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Retrovirus(Raymond B. Birge, PhD)
October 9th, 2008
Viruses, Cells, andDiseases
(CIOC5125Q)
Oncogenic VirusesDefinition: A virus that is able to take residence in a cell and alter cellular growth and give properties of neoplasia. These cells are referred to as “transformed”
cells
DNA Tumor Viruses RNA Tumor Viruses
DNA Viral Genome
Viral mRNA
Viral Protein
DNA-dependent DNA polymerase
(Viral or Host)
RNA polymerase
(Host)
Examples:
Papovaviruses (Papillomavirus, SV40)
Adenoviridae (Adenovirus)
Herpesviridae (Epstein-Barr virus)
RNA Viral Genome
Viral DNA Genome (integrated)
Viral RNA Genome
Viral Protein
Reverse transcriptase
(Viral)
DNA-dependent RNA polymerase
(Host)
RNA Splicing
(Host)
Examples:
Oncovirinae (RSV, MuLV, HTLV-1,2)
Lentivirinae (HIV)
TERMINOLOGY
Oncogene: A viral or cellular gene directly responsiblefor the induction of abnormal cell proliferation.
Proto-Oncogene: A cellular gene that has the potential tobe an oncogene if its encounters mutation/de-regulation.
Tumor Suppressor Gene: A cellular gene involve in growth suppression. These genes are inactivated by deletion, mutation, of interaction with viral proteins(for example, HPV gene products).
Oncogene Hypothesis: Malignant carcinoma’s arisefrom de-regulation or mutation of cellular genes.
Basic Structure of Retrovirus; Genome encodes three basic genes(Gag, Pol, and Env)
ALV (ASLV) Avian (Sarcoma) and Leukosis Virus
Coat proteins (surface glycoantigens); Encoded by Env
Outer lipid Envelope (Derived from the host membrane)
Can be trans-membrane or cleaved. Env variationsdetermine subgroups (A-E, and J).
Capsid core proteins (core shell, includes the Matrix (MA)Capsid (CA-most abundant), and Nucleocapsid (NC)
RNA genome (2 molecules)
Polymerase (several genes; protease RT, RNase H, Integrase)
RNA genome; both RNA molecules are single stranded (+) sense and5’ cap and 3’ poly A tail. They also have a small molecule of tRNA (usually
for Trp or Lys!)
Basic Structure of Retrovirus; Genome encodes three basic genes(Gag, Pol, and Env)
ALV (ASLV) Avian (Sarcoma) and Leukosis Virus
Coat proteins (surface glycoantigens); Encoded by Env
Outer lipid Envelope (Derived from the host membrane)
Can be trans-membrane or cleaved. Env variationsdetermine subgroups (A-E, and J).
Capsid core proteins (core shell, includes the Matrix (MA)Capsid (CA-most abundant), and Nucleocapsid (NC)
RNA genome (2 molecules)
Polymerase (several genes; protease RT, RNase H, Integrase)
Coat proteins also determine trophism
Coat proteins determine trophism;Ecotrophic=infects mouseXenotrophic=infects non mouse (rat, hamster)Amphotrophic=mouse and non-mouse (human)
Topology and classification of retrovirus
A-type: Non-enveloped particles, only seen inside cells(maybe they are partially expressed endogenous virus)
B-type: Enveloped particles, with condensed core and prominent envelope spikes (MMTV)
C-type: Enveloped particles, with condensed core and few envelope spikes (ALV, RSV, HIV, HTLV)
D-type: Enveloped particles, less condensed core, fewenvelope spikes (able to super-infect C-type virus)
Taxonomy of RNA Reverse Transcribing Viruses(Family=Retroviridae)
Alpharetrovirus ALV, RSV, CT10, Y73 Sarcoma Virus Vertebrate
Betaretrovirus MMTV, Squirrel monkey retrovirus Vertebrate
Gammaretrovirus FLV, Harvey MSV, Moloney MSV Vertebrates
Deltaretrovirus BLV, Primate T-lymphocytic retrovirus Vertebrate
Lentivirus HIV-1, HIV-2, SIV, HTLV Vertebrates
Spumavirus Chimp foamy virus Vertebrates
Genus Type Hosts
Classification taxonomy depend on various factors that include genome size, assembly mechanisms, subtype, malignancies, immunodeficiency, and homology.
