existence of multimers of the coxsackie B3 virus 2B protein in single living cells was explored by fluorescence resonance energy transfer (FRET) microscopy. FRET between fusion proteins 2B-ECFP and 2B-EYFP (enhanced cyan and yellow fluorescent variants of green fluorescent protein) was monitored by using spectral imaging microscopy (SPIM) and fluorescence lifetime imaging microscopy (FLIM). Both techniques revealed the occurrence of intermolecular FRET between 2B-ECFP and 2B-EYFP, providing evidence for the formation of protein 2B homomultimers. Putative models for the mode of action of the membrane-active 2B protein and the formation of membrane-integral pores by 2B multimers are discussed.
Department of Medical Microbiology, University Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands. f.vankuppeveld@ncmls.kun.nl
Frank J M
FJ
van Kuppeveld
Willem J G
WJ
Melchers
Peter H G M
PH
Willems
Theodorus W J
TW
Gadella
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000502:physiology*; D019281:Dimerization; D004735:Energy Transfer; D029822:Enterovirus B, Human / Q000378:metabolism / Q000472:pathogenicity; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C097017:coxsackie B3 virus protein 2B; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins