Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM.
We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60 min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly.
Council for the Central Laboratory of the Research Councils, Daresbury Laboratory, Daresbury, Warrington WA4 4AD, United Kingdom.
Marisa
M
Martin-Fernandez
Samantha V
SV
Longshaw
Ian
I
Kirby
George
G
Santis
Mark J
MJ
Tobin
David T
DT
Clarke
Gareth R
GR
Jones
D000260:Adenoviruses, Human / Q000472:pathogenicity / Q000502:physiology* / Q000648:ultrastructure*; D016880:Anisotropy; D036022:Capsid Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology* / Q000821:virology*; D008856:Microscopy, Fluorescence / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D009038:Motion; D009862:Online Systems; D019065:Virus Assembly / Q000502:physiology
D036022:Capsid Proteins