The basic retrovirus lifecycle
The retrovirus lifecycle in more detail
1. Binding to a receptor (fusion/internalization)Note, there are four subtypes(A-type, B-type, C-type, D-type)
2. RNA (plus strand) is copied to DNA(minus)strand. ssDNA copies to dsDNA
3. DNA, called provirus) is integrated intohost chromosome randomly
4. Full-length genomic RNA is copied from integrated DNA by pol II.
5. RNA is spliced and translated into protein
6. Virus particles assemble and bud from plasma membrane
The lifecycle in more detail
1. Binding to a receptor (fusion/internalization)Note, there are several subtypes(A-type, B-type, C-type, D-type etc)
2. RNA (plus strand) is copied to DNA(minus)strand. ssDNA copies to dsDNA
3. DNA, called provirus) is integrated intohost chromosome randomly
4. Full-length genomic RNA is copied from integrated DNA by Pol II.
5. RNA is spliced and translated into protein
6. Virus particles assemble and bud from plasma membrane
Retrovirus lifecycle is somewhatuneventful, consuming about 1%of the total cellular energy.
Retrovirus replication and integration:
5’Repeat
3’Repeat
5 ‘Unique
3’Unique
Primer binding
site
PolypurineTract
Single stranded RNA
Major elements of the retrovirus sense strand.
Direct repeats at both ends of the genome ‘terminally redundant”Primer binding site, uses a specific tRNA (15-20 nucleotides C’ to 3’ end)Polypurine Tract; Short stretch of A and G residues for initiating (+) strand
synthesisUnique 3’ region responsible which forms the promoter for the viral mRNA
5 Cap (AAAA)n
RT
Retrovirus replication:
5’Repeat
3’Repeat
5 ‘Unique
3’Unique
Primer Binding Site
Single stranded RNA
RT
RT
RT
RT
tRNA primer binds to PBS; RT extends in 3’ direction
(only virus that uses tRNA for replication!)
RNase H degrades ds RNA
(part of RT enzyme; degrades RNA/DNA duplex to ssDNA
SS DNA jumps to opposite
complementary Strand
tRNA primer binds to PBS; RT extends in 3’ direction
Polypurine Tract
Note, this sense strand does
not serve directly as mRNA
Retrovirus replication :
RTtRNA primer binds to PBS; RT
extends in 3’ direction
RNase H degrades all ds RNA except for the polypurine tract
RT
PolypurineTract
RT now extends in the sense direction
RT
RTRNA
RNase H removes remaining RNA
C’ sequences allow for
circularization
After circularization, RT acts as a DNA-dependent DNA polymerizationto make the ds DNA pro-virus
David Baltimore(MIT)
Howard Temin(U Wisconsin)
RSV virus particles contain an endogenous DNA polymerase activity
(incorporates deoxyribonucleside monophosphates into DNA and requires allfour deoxyribonucleotide triphosphates, a divalent cation,and is inactivated by RNAase)
1975 Nobel Prize in Physiology and Medicine
The next step in virus life is the DNA provirus integration, catalyzed by the integrase function of RT.
Integrase Function is part of RT, proviral insertion
Can occur with either linear of circular form of the provirus
The ends of the LTR’s have inverted repeats that are cleaved to form a staggered cut.
5’ LTR 3’ LTR
IN
IN also makes a cut in the host cell DNA, allowing permanent insertion of the entire ds provirus
Host DNA Host DNAViral DNA
DNA provirus
host hostLTR LTRgag pol env
5’ 3’gag pol env
Transcription (through cellular transcriptionalmachinery)
gag-pol polyprotein
gag polyprotein
Virion structuralproteins
Integrase; RT;RHase H, protease
Envelope glycoproteins(SU and TM)
env
5’ 3’envSubgenomic env mRNA
“A transmissible sarcoma of the chicken has been under observation in this laboratory for the past fourteen months, and it has assumed of late a special interest because of its extreme malignancy and a tendency to wide-spread metastasis. In a careful study of the growth,tests have been made to determine whether it can transmitted by a filtrate free of the tumor cells…small quantities of a cell-free filtrate have sufficed to transmit the growth to susceptible fowl” (Rous, Nature , 1911). The cancer was named Rous' sarcoma, and Rous won the Nobel Prize in 1966 for his achievement.
