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Oncogenic Viruses “There is no single mechanism by which viruses cause tumors” 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 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 Cell cycle control proteins • Activation of cell cycle progression cyclins, cyclin dependent protein kinases (Cdks), Cdk inhibitors • Inhibitors of cell cycle progression - tumor suppressors Tumor suppressor Rb • Rb binds to transcription factor E2F and prevents gene expression of proteins needed to go to S phase 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 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 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) 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 (vonc) 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 oncogenes • Cell gene is called protooncogene – 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. Constitutive - agonist independent receptors 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) % transformed time 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 • Trans-acting transcriptional activation • Usually poor efficiency Virus gene • Must require additional factors product C-onc 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 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?) 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 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 How should these proteins be similar? Amino acid sequence similarities in Rb binding site AdE1a V P E V I D L T C H E A G F P P S D D HPV E7 Q P E T T D L Y C Y E Q L N D S S E E Sv40 Tag F N E E - N L F C S E E M - P S S D D L X C X E HPV E7 sequences differ in low and high risk strains 6/11 P V G L H C Y E Q L N D 16 T T D L Y C Y E Q L N D 18 P V D L L C H E Q L S D 31 A T D L H C Y E Q L P S 33 P T D L Y C Y E Q L S D Affects binding affinity to Rb What happens to virus DNA? • Oncogenes are integrated (adeno, papova) and retained • May require more than one viral gene (Rb and p53) Cotransfection of adenovirus E1A and other genes on Neo vector focus Plating after 4 weeks G418 is a neomycin-type drug Cells are transformed with E1A but only E1B/neo is maintained Immunoblots (a-c) and PCR (d-f) Cells transformed but don’t need viral genes to remain “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 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? • Engineer antisense plasmid (also could use siRNA) • What happens to cells? • Square = AS • Circle = untransfected • Triangle = vector control Is death due to apoptosis? • A) DNA gel sizes • B) ELISA - ab against nucleosome bound cytoplasmic DNA 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? Western blot What is happening with telomerase activity? Needed against senescence • Luciferase as a marker for gene activity HCV core protein expression (+) and apoptosis genes • Hep 191 cells engineered with core gene under induction control • HepRXR cells w/o core KSHV and Kaposi’s sarcoma Cells transfected with GPCR • Virus expresses constitutive G protein coupled receptor Blood vessels 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) 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 (4cd)