Mechanisms of Transformation by Retrovirues

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Mechanisms of Transformation by Retrovirues

• Virology 324A
• Dept. of Microbiology and Immunology
• McGill University
• Dr. John Hiscott
• 340-8222 ext. 5265
• John.hiscott_at_mcgill.ca

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Human Cancer Viruses

• Contributing factor in at least 15 of human
  cancers worldwide
• Major cause of liver cervical cancer

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Taxonomy of Tumor Viruses

• DNA viruses papovaviruses hepadnaviruses
  herpesviruses
  adenoviruses poxviruses
• RNA viruses retroviruses flaviviruses

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Human Viruses and Associated Malignancies

• HPV 16, 18, 31, 33, 45 Cervical Carcinoma
• Hepatitis BC viruses Hepatocellular Carcinoma
• HTLV1 Adult T cell Leukemia
• Epstein-Barr virus (HHV-4) Burkitts Lymphoma
• Hodgkins Disease
• PTLD
• Nasopharyngeal Carcinoma
• Gastric Carcinoma?
• Kaposi sarcoma-associated Kaposis Sarcoma
• herpesvirus (KSHV, HHV-8)

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How do viruses transform cells?

• Virus infection provides a hit towards the
  genesis of cancer.
• Act as a mutagen
• Other cofactors (genetic, immunological, or
  enviromental) may be needed for development of
  cancer
• Cell transformation is accompanied by the
  persistence of all or part of the viral
  genome and continual expression of a limited
  number of viral genes.
• Viral oncogenes are expressed that alter normal
  cellular gene expression and signal transduction
  pathways.

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(No Transcript)
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Generalization about Viral Transfomration

• RNA viruses activate oncogenes
• DNA viruses negate tumor suppressors

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Evidence for classifying a tumor virus

• Presence of part of viral genome in tumors and
  expression of some viral genes.
• In vitro infection of cells leads to
  transformation
• Tumorigenic assays
• Growth in low serum (reduced growth factor
  requirements)
• Growth in soft agar (anchorage independent
  growth)
• Identification of viral genes that transform
  cells in culture
• Infection of animal model system results in
  tumors
• No possible for human viruses
• Vaccination prevents tumor formation

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RNA TUMOR VIRUSES
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Retrovirus Lifecycle
Simple retrovirus

• LTR-gag-pol-env-LTR

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Retroviruses

• RNA tumor viruses create oncogenes by
  acquiring, modifying, deregulating cellular genes
  (proto-oncogenes)
• v-onc not essential viral gene unrelated to
  strategy of viral replication
• Replication of RNA viruses is not cytocidal nor
  is it required for tumorigenesis

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Mechanisms of cell transformation by retroviruses

• 1) Retroviral transduction of oncogene
  (transducing retrovirus)
• 2) Oncogene activation by retroviral insertion
  (cis-acting / nontransducing retrovirus)
• 3) Oncogenesis mediated by essential retrovirus
  proteins (trans-activating / nontransducing
  long-latency retrovirus)

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Transducing retroviruses

• Viral acquisition of cellular proto-oncogene with
  capacity to transform if deregulated, usually
  replacing viral coding sequences (exception is
  RSVsrc oncogene)
• Overexpression versus structural change in v-onc
• mos vs src
• Becomes replication defective, secondary to the
  loss of viral coding information requires helper
  virus

Host DNA
cell
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Mechanism of Acquisition of cellular sequences
Model for retroviral transduction of a cellular
proto-oncogene (cONC) to form an acute
transforming virus. A provirus is integrated
upstream of a cONC, the insertion may increase
the level of transcription of the oncogene.
Either a viral oncogene readthrough transcript is
made (A) or the provirus and the cONC gene are
fused by a deletion (B) Either event gives rise
to a hybrid RNA transcript initiating in the 5
LTR of the provirus and extending into the
oncogene. Additional proviruses integrated
elsewhere in the cellular genome can provide
helper function, forming virion particles (C)
that contain both helper and hybrid RNAs.
Recombination between these two RNAs during the
process of reverse transcription (D) joins the
ends of the viral genome to the hybrid RNA.
Either one or two crossovers are required
depending on the structure of the starting RNA.
Reverse transcription gives rise to a fully
transmissible retroviral genome carrying the
oncogene. Subsequent transmission of the new
genome (E) from doubly infected cells can occur
at high efficiency without further rearrangements.
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Acquired Genes Are Components of Signaling
Networks

