Rust et al 2004Troels Scheel
Single molecule biophysics
Assembly of endocytic machinery around individual influenza viruses during viral entry
Influenza Virus – www.medizin.de
Troels Scheel Rust et al 2004Influenza virus
life cycle
www.northwestern.edu
Troels Scheel Rust et al 2004Aim of study
• Aims:– To characterize endocytic mechanism of
Influenza virus infection - on a single-virus level– To elucidate through which pathways
endocytosis occurs• Clathrin-mediated• Clathrin- and caveolin-independent
– Formation of clathrin coated pits
Dr. Helge Gad, Karolinska Institute, Stockholm
Troels Scheel Rust et al 2004CCP / CCV
flourescence
A: Immunoflourescence (Ab) of clathrin-coated pits
B: EYFP-clathrin, transfected cell
C: overlay A/B (yellow)
D: Transferrin (red) internalization in EYFP-clathrin (green) cell
Test of EYFP incorporation into clathrin-coated pits
Troels Scheel Rust et al 2004Visualizing influenza
• Virus labelling– DiD (lipophilic)
Rust et al 2004 video 1.mp4
Troels Scheel Rust et al 2004Entry pathways
• 65%: clathrin-associated• 35%: no association
Rust et al 2004 video 3.mp4 Rust et al 2004 video 5.mp4
• 3-stage process– I: actin-dependent surface movement– II: rapid unidirectional, towards perinuclear region– III: bidirectional microtubule-dependent
Troels Scheel Rust et al 2004Formation
of CCPs
• Formation of CCPs:– 94% de novo
formation of CCPs– Prevalence at
random sites ~1:20
– Prevalence at earlier binding sites ~1:20
– Formation at mobile hospots should cause exp decay
Troels Scheel Rust et al 2004Dynamics of CCPs
• Dynamics of CCPs– On average
• Initiates 190s after virus binding
• Persists for 70s• CCV uncoating in few
seconds• Proceed to stage II
within 40s– Longer time before
stage II could be caused by recycling and a second endocytosis
Troels Scheel Rust et al 2004Neuraminidase effect
• Neuraminidase effect– Cleave multivalent
bands between HA and cell surface sialic acid
– Thereby facilitating viral movement?
– NA inhibitor no effect on endocytosis
Troels Scheel Rust et al 2004Viral fusion
• Test for viral fusion in both pathways– Quenching by high
density viral surface DiD
– 65% clathrin-associated
– 35% non-associated
Rust et al 2004 video 6.mp4 Rust et al 2004 video 7.mp4
Troels Scheel Rust et al 2004Trafficking kinetics
• Equal binding time– Pathways
competing for viral entry
• Equal distance to nucleus
• Equal stage II time– Convergence
of endocytic trafficking
Troels Scheel Rust et al 2004Conclusions
• Influenza viruses enter cells by at least two endocytic pathways
• ~2/3 enters by clathrin-mediated endocytosis
• Allmost all CCPs are formed de novo• Pathways have equal kinetics• No NA effect on endocytosis• Signal to clathrin?
– Curvature of membrane by multivalent HA binding
Yoshihiro Kawaoka, University of Wisconsin-Madison
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