1. Oncogenic Viruses “There is no single mechanism by which viruses cause tumors”

2. Transformation and potential tumorigenesis Transformation - alteration in a cell’s properties that leads to immortalization and different growth patterns that result from alteration in cell cycle –Loss of anchorage dependence –Loss of contact inhibition (foci) –Decreased requirements for growth factors Tumorigenesis (oncogenicity) - in vivo development of tumors

3. Cell cycle M- mitosis G1 - cells grow S - DNA synthesis G2 - growth and preparation for mitosis G1/S decision point for going to dividing state Problem for DNA viruses that need S phase machinery

4. Cell cycle control proteins Activation of cell cycle progression - cyclins, cyclin dependent protein kinases (Cdks), Cdk inhibitors Inhibitors of cell cycle progression - tumor suppressors

5. Tumor suppressor Rb Rb binds to transcription factor E2F and prevents gene expression of proteins needed to go to S phase

6. Tumor suppressor p53 P53 halts progression when DNA damaged –to give cell time to repair or –triggers apoptosis of damaged cell by activating Bcl-2 causing mitochondria to release cytochrome C and activate caspase system If damaged (mutated) cell moves to S phase then it may replicate

7. Oncogenic viruses may be RNA or DNA 20% of human cancers believed to be of viral origin These include: –Cervical cancer –Burkitt’s lymphoma –Hepatocarcinoma –Kaposi’s sarcoma Virus is not only factor

8. Viruses cannot kill cell to be tumorigenic Therefore may depend on host cell May –Integrate as part of their cycle (retroviruses) –Viral ORI and genes push cell to S phase (herpes, papilloma)

9. RNA transforming viruses are retroviruses so far… (hepC) Permissive cells are transformed Integration of viral cDNA genome Requires expression of oncogenes –cell genes (c-onc) –modified viral versions (v- onc) whose expression promotes transformation and tumors HepC (no DNA phase) - chronic inflammation and repair –Viral proteins interact with p53 and lead to cell proliferation and prevent apoptosis

10. oncogenes Cell gene is called proto- oncogene –can induce transformation only after being altered (mutation or coming under the control of a highly active promoter). –usually encodes a protein that affects DNA replication or growth control at some stage of the normal development of the organism.

11. Constitutive - agonist independent receptors

12. V-onc genes - transducing Virus LTR is a strong promotor V-onc is altered form of c-onc rapid onset, high efficiency tumorigenesis (acute transforming) time % transformed

13. Cis-acting insertions are low efficiency tumor viruses Nondefective viruses Near c-onc and LTR activation Insertional inactivation of tumor suppressor genes Chronic-transforming % transformed

14. Trans-acting transcriptional activation Usually poor efficiency Must require additional factors C-onc Virus gene product

15. Identifying c-onc in mouse tumors Tumor cell DNA (mouse) Restriction fragments used to form circles PCR based on viral genome primers Sequence adjacent genes and compare to mouse genome and human equivalents Identified known sites and several new ones

16. Hepatitis B DNA virus with RNA intermediate In tumors virus is integrated with little gene expression Believed to be from chronic liver damage/loss and replacement causing increased mutations (similar to SOS response?)

17. DNA transforming viruses can be found in all families Papova - circular DNA Adeno, Herpes - linear Oncogenic efficiency is low Typically nonproductive infections - nonpermissive cells or mutant virus Oncogenes are normal virus early genes (used in replication) –Virus gets stuck in early phase and produces high concentrations –No cellular homologs

18. How are cells transformed? Cell cycle control changes due to viral genes that –Interact directly with the proteins in the cycle Bind to and inhibit or degrade –Interfere with expression of host cell cycle control genes

19. How should these proteins be similar?

20. Amino acid sequence similarities in Rb binding site VPEVIDLTCHEAGFPPSDD QPETTDLYCYEQLNDSSEE FNEE-NLFCSEEM-PSSDD LXCXE AdE1a HPV E7 Sv40 Tag

21. HPV E7 sequences differ in low and high risk strains 6/11PVGLHCYEQLND 16TTDLYCYEQLND 18PVDLLCHEQLSD 31ATDLHCYEQLPS 33PTDLYCYEQLSD Affects binding affinity to Rb

22. What happens to virus DNA? Oncogenes are integrated (adeno, papova) and retained May require more than one viral gene (Rb and p53)

23. Cotransfection of adenovirus E1A and other genes on Neo vector Plating after 4 weeks G418 is a neomycin-type drug Cells are transformed with E1A but only E1B/neo is maintained focus

24. Immunoblots (a-c) and PCR (d-f) Cells transformed but don’t need viral genes to remain

25. “Hit and run” mechanism Virus thought to cause mutation in cell genes and then virus is no longer needed Similar results with CMV Tumors may start with virus but leave no evidence of infection

26. The issue of HCV Core protein is a transcriptional regulator of cell promoters for p53, p 21 etc Can immortalize hepatocytes if engineer cell with core on plasmid What is the affect on immortalized cells of eliminating core protein? How can you do this?

27. Engineer antisense plasmid (also could use siRNA) What happens to cells? Square = AS Circle = untransfected Triangle = vector control

28. Is death due to apoptosis? A) DNA gel sizes B) ELISA - ab against nucleosome bound cytoplasmic DNA

29. Is expression of p53, p21 affected by core AS? RNA protection assay –Isolate mRNAs and add AS then RNAase –Run gel on protected fragments How about protein levels?

30. Western blot

31. What is happening with telomerase activity? Needed against senescence Luciferase as a marker for gene activity

32. HCV core protein expression (+) and apoptosis genes Hep 191 cells engineered with core gene under induction control HepRXR cells w/o core

33. KSHV and Kaposi’s sarcoma Virus expresses constitutive G protein coupled receptor Cells transfected with GPCR Blood vessels

34. Human Umbilical Vein Endothelial cells (HUVECs) Grew transformed +/- GPCR 3T3 cells and collected medium. Added it to HUVECS and counted (3a) Concentration dependent (3b) Angiogenicity - microtubule formation HUVECs added on top of gel-like material and conditioned medium added (3c) Coculture expt - gel added on top of transfected cells and HUVECs added on top (3d)

35. VEGF is a major angiogenic inducer Transfected cells w/ or w/o GPCR and measured VEGF in medium by ELISA (4a) Used antibody to VEGF to mitogenicity (4b) grey bar = anti-vegf; white bar = control ab Repear angiogencity expts (4c- d)