virology15tumorvir

Oncogenic Viruses,“There is no single mechanism by which viruses cause tumors”,Transformation and potential tumorigenesis,Transformation - alteration in a cells properties that leads to immortalization and different growth patterns that result from alteration in cell cycleLoss of anchorage dependenceLoss of contact inhibition (foci)Decreased requirements for growth factorsTumorigenesis (oncogenicity) - in vivo development of tumors,Cell cycle,M- mitosisG1 - cells growS - DNA synthesisG2 - growth and preparation for mitosisG1/S decision point for going to dividing stateProblem for DNA viruses that need S phase machinery,Cell cycle control proteins,Activation of cell cycle progression -cyclins, cyclin dependent protein kinases (Cdks), Cdk inhibitorsInhibitors 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 ortriggers apoptosis of damaged cell by activating Bcl-2 causing mitochondria to release cytochrome C and activate caspase systemIf 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 originThese include:Cervical cancerBurkitts lymphomaHepatocarcinomaKaposis sarcomaVirus is not only factor,Viruses cannot kill cell to be tumorigenic,Therefore may depend on host cell MayIntegrate 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 transformedIntegration of viral cDNA genomeRequires expression of oncogenes cell genes (c-onc) modified viral versions (v-onc) whose expression promotes transformation and tumorsHepC (no DNA phase) - chronic inflammation and repairViral proteins interact with p53 and lead to cell proliferation and prevent apoptosis,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.,Constitutive - agonist independent receptors,V-onc genes - transducing,Virus LTR is a strong promotorV-onc is altered form of c-oncrapid onset, high efficiency tumorigenesis (acute transforming),time,% transformed,Cis-acting insertions are low efficiency tumor viruses,Nondefective virusesNear c-onc and LTR activationInsertional inactivation of tumor suppressor genesChronic-transforming,% transformed,Trans-acting transcriptional activationUsually poor efficiencyMust require additional factors,C-onc,Virus gene product,Identifying c-onc in mouse tumors,Tumor cell DNA (mouse)Restriction fragments used to form circlesPCR based on viral genome primersSequence adjacent genes and compare to mouse genome and human equivalentsIdentified known sites and several new ones,Hepatitis B,DNA virus with RNA intermediateIn tumors virus is integrated with little gene expressionBelieved 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 DNAAdeno, Herpes - linearOncogenic efficiency is lowTypically nonproductive infections - nonpermissive cells or mutant virusOncogenes are normal virus early genes (used in replication)Virus gets stuck in early phase and produces high concentrationsNo cellular homologs,How are cells transformed?,Cell cycle control changes due to viral genes that Interact directly with the proteins in the cycleBind to and inhibit or degradeInterfere with expression of host cell cycle control genes,How should these proteins be similar?,Amino acid sequence similarities in Rb binding site,AdE1a,HPV E7,Sv40 Tag,HPV E7 sequences differ in low and high risk strains,Affects binding affinity to Rb,What happens to virus DNA?,Oncogenes are integrated (adeno, papova) and retainedMay require more than one viral gene (Rb and p53),Cotransfection of adenovirus E1A and other genes on Neo vector,Plating after 4 weeksG418 is a neomycin-type drug,Cells are transformed with E1A but only E1B/neo is maintained,focus,Immunoblots (a-c) and PCR (d-f),Cells transformed but dont need viral genes to remain,“Hit and run” mechanism,Virus thought to cause mutation in cell genes and then virus is no longer neededSimilar results with CMVTumors 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 etcCan immortalize hepatocytes if engineer cell with core on plasmidWhat 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 = ASCircle = untransfectedTriangle = vector control,Is death due to apoptosis?,A) DNA gel sizesB) ELISA - ab against nucleosome bound cytoplasmic DNA,Is expression of p53, p21 affected by core AS?,RNA protection assayIsolate mRNAs and add AS then RNAaseRun gel on protected fragmentsHow about protein levels?,Western blot,What is happening with telomerase activity?,Needed against senescenceLuciferase as a marker for gene activity,HCV core protein expression (+) and apoptosis genes,Hep 191 cells engineered with core gene under induction controlHepRXR cells w/o core,KSHV and Kaposis sarcoma,Virus expresses constitutive G protein coupled receptor,Cells transfe