BMD 602
微生物學 – 病毒學
RNA Tumor Viruses
Robert Yung-Liang Wang
王永樑
[email protected]
生物醫學研究所
微生物組
1
Teaching Objectives :
¾ To learn which viruses can cause cancer in humans
¾ To learn how cells become transformed by the virus
¾ To learn the differences between DNA and RNA tumor virus.
¾ To learn the group of Retrovirus.
¾ To understand the replication of Retrovirus
¾ To understand the discovery of cellular proto-oncogenes
¾ To understand how RNA viral oncogenes result in cell
transformation
¾ HCV
2
¾ WHAT IS CANCER?
Cancer:
The American Cancer Society
Characteristics of Cancer :
9
9
9
9
4
Tumor Viruses
For most viruses:
Genome
viral proteins
Replication
Progeny virions
Lytic Life Cycle
5
Tumor Viruses
Latent Life Cycle
Virus
Cell
Integration (usually)
Virus-specific proteins expressed Changes in the properties of host cell -
6
Tumor Viruses
Transformation:
Loss of
Ability to form
- viral genes interfere with
control of cell replication
7
TRANSFORMATION
Both DNA and RNA tumor viruses can transform cells
Integration occurs (usually)
Similar mechanisms
VIRAL TRANSFORMATION
The changes in the biological functions of a cell that result
from
of the cell’s metabolism by viral genes and that confer on the
infected cell certain properties characteristic of
These changes often result from the integration of the viral
genome into the host cell DNA
8
TRANSFORMATION
Among the many altered properties of the
TRANSFORMED CELL are:
• Loss of
in cultured cells)
•
(loss of contact inhibition
formation
•
• Reduced
• Transformed cells frequently exhibit
chromosomal aberrations
9
Two Major Classes of Tumor Viruses
DNA Tumor Viruses
DNA viral genome
Viral mRNA
Viral protein
10
RNA Tumor Viruses
Viral RNA genome
(Virusencoded)
Viral DNA genome (integrated)
IMPORTANT
(Host RNA
pol II)
Viral genomic RNA
(Host splicing enzymes)
messenger RNA
viral protein
Virus
Important: Use
RNA polymerase
to make its genome
An enzyme that
normally
makes mRNA 11
DNA Tumor Viruses
DNA genome
mRNA
protein
virus
OR TRANSFORMATION
In transformation usually only EARLY functions are expressed
12
How cells become transformed by the virus?
If a virus takes up
in a cell and
alters the properties of that cell, the cell is
said to be
.
Transformation by a virus may be defined as: changes in
the biologic functions of a cell that result from regulation of
the cell by viral genes and that confer on the infected cell
certain properties characteristic of neoplasia. these
changes often (but not always) result from integration of
the viral genome into the host cell genome
13
RNA Tumor Viruses
RNA Genome - Retroviruses
RNA-dependent DNA Polymerase encoded by virus
REVERSE TRANSCRIPTASE
RNA genome
Reverse transcriptase
DNA genome
Integrase
Integrates
Host RNA polymerase II
RNA genome
14
RNA Tumor
Viruses
15
RNA Tumor Viruses
A normal retrovirus has:
3 genes
: internal proteins
: Envelope glycoproteins
: Enzymes
Reverse transcriptase
Integrase
Protease
16
RNA Tumor Viruses
RNA is:
• Diploid
•
Capped and polyadenylated
sense (same as mRNA)
Viral RNA cannot be read as mRNA
New mRNA must be made
Virus must make
before proteins are
made
Therefore virus must carry
into the cell
17
RNA Tumor Viruses
18
RNA Tumor Viruses
Groups of Retroviruses
• Oncovirinae
Tumor viruses and similar
• Lentiviruses
Long latent period
Progressive chronic disease
Visna
HIV
• Spumavirinae
19
RNA Tumor Viruses
Retroviruses known to cause human cancer
• Human T cell lymphotropic virus -1 (HTLV-1)
Adult T cell leukemia, Sezary T-cell leukemia
Africa, Caribbean, Some Japanese Islands
• Human T cell lymphotropic virus -2 (HTLV-2)
Hairy cell leukemia
• HIV?
