Mechanisms of Transformation
by Retrovirues
Virology 324A
Dept. of Microbiology and Immunology
McGill University
Dr. John Hiscott
340-8222 ext. 5265
John.hiscott@mcgill.ca
Human Cancer Viruses
• Contributing factor in at least 15% of
human cancers worldwide
• Major cause of liver & cervical cancer
Taxonomy of Tumor Viruses
• DNA viruses: papovaviruses
hepadnaviruses
EBV
herpesviruses
KSHV
adenoviruses
poxviruses
• RNA viruses: retroviruses
flaviviruses
HBV
HPV
SV-40
BK, JC
HTLV-1
Hepatitis C virus
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
herpesvirus (KSHV, HHV-8)
Kaposi’s Sarcoma
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
Transfomration
• RNA viruses activate oncogenes
• DNA viruses negate tumor suppressors
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
RNA TUMOR
VIRUSES
Retrovirus Lifecycle
Simple retrovirus
•LTR-gag-pol-env-LTR
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
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)
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
v-ONC
Host DNA
c-ONC
cell
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
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)
Structural Changes in an Acquired vOnc
c-Erb B (EGFR)
Epidermal growth factor
receptor
v-Erb B
Transduced retroviral
version
Ligand binding
domains
Viral gag
membrane
Kinase
domain
P
P
P
P
P
Regulatory
domain
P
P
P
P
Altered v-Erb B
functions as a
constitutively
activated EGFR
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
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)
Cis-acting retroviruses
• Do not carry oncogenes
• Retain all viral genes
• Are replication-competent
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)
LTR
Exon 1
Host
DNA
LTR
ALV
Exon 2
Exon 3
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
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)
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.
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
•
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
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
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
pro
tax
env
rev
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 coexpressed with ras
• Tax transgenic mice develop tumors
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, GMCSF,platelet-derived growth factor, tumor growth factor 1,
MHC class I, c-myc, c-fos, parathyroid hormone-related
protein
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
Tax 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.
↓ p53
CBP/p300
p18INK4c
↑ Cell cycle
Tax
progression
↓ DNA repair
↑ Transcription
factors, proto-oncogenes
Apoptosis
Mechanisms of cell
transformation by retroviruses
Virus
category
Tumor
latency
period
Efficiency
of tumor
formation
Oncogenic
effector
Infecting
viral
Genome
Transform
cultured
cells?
Viral-cellular
chimera,
replication
defective
Yes
Transducing
retrovirus
Short (days)
High (can
reach 100%
of animals)
Cell-derived
oncogene carried
in viral genome
Cis-acting/
nontransducing
Intermediate
(wk, mo)
High to
intermediate
Cellular oncogene Intact,
activated in situ by replication
provirus insertion
competent
No
Transactivating/
nontransducing
long latency
Long
(mo, yr)
Very low
(<5%)
Virus-coded
Transcriptional
regulatory protein
No
Intact,
replication
competent
DNA TUMOR
VIRUSES
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
E1A
E1B
Rb
p53
SV40
Polyomavirus
Large T antigen
Large T antigen
Middle T antigen
Rb, p53
Rb
Src, PI3K
Papillomavirus
E7
E6
E5
Rb
p53
PDGF receptor
Mechanism of Rb inactivation
E2F
Rb
E1A
T ag
E7
Transcription of
E2F responsive
genes
E1A
E2F
Release of Rb
cell cycle brake
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
p53
p53
T ag
E6
Tag
Stabilizes p53 in
an inactive state
p53
Ub
Ub
Ub
p53
E6AP
E6
E6AP:
E3 Ub ligase
E4
p53
p53
p53
E1B
p53
E1B
Converts p53
from activator to
repressor of
transcription
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
Macrophage inflammatory
factors
v-G protein coupled
receptor
vIL-6
v-interferon regulatory
protein
Cell cycle
v-Bcl2
v cyclin D
KSHV Proteins Interact with TumorSuppressor 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 ligandindependent 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