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