1. The Many Faces of Phosphatidylserine (PS) In Virus Entry David Coil Miller Lab

2. Why study Virus Entry? + = + = Basic Biology Disease Gene Therapy

3. Lipid bilayer Basic anatomy of a enveloped virion Virus capsid Envelope protein Viral genome

5. Characterized Virus Receptors Single Transmembrane Proteins Multiple Membrane-Spanning Proteins GPI-anchored Proteins Carbohydrates

7. Vesicular Stomatitis Virus (VSV) From Fields Virology p. 1122 From F.A. Murphy, UC Davis

8. Cellular Receptor for VSV -Infectable cell extracts still inhibited binding after treatment with: -trypsin -pronase -Neuraminidase -heating to 100 ºC -However the inhibitory factor was soluble in chloroform-methanol and sensitive to PLPC. Conclusion: Phospholipid -Attempted to inhibit VSV binding with various purified lipids and only phosphatidylserine (PS) totally inhibited binding

9. Phosphatidylserine (PS) -Ubiquitous membrane lipid -Primarily found on inner leaflet of the plasma membrane -Exposure of PS is a hallmark of apoptosis

10. Annexin-V (binds to PS)

11. Annexin-V Staining Frog Unstained Cells Cell incubated with Annexin-V Minnow Zebrafish Mosquito

12. PS level (relative fluorescence units) VSV - (iu/ml) 50 100 150 250 200 350 300 0 10 7 10 8 10 9 10 6 10 5 10 4 <10 3 10 10 400 Chicken Human Dog Quail Hamster Minnow Mosquito Zebrafish Frog PS level (relative fluorescence units) VSV - GFP titer 50 100 150 250 200 350 300 0 10 7 10 8 10 9 10 6 10 5 10 4 <10 3 10 10 400 Chicken Human Dog Quail Hamster Minnow Mosquito Zebrafish Frog PS levels on cells versus VSV Infection

13. VSV - G - GFP binding per unit cell surface area 20 40 60 100 80 120 0 0 2 4 8 6 Minnow Hamster Mosquito PS levels per unit cell surface area VSV - G - GFP binding per unit cell surface area 20 40 60 100 80 120 0 0 2 4 8 6 Minnow Hamster Mosquito PS levels on cells versus VSV Binding Quail Chicken Zebrafish Dog Human Frog 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 3 4 1.2 Quail Chicken Zebrafish Dog Human Frog 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 3 4 1.2

14. Cell counts Annexin-V binding Preincubation with annexin-V Preincubation without annexin-V Unlabeled cells Saturation of cell-surface PS with annexin-V Zebrafish Cells

15. Effect of annexin-V saturation on VSV infection 0 200 400 600 800 1000 1200 # of Infected Cells (zebrafish) VSV alone With annexin-V

16. Annexin Interference with Binding VSV Binding Cell counts Unlabeled cells Preincubation with annexin-V Preincubation without annexin-V

17. PS as a “Fusion Receptor” for VSV? -Some viruses such as HIV, SIV and FELV-T require two component receptors -Characterization of a small region of VSV-G that interacts with target membranes at low pH, (Durrer et al 1995) -Increased PS in target membrane enhances VSV fusion in vitro (Carneiro et al 2002) -A peptide within this region has been shown to have PS binding capability in vitro (Coll 1997)

18. Summary of VSV Results -VSV infection does not correlate with PS levels -VSV binding does not correlate with PS levels -Saturating concentrations of annexin-V do not inhibit VSV infection -Saturating concentrations of annexin-V do not inhibit VSV binding -Potential role for PS as a secondary receptor for VSV

19. Generation of PS liposomes -comes as free lipid in chloroform/methanol -dry under nitrogen -resuspend in PBS -sonicate to generate uniform vesicles (bilayer liposomes formed with preference to micells)

20. Blue = unstained negative control Green = Annexin-V stained cells Red = Annexin-V stained cells (with PS) PS liposome addition to cells increases cell surface PS levels (8-fold change) Mouse Cells

21. VSV - G None VSV - G + PS 20 25 10 15 30 5 0 GFP Vector Env: Liposomes: VSV - G None VSV - G + PS 20 25 10 15 30 5 0 -positive cells per 10 3 cells Vector Env: Liposomes: Effect of PS on Virus Infection RD114 None RD114 + PS RD114 None RD114 + PS

22. Enhancement of enveloped virus infection following treatment of cells with PS Virus Fold increase Maximum Cell type envelope in infection n titer 3-6 4 1.8 x 10 4 RD114 8-11 4 2.4 x 10 4 GALV 3 2 6.6 x 10 4 A-MLV 3-5 2 1.3 x 10 5 RD114 2-5 5 3.0 x 10 6 JSRV 4-8 3 3.4 x 10 4 JSRV 2-7 9 6.0 x 10 3 RD114 10-20 10 4.5 x 10 5 ZF4 HTX Rat-2/Hyal2 NIH 3T3/RDR NIH 3T3/Pit1 GALV 3-8 2 1.8 x 10 4 VSV-G

