Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.
Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.
Toivola
Jouni
J
University of Jyväskylä, Department of Biological and Environmental Science, PO Box 35, FIN-40014 University of Jyväskylä, Finland.
Ojala
Kirsi
K
Michel
Patrik O
PO
Vuento
Matti
M
Oker-Blom
Christian
C
D000465:Algorithms; D000818:Animals; D016367:Baculoviridae / Q000737:chemistry* / Q000378:metabolism*; D003902:Detergents; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D014760:Viral Fusion Proteins / Q000378:metabolism
D003902:Detergents; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D014760:Viral Fusion Proteins; D049452:Green Fluorescent Proteins