1. Rust et al 2004Troels Scheel Single molecule biophysics Assembly of endocytic machinery around individual influenza viruses during viral entry Influenza Virus – www.medizin.de

2. Troels ScheelRust et al 2004 Influenza virus life cycle www.northwestern.edu

3. Troels ScheelRust et al 2004 Aim 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

4. Troels ScheelRust et al 2004 CCP / 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

5. Troels ScheelRust et al 2004 Visualizing influenza Virus labelling –DiD (lipophilic)

6. Troels ScheelRust et al 2004 Entry pathways 65%: clathrin-associated 35%: no association 3-stage process –I: actin-dependent surface movement –II: rapid unidirectional, towards perinuclear region –III: bidirectional microtubule-dependent

7. Troels ScheelRust et al 2004 Formation 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

8. Troels ScheelRust et al 2004 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

9. Troels ScheelRust et al 2004 Neuraminidase effect –Cleave multivalent bands between HA and cell surface sialic acid –Thereby facilitating viral movement? –NA inhibitor  no effect on endocytosis

10. Troels ScheelRust et al 2004 Viral fusion Test for viral fusion in both pathways –Quenching by high density viral surface DiD –65% clathrin- associated –35% non-associated

11. Troels ScheelRust et al 2004 Trafficking kinetics Equal binding time –Pathways competing for viral entry Equal distance to nucleus Equal stage II time –Convergence of endocytic trafficking

12. Troels ScheelRust et al 2004 Conclusions 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