A cancer-causing virus in chicken
RSV-Malignant sarcoma’s within 2 weeks
ALV-Weakly transforming virus (tumors in 2-3 months)
Growth characteristic Normal cells Tumor cells
Density dependent inhibition of growth Present Absent Growth factor requirement High LowAnchorage dependence Present AbsentProliferative life span Finite IndefiniteContact inhibition of motility Present AbsentMorphology Flat RoundedColonies in agar NO YES
Normal cells RSV transformed cells
ALV and RSV could be propagated and isolated cultured cells
How can ALV, with a relatively uneventful infectionlifecycle, induce cell transformation and neoplasia?
The DNA Provirus Hypothesis (1965-74)
RNARSV DNARSV
Infecting virus
RNARSV
Provirus Progeny Virus
The Oncogene Hypothesis(The protovirus hypothesis for
origin of cancer genes�)
DNA Altered DNADNARSV
Provirus
Hidesaburo Hanafusa:Defectiveness of Rous sarcoma virus; Virtually all acutely transforming retroviruses of animals are mixtures of replication competent helper virus and replication defective transforming virus.Replication function is provided bythe helper virus in trans
Hybridization experiments. PNAS (1970). “These results indicate that both cell types (chicken cells that contain RAV-60 in a replicating form or do not appear to contain a replicating form) contain DNA that is complementary to RNA from the avian tumor virus”.
(~1971)
ALV RSV
Weakly transforming virus (3-6 months)Genome size ~8.5 kbReplication competent
Acutely transforming virus (1-2 weeks)Genome size ~10 kbReplication deficient
What is the difference between weak and acutely transforming viruses?
Origin of Retroviral Transforming Genes
RSV (gag pol env src)r.t.
gag, pol etcsrc
cDNA
DENATURE
AL V (gag,pol,env)
isolate genomic RNA
gag, pol, env
HYBRIDIZE
unhybridizedsequences
hybridizedsequences
gag, pol, envsrc
Stehelin, Bishop and Varmus
src
Origin of Retroviral Transforming Genes
Specific src probe
HYBRIDIZE
RSV-InfectedCEF (+ control)
“Normal”chick DNA
MOUSEDROSOPHILA
HUMAN
+ + + + +
Stehelin, Bishop and Varmus
Thus: a proto-oncogene is the NORMAL progenitor gene of a viral oncogene
J.Michael Biship Harold Varmus
1989 Nobel Prize in Physiology and Medicine
Retrovirus and the Cancer Connection
ALV RSV
Weakly transforming virus (3-6 months)Genome size ~8.5 kbReplication competent
Acutely transforming virus (1-2 weeks)Genome size ~10 kbReplication deficient
Whats the difference between weak and acutely transforming viruses?
gag envpolΔenv
gag pol env src
LTR LTRgag pol env
LTR LTRgag pol
env src
8.5 kb
10 kb
ALV DNA
RSV DNA
pol
env src
gag
gag & pol proteins
Env proteins
Src proteins*
Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)
*Overexpression of mutated Src tyrosine kinase leads to cell transformation
Avian Leukosis Virus Leukemogenesisby Promoter Insertional mutagenesis
ALV
ALV Integration Site5’ or 3’ of gene
c-myc gene
transcription of myc from LTR
ABERRANT REGULATION OFmyc TRANSCRIPTIONMMTV ‐int‐1
int‐2
etc.
LTR LTRgag pol env
LTR LTRgag pol
env src
8.5 kb
10 kb
ALV DNA
RSV DNA
pol
env src
gag
gag & pol proteins
Env proteins
Src proteins*
Genomes of Avian Leukosis Virus (ALV) and Rous Sarcoma Virus (RSV)
*Overexpression of mutated Src tyrosine kinase leads to cell transformation
Src and tyrosine phosphorylation (Brugge and Hunter)
RSV encodes a 60 kD protein called Src
Injected RSV-bearing tumors into rabbits--generatedanti-sera against 60 kD protein
Using a technique called Immunoprecipitation, investigators showed that this anti-sera bound a protein with an unusual kinase specificity--
Shortly thereafter, many oncogenes identified in RNA viruses encoded tyrosine kinases.