• External signal molecules or growth factors
  (receptor ligands) (sis)
• Cellular receptors (erbB, fms, kit)
• Second messengers in signaling cascade (kinases
  src, abl, fgr, yes mos raf)
• Transcription factors (jun, fos, myc, myb, ets,
  rel)

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Structural Changes in an Acquired vOnc
c-Erb B (EGFR) v-Erb B
Epidermal growth factor receptor
Transduced retroviral version
membrane
Altered v-Erb B functions as a constitutively
activated EGFR
PP PP P
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Outcome of Retroviral Transduction

• Single hit carcinogenesis (one event)
• Polyclonal tumor growth initiated in every
  infected cell
• Tumors form within days
• Characteristic of animal retroviruses

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Mechanisms of cell transformation by retroviruses

• 1) Retroviral transduction of oncogene
  (transducing retrovirus)
• 2) Oncogene activation by retroviral insertion
  (cis-acting / nontransducing retrovirus)
• 3) Oncogenesis mediated by essential retrovirus
  proteins (trans-activating / nontransducing
  long-latency retrovirus)

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Cis-acting retroviruses

• Do not carry oncogenes
• Retain all viral genes
• Are replication-competent

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Mechanism of cell transformation for cis-acting
retroviruses

• Random retroviral integration into cell DNA
• Insertional activation (or inactivation)
• Cis activation by promoter or enhancer insertion
  next to proto-oncogene (encoded by exons 1-3)

ALV
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Outcome of Oncogene Activation by Retrovirus
Insertion

• Cell transformation rare event because insertion
  near potential oncogenes is infrequent
• Monoclonal tumors proviral sequences integrated
  at same chromosomal site
• Tumors induced more slowly (months) since tumor
  derived from single cell

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Mechanisms of cell transformation by retroviruses

• 1) Retroviral transduction of oncogene
  (transducing retrovirus)
• 2) Oncogene activation by retroviral insertion
  (cis-acting / nontransducing retrovirus)
• 3) Oncogenesis mediated by essential retrovirus
  proteins (trans-activating / nontransducing
  long-latency retrovirus)

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Human T cell Leukemia Virus type I (HTLV-I)

• Associated with 2 fatal human diseases
• Adult T cell leukemia (ATL)
• clonal malignancy of infected mature CD4 T cells
• Tropical spastic paraparesis/HTLV-1 associated
  myelopathy
• neurodegenerative disease
• Endemic in parts of Japan, South America, Africa,
  and the Caribbean
• With an estimated 10-20 million people infected
  worldwide
• Asymptomatic in majority of individuals with
  approximately 2-5 of HTLV-I carriers developing
  disease 20-40yrs post infection.
• The long clinical latency and low percentage of
  individuals who develop leukemia suggest that
  T-cell transformation occurs after a series of
  cellular alterations and mutations.
• Infects primarily CD4 T cells.

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HTLV 1 Transmission

• Extended close contact (cell-associated virus)
• Sexual (60 male to female versus 1 female to
  male transmission)
• Blood products (screening of blood supply since
  1988)
• Mother to child (breast feeding 20 children
  with seropositive mothers acquire virus)

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HTLV-I and ATL

• 1980 Gallo isolated type C retrovirus (HTLV1)
  from patient with cutaneous T cell lymphoma
• The provirus is present in all cases ATL
• Integration occurs at the same site in all cells
  derived from an ATL tumor (monoclonal).
• Integration site varies in different patients
• Integration does not occur at a preferred
  chromosomal site (no cis-activation of
  oncogenes).

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Oncogenesis Mediated by Essential Retrovirus
Protein

• Exception to paradigm of retroviral oncogenesis
  (HTLV-1)
• HTLV-1 does not carry cell-derived oncogene nor
  does it mediate cis-activation of oncogene
• HTLV-1 oncogenesis involves nonstructural viral
  regulatory protein (Tax)
• Tax essential to viral replication

Atypical flower cells of ATL
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HTLV-I genome

• 9 kilobase RNA genome
• HTLV-I does not carry a cellular-derived
  oncogene
• Unique regulatory proteins Tax and Rev
• Essential for viral replication
• Function in viral gene expression

LTR
LTR
gag
pol
tax
pro
env
rev
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Tax and Oncogenesis

• Tax essential to viral replication
• 40kda phosphoprotein
• Transcriptional activator for HTLV-I genome
• Targets viral LTR to dramatically activated
  viral gene expression in concert with cellular
  factors
• Interacts with cellular transcription factors
  and signaling molecules to enhance or repress
  cellular gene expression
• Tax can transform fibroblasts in culture when
  co-expressed with ras
• Tax transgenic mice develop tumors