23. PS treatment does not allow infection when a functional receptor is not present Virus Envelope Cell Type Titer Titer with PS (iu/ml) (iu/ml) MoMLV (ecotropic) 293 <1 <1 HTX <1 <1 JSRV NIH 3T3 <1 <1 208F <1 <1 NRK <1 <1 Rat-2 <1 <1 GALV NIH 3T3 <1 <1 AKR6 (xenotropic MLV) CHO-K1 <1 <1

24. PS treatment does not allow infection when a functional receptor is not present Virus Envelope Cell Type Titer Titer with PS (iu/ml) (iu/ml) MoMLV (ecotropic) 293 <1 <1 HTX <1 <1 JSRV NIH 3T3 <1 <1 208F <1 <1 NRK <1 <1 Rat-2 <1 <1 GALV NIH 3T3 <1 <1 AKR6 (xenotropic MLV) CHO-K1 <1 <1

25. Can related phospholipids enhance virus infection? Phosphatidylserine Phosphatidylcholine Phosphatidylglycercol Phosphatidylethanolamine

26. Liposomes 0 40 80 120 160 None PS Liposomes 0 40 80 120 160 None PS JSRV Infection of human cells (foci/well) Enhancement of infection is specific to PS PC PE PG PC PE PG

27. Summary of PS effects -Increases enveloped virus infection 2 to 20-fold -Receptor-dependent -Specific to PS -Does not affect receptor levels or virus binding -Does not enhance non-enveloped virus infection -Rapid timecourse

28. Model for PS Fusion Effect (normal cells) Virus Membrane Target Cell Membrane High Energy

29. Virus Membrane Target Cell Membrane Lower Energy Model for PS Fusion Effect (PS treated cells)

30. PS is a powerful tool used to enhance virus infection -generated in large batches -freezes well - synergistic effects with Polybrene (PB) -effects in vivo ?

31. Receptor present Receptor absent Infection No infection Glycosylation-Blocked Receptors Receptor blocked by glycosylation No infection

32. Target Cells Virus Type PS treatment Titer (iu/ml) Mouse RD114 - <1 RD114 + Hamster MoMLV - <5 MoMLV + Infection of cells containing glycosylation-blocked receptors 2.3 x 10 4 2.3 x 10 3 Hypothesis: PS treatment affects the glycosylation machinery of the cell.

33. RD114 Receptor (glycosylated) RD114 Receptor (unglycosylated) PNGaseF PS − − + − Effect of PS treatment on receptor glycosylation

34. RD114 Receptor (glycosylated) RD114 Receptor (unglycosylated) PNGaseF PS − − + − + + + − Effect of PS treatment on receptor glycosylation Conclusion: PS treatment does not affect the glycosylation machinery of the cell.

35. Glycosylation-Blocked Receptors Receptor present Receptor absent Infection No infection Receptor present Receptor absent Infection No infection PS treatment allows infection No infection No infection +PS Huh? Receptor blocked by glycosylation No infection Receptor blocked by glycosylation No infection Receptor blocked by glycosylation No infection Infection

36. Normal mouse cells No infection Overexpressed RD114 Receptor RD114 virus Mouse cells overexpressing the human RD114 receptor (RDR) Infection

37. PS Addition Timecourse (24 hours) PS Levels Time after PS addition (hours) Annexin-V staining Threshold effect? Normal mouse cells Time after PS addition (hours) RD114 infection Mouse cells with hRDR Time after PS addition (hours) RD114 infection

38. PS Level 200 µM 400 µM Annexin-V binding (fluorescence units) 0 40 80 120 0 5 10 15 20 25 30 PS Exposure Time (h) RD114 Infection (mouse cells) 400 µM 200 µM 200 400 600 0 RD114 Infection (average foci/well)

39. Standard Glycosylation Block Model Virus Cell No recognition

40. Requirement for multi-valent contact Virus Cell Association prevented

41. Reduced requirement for multi-valent contact with PS treatment Virus Cell +PS

42. Summary -Treatment of target cells with PS enhances infection by enveloped viruses, most likely through an effect on virus fusion -Utility of PS as a tool for virus infection -PS is not the cell-surface receptor for VSV -PS treatment allows certain viruses to overcome glycosylation-blocked receptors No infection No infection Infection No infection No infection

43. Acknowledgments Thesis Committee: Dusty Miller Michael Emerman Larry Rohrschneider Stan Mcknight John Albers Michele Karantsavelos MaryEllin Robinson Human Biology Office Miller Lab: John Alfano Josh Danke Clarissa Dirks Christine Halbert Neal Van Hoeven Shan-Lu Liu Siu Ling Lam Vladimir Vigdorovich Sarah Wooten Strong Lab Geballe Lab Funding: MCB VOTG Training Grant Dusty Miller

44. Dusty Miller

45. Isabelle