Mechanisms of Transformation by Retrovirues Virology 324A Dept. of Microbiology and Immunology McGill University Dr. John Hiscott 340-8222 ext. 5265 [email protected].

Mechanisms of Transformation Mechanisms of Transformation by Retroviruesby 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

• Major cause of liver & cervical cancer

Taxonomy of Tumor VirusesTaxonomy of Tumor Viruses

• DNA viruses: papovaviruses hepadnaviruses herpesviruses

adenoviruses poxviruses

• RNA viruses: retrovirusesflaviviruses

HPV SV-40 BK, JCHBV

EBV KSHV

HTLV-1

Hepatitis C virus

Human Viruses and Associated Human Viruses and Associated MalignanciesMalignancies

• 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 LymphomaHodgkin’s DiseasePTLDNasopharyngeal

CarcinomaGastric Carcinoma?

• Kaposi sarcoma-associated Kaposi’s Sarcoma herpesvirus (KSHV, HHV-8)

How do viruses transform cells?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.

Generalization about Viral Generalization about Viral TransfomrationTransfomration

• RNA viruses activate oncogenes

• DNA viruses negate tumor suppressors

Evidence for classifying a tumor Evidence for classifying a tumor virusvirus

• 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

RNA TUMOR RNA TUMOR VIRUSESVIRUSES

Retrovirus Lifecycle

•LTR-gag-pol-env-LTR

Simple retrovirus

RetrovirusesRetroviruses

• 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

Mechanisms of cell Mechanisms of cell transformation by retrovirusestransformation 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)

Transducing retrovirusesTransducing 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

c-ONC cellHost DNA

v-ONC

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.

Acquired Genes Are Components of Acquired Genes Are Components of Signaling NetworksSignaling 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)

Structural Changes in an Acquired vOncStructural Changes in an Acquired vOnc

PP

PP

P

Ligand binding domains

Viral gag

Kinase domain

Regulatory domain

PP

PP

c-Erb B (EGFR) v-Erb B

Altered v-Erb B functions as a constitutively activated EGFR

Epidermal growth factor receptor

Transduced retroviral version

membrane

Outcome of Retroviral TransductionOutcome of Retroviral Transduction

• “Single hit” carcinogenesis (one event)

• Polyclonal: tumor growth initiated in every infected cell

• Tumors form within days

• Characteristic of animal retroviruses

Mechanisms of cell Mechanisms of cell transformation by retrovirusestransformation 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)

Cis-acting retrovirusesCis-acting retroviruses

• Do not carry oncogenes

• Retain all viral genes

• Are replication-competent

Mechanism of cell transformation for Mechanism of cell transformation for cis-acting retrovirusescis-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)

ALVExon 1 Exon 2 Exon 3

Host DNA

LTR LTR

Outcome of Oncogene Activation Outcome of Oncogene Activation by Retrovirus Insertionby 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

Mechanisms of cell Mechanisms of cell transformation by retrovirusestransformation 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)

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

• 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 1 TransmissionHTLV 1 Transmission

HTLV-I and ATLHTLV-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).

Oncogenesis Mediated by Oncogenesis Mediated by Essential Retrovirus ProteinEssential 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

HTLV-I genomeHTLV-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

env

tax

rev

pro

Tax and OncogenesisTax 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

Tax is a Promiscuous TransactivatorTax is a Promiscuous Transactivator

•Binds cellular transcription factors to enhance their binding to cellular promoters

•Dissociates NF-B/IB 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

Tax Targets Cell Cycle Regulatory Tax Targets Cell Cycle Regulatory ProteinsProteins

•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

Tax represses DNA repairTax represses DNA repair

•Represses DNA pol involved in base and nucleotide excision DNA repair

•HTLV-I transformed lymphocytes demostrate wide range of chromosomal abnormalities, rearrangements, duplications and euploidy.

Tax

↑ Transcription factors, proto-oncogenes

↓ DNA repairApoptosis

↓ p53CBP/p300

p18INK4c

↑ Cell cycleprogression

Mechanisms of cell Mechanisms of cell transformation by retrovirusestransformation 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 (<5%)

Virus-coded Transcriptional regulatory protein

Intact, replication competent

No

DNA TUMOR DNA TUMOR VIRUSESVIRUSES

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 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.

DNA tumor viruses target tumor suppressors

Virus Gene Product Cellular target

Adenovirus

SV40Polyomavirus

Papillomavirus

E1A E1B

Large T antigen Large T antigen Middle T antigen

E7 E6 E5

Rb p53

Rb, p53 Rb Src, PI3K

Rb p53 PDGF receptor

Mechanism of Rb inactivation

E2F

Rb

E1AT agE7

E2F

Rb

E1A

Transcription ofE2F responsive genes

Release of Rb cell cycle brake

•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

p53T ag

p53Tag Stabilizes p53 in

an inactive state

p53E6

p53E6

E6APUbUbUb

E6AP: E3 Ub ligase

p53 p53E4

p53 p53E1B Converts p53 from

activator to repressor of transcription

E1B

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

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

• 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

KSHV Genome Encoding Genes Homologous to Cell-Signaling and

Regulatory Pathway Proteins

Chemokines Signaling molecules Cell cycleMacrophage inflammatory factors

vIL-6

v-G protein coupled receptor

v-interferon regulatory protein

v-Bcl2

v cyclin D

KSHV Proteins Interact with Tumor- Suppressor Pathways governed by Rb and

p53

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

Papilloma E5 mimics PDGF ligand

Ligand binding domain

Kinase domain

PDGF mediated receptor dimerization

BPV E5 ligand-independent dimerization

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.

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

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

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

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

Learning Objectives

• Understand how RNA tumor viruses mediate oncogenesis

• Understand how DNA tumor viruses mediate oncogenesis

• Be able to identify viruses that mediate oncogenesis

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)

Retrovirus Structure