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Tax is a Promiscuous Transactivator

• Binds cellular transcription factors to enhance
  their binding to cellular promoters
• Dissociates NF-?B/I?B complexes
• Upregulates IL-2, IL-2 receptor ?, IL-1, IL-3,
  IL-6, GM-CSF,platelet-derived growth factor,
  tumor growth factor ?1, MHC class I, c-myc,
  c-fos, parathyroid hormone-related protein

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Tax Targets Cell Cycle Regulatory Proteins

• Inactivate p53 (G1/S restriction control point)
• Activates cyclin D, cdk2, 4, and 6 which
  phosphorylate Rb to induce G1/S transition.
• Binds MAD1 (mitotic arrest-defective protein),
  interfering with G2/M phase of cell cycle
  progression, chromosomal segregation, and
  post-mitotic nuclear assembly

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Tax represses DNA repair

• Represses DNA pol b involved in base and
  nucleotide excision DNA repair
• HTLV-I transformed lymphocytes demostrate wide
  range of chromosomal abnormalities,
  rearrangements, duplications and euploidy.

? p53 CBP/p300 p18INK4c
? Cell cycle progression
Tax
? DNA repair Apoptosis
? Transcription factors, proto-oncogenes
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Mechanisms of cell transformation by retroviruses
Virus category Tumor latency period Efficiency of tumor formation Oncogenic effector Infecting viral Genome Transform cultured cells?
Transducing retrovirus Short (days) High (can reach 100 of animals) Cell-derived oncogene carried in viral genome Viral-cellular chimera, replication defective Yes
Cis-acting/ nontransducing Intermediate (wk, mo) High to intermediate Cellular oncogene activated in situ by provirus insertion Intact, replication competent No
Trans-activating/ nontransducing long latency Long (mo, yr) Very low (lt5) Virus-coded Transcriptional regulatory protein Intact, replication competent No
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DNA TUMOR VIRUSES
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DNA tumor viruses

• Diverse group of viruses with different
  structures, genome organization, and strategies
  of replication
• Some induce tumors in natural host
• Papilloma
• EBV, KSHV
• Hepatitis B
• Others induce tumors in experimental systems
• Adenovirus
• Polyomaviruses , SV40

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DNA tumor viruses

• Oncogenic potential linked to virus replication
  strategy
• Oncogenes are essential viral genes without
  cellular homologues (for small DNA tumor viruses)
• Transformation occurs ONLY in aborted viral
  life cycle (early genes expressed but
  replication, which is cytocidal, does not occur)
• Adenovirus, SV40, and polyomavirus frequency of
  transformation is less than 1 in 105 infected
  cells.
• For small DNA tumor viruses, integration of viral
  genome may enable abortive viral lifecycle.

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DNA tumor viruses target tumor suppressors
Virus Gene Product Cellular target
Adenovirus SV40 Polyomavirus Papillomavirus
E1A E1B
Rb p53
Large T antigen Large T antigen Middle T antigen
Rb, p53 Rb Src, PI3K
E7 E6 E5
Rb p53
PDGF receptor
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Mechanism of Rb inactivation
Transcription of E2F responsive genes Release of
Rb cell cycle brake
E1A
E1A T ag E7
E2F
E2F
Rb
Rb

• Investigation on mode of action of E1A lead to
  the discovery of E2F transcription factor and its
  interactions with Rb.
• Important for transcription of Adenovirus E2 gene

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Mechanisms of p53 inactivation
Tag
T ag
Stabilizes p53 in an inactive state
p53
p53
Ub
Ub
E6
p53
p53
E6AP
Ub
E6AP E3 Ub ligase
E6
E4
p53
p53
p53
p53
E1B
Converts p53 from activator to repressor of
transcription
E1B
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DNA Virus Transforming Activities via Cellular
Homologues

• EBV LMP1 mimics CD40 (tumor necrosis factor
  receptor)
• E5 gene of bovine papillomavirus is molecular
  mimic of growth factor (activates PDGF receptor
  signaling cascade)
• Polyomavirus middle T src signaling pathway
• HHV 8 Encodes viral D cyclin, vIL-6

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Epstein-Barr Virus LMP1

• One of several EBV genes implicated in
  immortalization of B cells.
• LMP1 signaling leads to increased expression of
  adhesion molecules
• Induces transformed phenotype in rodent
  fibroblasts

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Epstein-Barr Virus and Cancer

