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 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 Burkitts lymphoma Hepatocarcinoma Kaposis 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

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 Must require additional factors

gene 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 HPV E7 V P E V I D L T C H E A G F P P S D D 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 16 18 31 33 P T P A P V T V T T G D D D D L L L L L H Y L H Y C C C C C Y Y H Y Y E E E E E Q Q Q Q Q L L L L L N N S P S D D D 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 dont 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 Kaposis 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)