20
HIV is a lentivirus, a class of retrovirus
¾ Lentiviruses integrate into the host cell genome
as a
.
¾ HIV can lie dormant (
) in the proviral
Form within a cell for many years, especially in
resting (memory)
lymphocytes.
¾ HIV can set up a lifelong infection.
¾ HIV is not transmitted through the germ line.
21
Provirus
22
What are the major mechanisms of HIV
transmission in different regions of the world?
23
First report of hemophilia-related immune deficiency in
July 1982
Kaposi's sarcoma (skin).
Skin showing AIDS-associated Kaposi's sarcoma
24
Acute infection (acute retroviral syndrome)
This period lasts for 6 to 12 weeks after initial infection
until anti-HIV antibodies are detectable.
cells are the major target been infected.
25
Virus titer, CD4 cell number and anti-gp120 titer during the course
of HIV infection
Rate of progression to AIDS associated with homosexuality
and hemophilia
HIV-HS: HIV negative homosexual.
HIV+ HS: HIV positive homosexual
Loss of the specific immune response to HIV
¾ responding CD4+ cells become infected. Thus, there is clonal
deletion leading to tolerance. The cells that proliferate to respond
to the virus are infected and killed by it.
¾ epitope variation can lead to escape of HIV from the immune
response
¾ activated CD4+ T cells are susceptible to apoptosis.
Spontaneous apoptosis of uninfected CD4+ and CD8+ T cells
occurs in HIV-infected patients. Also there appears to be selective
apoptosis of HIV-specific CD8+ cells
¾ the number of follicular dendritic cells falls over time, resulting in
diminished capacity to stimulate CD4+ cells
HIV LOAD BUT NOT CD4+ T CELL NUMBER IS A GOOD
INDICATOR OF RAPID PROGRESSION TO AIDS
Cells that are infected by HIV
Entry of HIV via the mucosal route and transit
via dendritic cells to the lymph nodes
RNA Tumor Viruses
Retrovirus Life Cycle
Endocytosis
Fusion of membranes
Release of
to cytoplasm
Nucleus
35
The Structure of Retrovirus
Retrovirus Structure
1. The envelope comes from the host cell plasma
membrane
2. The envelope gene encodes the
surface glycoprotein (SU) –
transmembrane (TM) polyprotein.
Retrovirus Structure
3. The gag (group specific antigen) gene encodes the
viral matrix (MA) capsid (CA) and nucleoproteins
(NC).
Retrovirus Structure
The pol gene products are:
a)
(a polymerase that copies RNA
to DNA)
b)
(integrates the viral genome into the host
genome)
c)
(cleaves the RNA as the DNA is transcribed
so that reverse transcriptase can make the second
complementary strand of DNA)
d)
(cleaves the polyproteins translated from
mRNAs from the gag gene and the pol gene itself).
This virally encoded protease is the
target of a new generation of anti-viral drugs.
Retrovirus Structure
1. The envelope comes from the host cell plasma membrane
2. The envelope gene encodes the surface glycoprotein (SU) –
transmembrane (TM) polyprotein.
3. The gag (group specific antigen) gene encodes the viral matrix (MA)
capsid (CA) and nucleoproteins (NC).
4. The pol gene products are:
The replication of RNA virus :
Video: HIV replication and life cycle !!
The origin of viruses?
病毒的起源?
46
RNA Tumor Viruses
Parental RNA
RNA/DNA Hybrid
Linear DNA/DNA duplex
Circular Duplex DNA
Integration
cell)
Replication (DNA genome in
47
RNA Tumor Viruses
Drawback to this lifestyle
Genomic RNA
Reverse transcriptase
DNA
Genomic RNA
Pol II is a host enzyme that, in the uninfected cell, makes mRNA
When making mRNA, pol II
entire gene to RNA
48
Problem of using RNA pol II to copy a gene
RT
primer
Viral
genomicRN
Reverse
A
transcriptase
dsDNA
RNA synthesis
initiation site
promotor
RNA pol II
RNA synthesis termination
site
49
Result: New copy of viral RNA is shorter - lacks control sequences
RNA Tumor Viruses
RNA polymerase II will not copy
Upstream sequences from transcription initiation site
• Promotors / Enhancers
Down stream sequences from transcription termination site
• Enhancers / Poly A site / termination site
?