In cells transformed by many Src and Abl (and other TK oncogenes)There is dramatic increase in protein tyrosine phosphosprylations
Structure of v-Src gene
SH3 SH2 SH1 (PTK catalytic)p60src (RSV)
Structure of v-Src vs c-Src genes
SH3 SH2 SH1 (PTK catalytic)v-Src
c-SrcTyr527
Structure of v-Src vs c-Src genes
SH3 SH2 SH1 (PTK catalytic)v-Src
c-Src
Tyr527
(open-high activity)
(closed-suppressed activity)
Non-receptor tyrosine kinases are generally maintained inauto-inhibited clamped structures, but very sensitive to
mutational activation and global destabilization.
Kuriyan, Rockefeller University, UC Berkely
Abelson leukemia virus encodes a cellular oncogene called abl, anothernon-receptor tyrosine kinase (like Src)
Abelson murine leukemia virus (A-MuLV)
Derived from Moloney murine leukemia virus (Mo-MuLV)Genus= gamma-retrovirusCauses a rapidly progressive lymphosarcoma in mice
6 kB
LTR LTRgag abl
9 kB
LTR LTRgag
p120 Gag-Abl
pol env
A-MuLV
Mo-MuLV
Helper or Dependovirus
Tyrosine kinases are regulated by ‘auto-clamped’structures using their SH2 and SH3 domains
Gag
Bcr CML, ALL
c-ablc-Abl
v-ablv-Abl
v-Src and v-Crk oncogene products
SH3 SH2 SH1 (PTK catalytic)
viral gag
p60src (RSV)
p47gag-crk (CT10)
PLC-γ II catalytic-1 catalytic-2
Crk was originally identified in the retroviral genome of avian retrovirus CT10
SH2gag SH31 440208 248 340 373 424
PhosphotyrosinepYxxP
Proline-rich sequencePx(L)PxK,R
p130 Cas
Paxillinc-CblEGF-RGab1XWee1
c-AblC3GSOSDOCK180Esp15JNKPI3 Kinase
v-Crk ; CT10 regulator of kinase
p47gag-crk
97
66
45
kDa
.
116
CEF
/ v-
Crk
Blot : α- P-Tyr
CEF
31
200p130 Cas
Paxillin
Binding partners* Binding partners*
* Partial List
AFAP
cAbl/Bcr-Abl
SH2 SH3-CSH3-N Y
PP
222c-Crk II
SH2 SH3-N c-Crk I
SH2 SH3-NGag v-Crk
SH2 SH3-CSH3-N YP
P Crk-L207
Crk family of adaptor proteins
Harvey and Kirsten murine sarcoma viruses Originally discovered in 1960’s by Jennifer Harvey and Weiner Kirsten (Harvey murine sarcoma virus and the Kirsten sarcoma virus)
Both viruses encoded retroviral genomes with the h-ras or k-rascellular genes. The protein products were 21 kD GTPases and later shown to have G12V mutations.
Ras-GDP Ras-GTP
Guanine-nucleotide exchange factor
GTPase activating protein (GAP)
Guanine-nucleotide exchange factor
GTPase activating protein (GAP)
G12V RasG12V Ras98% 2% 100%0%
The Field Comes Of Age
Raf
ERK
AP-1
Activated Proto-oncogenes from DNA Transfection
RESULT: RAS “Activation”is due to a SINGLE point mutation (gly val) at codon 12
Use Aluprobe
Isolate Human DNA
RESULT: A SINGLE human gene is responsible for transformingcapability
Sequence
RESULT: The gene is the HUMANc-H-ras gene !!!
