Architecture of ebolavirus particles

1. Architecture of ebolavirus particles • GP mediates all of the steps in entry, including cell attachment and fusion between viral and cellular lipid bilayers

2. Prefusion structure of GP fp GP1 GP2 nhr chr msd Lee et al., 2008 C

3. Prefusion structure of GP

4. Deploying the GP fusion machine: Priming Steps Prime Furin cleavage furin cleavage site

5. Deploying the GP fusion machine: Triggering Step(s) Host Signals fp Trigger Acid pH nhr

6. Deploying the GP fusion machine: GP2 refolding cellular membrane fused membrane fp . . . . . . fp chr Trigger nhr Acid pH nhr Malashkevich, 1997 Weissenhorn, 1998

7. Key regulatory role for GP1 in membrane fusion? fused membrane Host Signals fp . . . . . . fp chr GP1 Trigger nhr GP2 Acid pH chr C msd • Hypothesis: GP1 senses host signal and “unclamps” GP2 to trigger membrane fusion

8. rbs head glycan cap base Base subdomain: N–terminal regions of GP1 and GP2 GP1 head subdomain: Insertion into base subdomain Glycan cap subdomain: C–terminal variable region in GP1 Structural subdomains of the prefusion GP trimer fp chr msd C

9. Can we generate mutant viruses that do not require cleavage by CatB?

10. VSV pseudotype system does not permit forward genetic studies of ebolavirus entry

11. Recombinant VSV expressing ebolavirus GP and EGFP GP BHK cells rVSV-GP WT Genomic plasmid Vaccinia- T7 virus

12. rVSV–GP plaque

13. rVSV-GP infection is blocked by a CatB inhibitor

14. Forward genetic strategy to identify CatB-independent viruses

15. Isolation of mutants resistant to CatB inhibitor

16. Isolation of mutants resistant to CatB inhibitor

17. Mutants require neither CatL nor CatB for entry ... • Viruses have not adapted to require CatL instead of CatB

18. ... but they do require cysteine cathepsin activity • Viruses can use least one non-CatL/CatB cysteine cathepsin in Vero cells

19. How do mutations affect generation of GP proteolytic intermediates?

20. VSV(GP–18K) VSV(GP–17K) Do GP proteolytic intermediates differ in their sensitivity to proteolysis? Cannot convert GP–18K to GP–17K GP cleavage CatL ? pH 5.5 Viral infectivity

21. Relationship between CatB dependence and GP–17K proteolytic stability Protease-sensitive 17K? Filovirus GP CatB–dep? Zaire Zaire mutants (some) Marburg Sudan Zaire GP1–Sudan GP2 Sudan GP1–Zaire GP2

22. Hypothesis: Some CatB-independent GP proteins generate “hair-trigger” entry intermediates WT: Mutants: Trigger Post-fusion conformation 18K 17K 17K* Protease Degradation • Entry inhibitors select for hair-trigger mutants with other class I fusion glycoproteins (e.g., HIV-1 Env mutants with altered CCR5 receptor usage)

23. Models for how mutants bypass the CatB requirement WT: Trigger Cat? CatL CatB Post-fusion conformation GP 18K 17K Class 1: Uses alternative protease to do 18K→17K step Class 2:Conformational change in 17K accelerates downstream step(s) Class 3: Conformational change in 18K allows bypass of 18K→17K step entirely

24. Exposes rbs Destabilizes pre-fusion conformer Protease plays role in triggering? What do mutants teach us about entry by WT GP? Post-fusion

25. Einstein Tony Wong Rohini Sandesara Nirupama Mulherkar Emily Miller Harvard Medical School Sean Whelan James Cunningham The Scripps Research Institute Erica Saphire Financial support NIH Keystone Symposia Organizers Acknowledgements

26. Lowering a glycan shield fp muc chr msd C Adapted from Lee et al., 2008