Perhaps virus could integrate downstream of a promotor etc so
that the cell provides sequences
OR
Virus provides its own promotors etc
BUT not copied!
50
¾ How can a retrovirus provide its own control
promotors and enhancers
The structure of the RNA genome of the mature retrovirus
The genome structure of the DNA proviral form of a retrovirus
RNA Tumor Viruses
Repeat
region
Clue: Difference in the two forms
Repeat
region
RNA
R
U5
GAG
POL
ENV
U3
R
DNA
U3
R
LTR
U5
GAG
POL
ENV U3
R
U5
52
R
U5
Viral RNA
U3
R
Reverse
transcriptase
U3
R
U5
U3
R
U5
Long terminal repeats are formed
promotor
POLII
POLII
RNA initiation
site
RNA termination
site
53
Retroviruses can have only
one promotor
Contained in U3
LTR
LTR
POLII
POLII
RNA initiation
site
RNA termination
site
Therefore only one long RNA can be made
U5
Therefore mRNA requires processing
Explains why RNA has to be positive sense
54
¾ The discovery of cellular proto-oncogenes
If the virus is to transform a cell, it must have
sequences that
and
provide the other functions that are typical of a
transformed cell. These are in addition to the
gag/pol/env genome.
Thus we also find an
(onc) in the viral
genome of many retroviruses that transform cells to
neoplasia. It should be emphasized that the
oncogene in RNA tumor viruses
.
It is an additional gene that gives the virus its
capacity to transform the host cell.
Some retroviruses have an
extra gene
“typical retrovirus”
R
U5
GAG
POL
ENV
U3
R
Rous Sarcoma Virus
R
U5
GAG
POL
ENV
SRC
U3
R
56
Some retroviruses have an oncogene
instead of their regular genes
Avian Myeloblastosis
Virus
R
U5
GAG
POL
MYB
U3
R
Feline Sarcoma Virus (FSV)
R
U5
dGAG
FMS
dENV
U3
R
Avian Myelocytoma Virus (MC29)
R
U5
dGAG
MYC
dENV
U3
R
57
RNA Tumor Viruses
Viral Oncogene
V-onc
Cellular Proto-oncogene
C-onc
58
RNA Tumor Viruses
Proto-oncogene
A cellular (host) gene that is homologous with a
similar gene that is found in a transforming virus
A cellular oncogene can only induce
transformation after
•
•
59
RNA Tumor Viruses
The discovery of the acutely transforming
retroviruses that contain
v-oncs explains how cancers may arise as a
result of infection
These viruses cause rapid cancer in animals in
the laboratory
60
RNA Tumor Viruses
In contrast:
Chronically transforming retroviruses
cause tumors inefficiently after prolonged period of
time
Avian Leukosis Virus (causes lymphomas)
R
U5
GAG
POL
ENV
U3
R
No oncogene! – How does it cause a
tumor?
61
Chronically transforming retroviruses do not have a v-onc
Oncogenesis by
Chronically transforming retroviruses do not have a v-onc
Oncogenesis by
RNA Tumor Viruses
ALV can integrate into the host cell genome at
MANY locations
but in tumor it is always at the SAME site (or
restricted number of sites)
Suggest tumor arose from one cell
• Something must be important about this site
for transformation
• Crucial event must be rare
64
RNA Tumor Viruses
What is special about this site?
Myelocytoma tumors from several birds all have
the oncogene close to this site
It is close to
C-myc!
Oncogenesis by promotor insertion
65
RNA Tumor Viruses
Could C-oncs be involved in NON-VIRAL
cancers?