Compare sequenceto NORMAL gene
Parada and Weinberg
Activated Proto-oncogenes from DNA Transfection
Human Bladder Tumorcell line DNA
Isolate high MWDNA
Isolate DNAfragments
restrictionendonuclease
TRANSFECTION
NIH 3T3fibroblasts
TRANSFORMATION
Isolate DNA (>99% mouse +8-10 human genes)
TRANSFORMATION
Isolate HumanDNA
Parada and Weinberg
• 1984- Avian erythroblastosis virus gene(v-erbB) is shown to be a truncated Epidermal Growth Factor receptor
• 1985- Simian sarcoma virus transforming gene (v-sis) is shown to be analogous to the β chain of PDGF receptor
• 1987- v-jun Avian oncogene is shown to be analogous to AP-1
Many of the Proto-oncogenes in viruses turned out to be involved in sporadic mutations in human cancers
Retrospective view of the Importance of Retroviral Oncogenes
About 25 naturally occurring oncogenes have been identified
Oncogenes in the 21st Century• STI 571 (Gleevec, Imatininb) is the first line therapy
for CML– The target protein is Bcr-Abl, a protein tyrosine kinase (Abl
was first known from a retrovirus)– Therapy resistance stems from changes in other proto-
oncogenes such as Src– Second line and combination therapies with Src inhibitors
are becoming standard• BUT-there are failures as well as successes-EGF-R
inhibitors are of limited value, so there is still much to learn
The EGF-R-Ras-Raf-Map kinase pathway is an important targeting module for cancer therapeutics
BAY439006 (Advanced kidney cancer)Raf
MAP-kinase
Transcription of growth gene
Src/PI3-kinase
(Tyrosine kinase; Abl)
EGF-R/HER2/Neu
Ras Ras InhibitorsRTK Inhibitors(MANY; ie Gefitinib)
Gleevac-STI571
AG490
Nu2048 (Myc)
v-Erb2v-Ha-Ras
v-Raf
v-Myc
v-Src
ABT-737v-p110
Memorial symposium for the contributions of Teruko Hanafusa (1998)
Nature Reviews Molecular Cell Biology 2; 467-475 (2001); THE HUNTING OF THE SRC
Homework Assignment: Due October 16th
1. How would you determine whether the virus is a retrovirus? What assays would you do, and how would you interpret your data?
Now that you are experienced virologists (after passing “Viruses, Cells, and Diseases”!),you have been invited to work on novel oncogenic virus (called C5125Q) isolated from a mouse populationin the Outback of Australia. Injection of serum or purified virus causes a acute leukemia, specific to T and B cells.
Using both specific knowledge (and your imagination) describe a scenario by which C5125Q may cause transformation. Remember, this is a novel retrovirus and not likely to behave exactly as the viruses we studied in class!
2.
1838. Recognized as a collection of disorganized, abnormal cells (Virchow)
1907-33. Cell-free filtrates induce tumors in chickens (Rous, Shope, Fujimani)
1910-25 Chromosomal abnormalities (Tyzzer, Boveri).
1915-41 Carcinogen-induced cancers, Multiple factorsinvolved--Initiation and Promotion factors (Yamigiwa, Berenblum)
1951-53 Mouse Leukemia Virus, Polyoma Virus (Gross, Friend, Stewart)
1964. Provirus Hypothesis (Temin)
1969-76. Normal cells suppress transformation--cell hybrids (Harris,Stanbridge)
1970. Reverse Transcriptase (Baltimore, Temin)
1971. Hereditary nature of retinoblastoma (Knudsen)
1976-80. Oncogene theory, cellular origin of viral transforming genes(Varmus, Bishop, Hanafusa, Vogt).
Milestones in Cancer Biology.
Milestones in Cancer Biology-part II
1979-1982. Discovery of Ras oncogene (Weinberg, Cooper, Wigler, Barbacid)
1982-1987. Activation of many genes by virus integration, many oncogenesidentified.
1988. Isolation of Retinoblastoma gene--concept of ‘tumor suppressor genes’developed. (Weinberg)
1988-1991. Multistep carcinogenesis models (Vogelstein, Liotta)
1992-1994. Programmed cell death, Bcl-2, p53 (Korsmeyer, Levine, Horvitz)
1996. Angiogenesis Inhibitors (Folkman)
1999. STI-571 (Glivac) Bcr-Abl Inhibitor, CML (Drucker, Kuriyan)
2001-06. Era of Genomics, Proteomics, to the cancer problem (Collins, Ventor,Lander)
2007. Cancer stem cell biology and tumor microenvironment (Bissell/Weinberg)