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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 [email protected] Human Cancer Viruses. Contributing factor in at least 15% of human cancers worldwide

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Mechanisms of transformation by retrovirues l.jpg

Mechanisms of Transformation by Retrovirues

Virology 324A

Dept. of Microbiology and Immunology

McGill University

Dr. John Hiscott

340-8222 ext. 5265

[email protected]


Human cancer viruses l.jpg
Human Cancer Viruses

  • Contributing factor in at least 15% of human cancers worldwide

  • Major cause of liver & cervical cancer


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HPV SV-40 BK, JC

HBV

EBV KSHV

HTLV-1

Hepatitis C virus

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 B&C viruses Hepatocellular Carcinoma

  • HTLV1 Adult T cell Leukemia

  • Epstein-Barr virus (HHV-4) Burkitt’s Lymphoma

    Hodgkin’s Disease

    PTLD

    Nasopharyngeal Carcinoma

    Gastric Carcinoma?

  • Kaposi sarcoma-associated Kaposi’s 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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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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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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v-ONC

c-ONC

Transducing retroviruses

  • Viral acquisition of cellular proto-oncogene with capacity to transform if deregulated, usually replacing viral coding sequences (exception is RSV=src 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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Ligand binding domains

Viral gag

Kinase domain

PP

PP

Regulatory domain

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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LTR

LTR

Host DNA

Exon 1 Exon 2 Exon 3

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)


    Htlv i and atl l.jpg
    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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    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


    Dna tumor viruses35 l.jpg
    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

    E1AE1B

    Rb p53

    Large T antigen Large T antigen Middle T antigen

    Rb, p53 Rb Src, PI3K

    E7 E6 E5

    Rb p53

    PDGF receptor


    Mechanism of rb inactivation l.jpg
    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


    Mechanisms of p53 inactivation l.jpg
    Mechanisms of p53 inactivation

    Tag

    T ag

    Stabilizes p53 in an inactive state

    p53

    p53

    Ub

    Ub

    E6

    p53

    Ub

    p53

    E6AP

    E6AP:

    E3 Ub ligase

    E6

    E4

    p53

    p53

    p53

    E1B

    p53

    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


    Epstein barr virus and cancer l.jpg
    Epstein-Barr Virus and Cancer

    • First human virus to be directly implicated in human tumors.

      • DNA identified in Burkitt’s 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

      • EBNA3A&B: Interfere with Notch signalling pathway

      • EBNA3C: Overcomes Rb cell cycle checkpoint

      • LMP1: constitutively active CD40=elevates 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



    Kshv and cancer l.jpg
    KSHV and Cancer governed by Rb and p53

    • 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 governed by Rb and p53

    Ligand binding domain

    Kinase domain

    PDGF mediated receptor dimerization

    BPV E5ligand-independent dimerization


    Papilloma and cervical cancer l.jpg
    Papilloma and Cervical Cancer governed by Rb and p53

    • 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 cell

    • 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

    • Hepatitis B vaccine (Taiwan 1984to1992): Childhood hepatitis B down from 10.5 to 1.7% Hepatocellular carcinoma down by factor of 4


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    DNA Tumor viruses cell

    • 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 cell

    • 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 cell

    • 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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