66
RNA Tumor Viruses
myb
mos
myc
Genes can be
assigned to
sites on
specific
chromosomes
mos and myc :
chromosome 8
fes
fes: chromosome 15
67
Cancers often result from gene
translocations
Burkitt’s Lymphoma
8:14 translocation
Break in chromosome
14 at q32
myc
Acute myelocytic leukemia
7:15
9:18
68
11:15:17
Oncogenesis by rearrangement
Tumor
c-onc
Burkitt’s lymphoma
myc
(8)
new promotor
Ig heavy (8 to 14)
Ig light (8 to 2)
T cell chronic lymphocytic
myc
T cell receptor (8 to 14)
B-cell chronic lymphocytic
bcl-1
Ig heavy (11 to 14)
leukemia
bcl-2
Ig heavy (18 to 14)
T cell chronic lymphocytic
tcl-1
T cell receptor
leukemia
leukemia
(14 inversion)
69
RNA Tumor Viruses
What do oncogenes encode?
Proteins that are involved in growth control and
differentiation
Growth factors
Growth factor receptors
Signal transduction proteins
Transcription factors
70
Oncogenes
Mutations in a proto-oncogene are dominant
“gain of function” mutations
However other oncogenic genes show recessive
mutations
Anti-Oncogenes
• Loss of function mutations
• Retinoblastoma
• p53
71
Retinoblastoma
Proto-oncogenes
Heterozygote
Dominant
mutations Homozygote
Allele 1
Allele 2
Allele 1
Allele 2
Normal
Mutant
Mutant
Mutant
Binds under
special
circumstances
Mutant
always
binds
Always binds
Mutant
always
binds
Mutant
always
binds
Always binds
73
Function gained
Function gained
Anti-Oncogenes
Recessive mutations
Mutation
Rb Gene
Mutant Rb
growth
Mutant Rb
Mutant Rb
Rb
Rb protein
Heterozygote
Rb
Binds and controls cell cycle
Homozygote
Function lost
No binding - Growth continues
74
Anti-Oncogenes
Retinoblastoma gene has normal
regulatory function in many cells
Involved in
Retinoblastoma
Lung carcinomas
Breast carcinomas
75
Anti-Oncogenes
P53
Inactivated by
• deletion
• point mutation
In a series of colorectal cancers all showed:
• Allele 1: partial or complete deletion
• Allele 2: Point mutation
76
DNA Tumor Viruses
Oncogenes
• Adenovirus
E1A region 2
• SV 40
Large T
• Polyoma
Large T
• BK virus
Large T
• Lymphotropic virus
Large T
• Human papilloma Virus-16
E7
All have a sequence in common
Mutations in this region abolish transformation capacity
77
Anti-Oncogenes
Retinoblastoma
Rb Gene
Rb
protein
Rb
Adenovirus E1A
105kD
Rb
Rb
Stops replication
Cell cycle continues
78
Anti-Oncogenes
p53
P53 gene
P53 gene
Hepatitis C
P53
P53
P53 gene
Papilloma
P53
Papilloma
proteolysis
P53
DNA
Stops replication
replication
replication 79
HCV: Hepatitis C virus
Characteristics of Hepatitis Viruses
Virus
A
B
C
D
E
RNA
DNA
RNA
RNA
RNA
Fecal-oral
Parenteral;
sexual;
vertical
Parenteral;
sexual
Like HBV
Fecal-oral
Acute
Acute and
chronic
Mild acute,
mostly
chronic
Acute or
chronic
Acute
2 – 6 weeks
3–5
month
2–9
weeks
Like HBV
1 month
Diagnostic tests
Antibody
Antibody,
Antigen
Antibody,
Antigen
Antibody,
Antigen
None
Virus secretion: site,
time after infection
Stool,
2-3 weeks
Blood,
3-4 weeks
Blood,
3-4 weeks
Blood,
3-4 weeks
Stool,
1 month
Genome
Transmission route
Disease course
Latency period for
disease
HCV: Hepatitis C virus
A positive-stranded RNA virus
HCV life cycle
HCV and Liver Cancer
HCC (hepatocellular carcinoma)
HCC (hepatocellular carcinoma)
Hepatocellular carcinoma (HCC, also called malignant hepatoma) is
a primary malignancy (cancer) of the liver.
Figure. Pathogenesis of human hepatocellular carcinoma
Lose of p53, pRb pathways leads to
genomic instability
Malignant Transformation
The Wnt pathway in the HCV-related (Hepatocellular carcinomas) HCC
Levrero M. 2006 Viral hepatitis and liver cancer