• First human virus to be directly implicated in
  human tumors.
• DNA identified in Burkitts lymphoma
• Experimental production of lymphomas in cottontop
  marmosets and owl monkeys
• Greater than 90 of adults persistently carry the
  virus
• Infection usually is asymptomatic, but causes
  infectious mononucleosis in adolescents.
• Encodes several viral proteins implicated in
  immortalization.
• EBNA1 maintenance of viral genome
• EBNA2 Transcriptional coactivator upregulates
  viral (LMP1) and cellular (c-myc) genes
• EBNA3AB Interfere with Notch signalling
  pathway
• EBNA3C Overcomes Rb cell cycle checkpoint
• LMP1 constitutively active CD40elevates bcl-2
  and A20
• LMP2 stimulates proliferation of epithelial
  cell

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KSHV Genome Encoding Genes Homologous to
Cell-Signaling and Regulatory Pathway Proteins
Chemokines Signaling molecules
Cell cycle
Macrophage inflammatory factors vIL-6
v-G protein coupled receptor v-interferon
regulatory protein
v-Bcl2 v cyclin D
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KSHV Proteins Interact with Tumor- Suppressor
Pathways governed by Rb and p53
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KSHV and Cancer

• Identified in 1994 as the infectious cause of
  Kaposi sarcoma.
• Also known as Human Herpesvirus 8 (HHV8)
• Infection is usually asymptomatic, but cancers
  develop in immunosuppressed individuals
• AIDS patients
• Transplant patients
• KSHV is the 3rd most common cancer caused by
  virus infection.
• In Africa due to AIDS epidemic, KS is the most
  common cancer

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Papilloma E5 mimics PDGF ligand
Ligand binding domain
Kinase domain
PDGF mediated receptor dimerization
BPV E5 ligand-independent dimerization
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Papilloma and Cervical Cancer

• Cervical cancer is a major cause of death among
  women in developing countries.
• In developed countries, mortality has decreased
  due to pap smear screening programs.
• 100 types of HPV divided into low, medium, and
  high risk types
• High risk 16, 18, 31, 33, 35, 39, 45,51, 52,
  56, and 86
• Low risk 6, 11, 40, 42, 43, 44, 54, 61, 70, 72,
  and 82
• HPV 16 (highest risk genotype) is detected in
  over 50 of cervical cancers
• An individual infected with HPV16 has a 5 chance
  of developing cervical cancer.

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Papilloma Replication Scheme replication in a
quiescent cell

• Virions penetrate epithelium thru microabrasions
  in skin
• Expression of E6 and E7 delays cell cycle arrest
  and differentiation
• Thickening of skin (wart)
• DNA replicates episomally
• Virus released from superficially epithelial
  cells to infect another individual
• Oncogenesis due to integration of virus. If
  integration disrupts E2 region (E2 represses txn
  of E6 and E7), overexpression of E6 and E7 ensues
• cells acquire extended lifespans, capacity to
  proliferate, and mutations

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Hepadnaviral (HBV) oncogenesis

• Liver is the major site of viral replication.
• Cause transient infection (3-12mo) and lifelong
  infections
• 0.1-25 of infections can become chronic
• 10 to 25 of chronic carriers are at risk of
  developing Heptocellular carcinoma (HCC)
• Long latency period (decades)
• Chronic infections leads to liver damage due to
  host anti-viral immune response
• Increased hepatocyte proliferation
• Increase concentrations of superoxides and other
  radicals
• Mutagenesis??
• Woodchuck animal model develop liver cancers by
  2-4 yrs of age
• HCC tumors usually harbor integrated virus
• HB X protein may be the viral oncoprotein
• Activates src tryosine kinase
• May inhibit p53 function

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DNA Tumor viruses

• DNA tumor viruses transform cells by
• Altering cell cycle progression
• Negate Rb and p53 cell cycle blocks to induce
  proliferation
• Encode cellular mimics to activate signal
  transduction pathways that enhance cell
  proliferation

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Learning Objectives

• Understand how RNA tumor viruses mediate
  oncogenesis
• Understand how DNA tumor viruses mediate
  oncogenesis
• Be able to identify viruses that mediate
  oncogenesis

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Historic Perspective

• 1908 Ellerman and Bang 1st showed that avian
  leukemia could be transmitted by filtered
  extracts.
• 1911 Peyton Rous demonstrated that sarcomas in
  chickens had a viral etiology
• 1933 Richard Shope discovered 1st DNA tumor virus
  (Papilloma in cottontail rabbits)

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Retrovirus Structure