virology15tumorvirppt课件.ppt

Transformation-alteration in a cells 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 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 Activation of cell cycle progression-cyclins,cyclin dependent protein kinases(Cdks),Cdk inhibitors Inhibitors of cell cycle progression-tumor suppressors Rb binds to transcription factor E2F and prevents gene expression of proteins needed to go to S phase 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 replicate20%of human cancers believed to be of viral originThese include:Cervical cancer Burkitts lymphoma Hepatocarcinoma Kaposis sarcomaVirus is not only factorTherefore may depend on host cell May Integrate as part of their cycle(retroviruses)Viral ORI and genes push cell to S phase(herpes,papilloma)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 repair Viral proteins interact with p53 and lead to cell proliferation and prevent apoptosisCell 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 Virus LTR is a strong promotorV-onc is altered form of c-oncrapid onset,high efficiency tumorigenesis(acute transforming)time%transformedNondefective virusesNear c-onc and LTR activationInsertional inactivation of tumor suppressor genesChronic-transforming%transformedTrans-acting transcriptional activationUsually poor efficiencyMust require additional factors C-oncVirus gene productTumor 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 onesDNA 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?)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 concentrations No cellular homologs 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 genesHow should these proteins be similar?V P E V ID L T C H E A G F P P S D DQ P E T T D L Y C Y E Q L N D S S E EF N E E-N L F C S E E M-P S S D DL X C X EAdE1aHPV E7Sv40 Tag6/11PVGLHCYEQLND16TTDLYCYEQLND18PVDLLCHEQLSD31ATDLHCYEQLPS33PTDLYCYEQLSDAffects binding affinity to RbOncogenes are integrated(adeno,papova)and retainedMay require more than one viral gene(Rb and p53)Plating after 4 weeksG418 is a neomycin-type drugCells are transformed with E1A but only E1B/neo is maintainedfocusImmunoblots(a-c)and PCR(d-f)Cells transformed but dont need viral genes to remain 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 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=ASCircle=untransfectedTriangle=vector controlA)DNA gel sizesB)ELISA-ab against nucleosome bound cytoplasmic DNARNA protection assay Isolate mRNAs and add AS then RNAase Run gel on protected fragmentsHow about protein levels?Needed against senescenceLuciferase as a marker for gene activityHep 191 cells engineered with core gene under induction controlHepRXR cells w/o coreVirus expresses constitutive G protein coupled receptorCells transfected with GPCRBlood vessels Grew transformed+/-GPCR 3T3 cells and collected medium.Added it to HUVECS and counted(3a)Concentration dependent(3b)Angiogenicity-microtubule formationHUVECs added on top of gel-like material and conditioned medium added(3c)Coculture expt-gel added on top of tran