1 / 24

Integrating studies of viral, receptor, antibody structures, functions, cell biology, & evolution.

Integrating studies of viral, receptor, antibody structures, functions, cell biology, & evolution. Example: Parvovirus capsids, receptors, and antibodies – controlling host range, cell infection and/or neutralization. Colin Parrish. Cornell Laura Palermo Christian Nelson Wendy Weichert

sue
Download Presentation

Integrating studies of viral, receptor, antibody structures, functions, cell biology, & evolution.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Integrating studies of viral, receptor, antibody structures, functions, cell biology, & evolution. Example: Parvovirus capsids, receptors, and antibodies – controlling host range, cell infection and/or neutralization. Colin Parrish Cornell • Laura Palermo • Christian Nelson • Wendy Weichert • Gail Sullivan • Karsten Hueffer Collaborators • Michael Rossmann • Susan Hafenstein • Mavis Agbandje-McKenna • Alan Simpson • Eddie Holmes • Laura Shackelton • Pamela Bjorkman

  2. Canine parvovirus (CPV) & feline panleukopenia virus (FPV) Non-enveloped, T =1 icosahedral capsid, 26 nm diameter. Single-stranded DNA genome 2 genes – 4 overlapping proteins. Capsid - 60 copies of VP2 and VP1 (90% VP2; 10% VP1). VP2 forms capsids; VP1 required for cell infection. ~5120 nts 3’ 5’ VP1 NS1 VP2 NS2

  3. Emergence and evolution of CPV. FPV MEV RPV CPV type-2a CPV type-2b 1990 1984 1970s 1 1979 1 5 17 1 1978- 1980 CPV type-2 • Questions: • What viral changes allowed the new canine host range? • How do changes alter virus-host interaction(s) (receptors)? • Other selections – antigenic; sialic acid binding;? • Epidemiology, selection and spread of mutants. • Evolutionary questions – population size, errors, bottlenecks, etc.

  4. 1978 ~1970 1979 VP2 gene evolution: CPV in a separate clade, divided into 2 subclades. Substitution rates: FPV to CPV: ~7 x 10-3 (nt/site/yr) CPV clade: 2 x 10-4(nt/site/yr) CPV ancestor ~1970. 9 changes 3 changes

  5. 3’-end DNA VP2 VP1 Methods Used: 1) Protein expression – baculovirus expression and cell infection. 2) X-ray crystallography and cryoEM (Rossmann lab). 3) Biochemistry – binding assays. 4) Cell biology and processes of cell entry. 5) Evolutionary biology – processes of cell selection.

  6. 32Å 20Å 30Å Residues controlling canine host range: Located on threefold spike of capsid: 323 299 93 323

  7. VP2 93 and 323 structures: Asp323Asn: Lys93Asn: 323

  8. VP2 “300 region” structures: controlling canine and feline host range E Block canine host range D G299 A300 G A/G568

  9. YTRF * Cell receptor = Transferrin Receptor 11nm Apical Helical Homodimeric type II membrane protein. Binds diferric transferrin. Highly expressed on dividing cells. Clathrin-mediated uptake. Protease-like FRTY Clathrin-AP2 *

  10. Transferrin binding Virus binding Virus TfR Tf Capsid binding to TfRs: Feline TfR: binds FPV & CPV. Canine TfR: only CPV Canine TfR Feline TfR FPV CPV-2 CPV- G299E

  11. Canine TfR and cell infection by FPV: Single changes in the canine TfR allow FPV infection. % infected cells 0 10 20 30 40 50 FPV Feline TfR CPV-2 CPV-2a Canine TfR Canine TfR N383K Canine TfR Ins205

  12. Virus binding to TfR: Determined by 1 face of the apical domain. 11Å 13Å 11Å

  13. A6 A8 A9 A10 A11 A6 A8 A9 A10 A11 Baculovirus Expression and purification of the TfR for in vitro binding studies. Viral stock High 5 Supernatant Insect cells Purification of dimeric TfR ectodomain Nickel column Purification of His-tag proteins Gel filtration Gel filtration

  14. In vitro binding to the feline TfR - VP2 93 and 323 have no effect on feline TfR binding. F P V C P V - 2 C P V - 2 b F P V - K 9 3 N / D 3 2 3 N C P V - N 9 3 K / N 3 2 3 D

  15. Low binding to canine TfR. Canine TfR-N383K distinguishes K93N/D323N mutants.

  16. Feline TfR:CPV-2 complex: Preliminary results: ~1-3 TfR per capsid. Attachment near top of 3-fold spike. See Susan Hafenstein’s Poster #20

  17. Global selection of CPV VP2 mutations 100 3045-T 3088-C 3685-G 80 3699-T (Global) 3045-A 60 3088-T 4062-G 3685-C (USA) 3699-G (Global) 3675-G (Germany) 40 3675-G 4062/4064-GT (USA) (Italy – Asia)) 20 4062-G (Germany) 4062/4064-GT (USA) 0 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 Year of Virus Isolation

  18. Antibody binding and neutralization: Two dominant antigenic sites on virus. Overlap the TfR binding sites. Host range mutations alter antigenicity. Site A Site B

  19. Testing 8 monoclonal Fabs:High or low neutralizing activity.

  20. Competition by Fabs for soluble feline TfR:All compete, highly neutralizing more efficient.

  21. CryoEM of antibody binding sites. CPV + 14 FPV + B • A site - around 3-fold axis. • B site - near two-fold axis. • MAb 8 and MAb F differ in neutralization. FPV + 8 FPV + F Susan Hafenstein & Christian Nelson

  22. J-Y Sgro Viruses of vertebrates compared to insect viruses Raised regions – targets of host antibodies? AAV2 CPV Artemis and Actaeon devoured by Artemis's dogs. Densovirus

  23. 3’-end DNA VP2 VP1 Conclusions: 1) The TfR-capsid interactions are optimized for each combination; affinity can differ widely. 2) Canine TfR binding controls both canine and feline host ranges. 3) Glycosylation of canine TfR important. 4) TfR binding required for cell infection. 5) Complex interplay between antibody and TfR binding.

More Related