LECTURE 3 + 4: Tumor Viruses Flashcards
Are all carcinogens mutagens? Are all mutagens carcinogens?
All mutagens ARE carcinogens
All carcinogens are NOT mutagens (eg. immune cell activators, asbestos)
Raus Sarcoma Virus
Chicken with sarcoma in breast tissue
removed, broken down, mixed with sand
filter
inject filtrate into young chicken
sarcoma develops
RNA tumor virus
virus with an RNA genome
Retrovirus
RNA virus
Replicates by reverse transcription (RNA -> DNA)
Inserts a DNA copy of its RNA genome into the DNA of the host cell
Doesn’t have to cause cancer
Sections of Retrovirus RNA genome
- Diploid viral genome - 2 copies of single stranded RNA
- gag: encodes core structural proteins of the virus, formed capsid protects the RNA
- pol: encodes enzymes - reverse transcriptase & integrase
- env: outer envelope protein
Retrovirus life cycle
- Reverse transcriptase (pol) synthesizes a complementary DNA strand using viral DNA as template (DNA-RNA hybrid)
- RNA strand degraded (ssDNA)
- Reverse transcriptase (pol) synthesizes a second DNA strand (Unintengrated dsDNA)
- Integrase drops it in the DNA of the host cell (integrated DNA)
Viral DNA is now a provirus.
viral DNA -> viral RNA -> viral proteins
regulatory regions at end of viral RNA
LTR/Long Terminal Repeat
Retrovirus RNA genome (w LTR)
*viral RNA *
5′ - [R] - [U5] - [Gag/Pol/Env genes] - [U3] -[R] - 3′
provirus
5′ - [U3] - [R] - [U5] - [Gag/Pol/Env genes] - [U3] - [R] - [U5] - 3′
LTR composition
U3 - Enhancer/promoter region
R - Repeating sequence of DNA
U5 - Initiation point for reverse transcriptase
Why was the discovery of reverse transcriptase so transformative?
- New understanding of evolutionary origin of DNA
- New tool for cloning genes
- Revealed how RNA viruses can cause cancer
Most RNA viruses __are/are not__ cytolytic
They are not cytolytic (DNA viruses are)
means they don’t kill the host cell they infect
How do RNA viruses impact cells they infect?
Can be non-transforming (ALV) or transforming (RSV)
transforming - permanently change the cell, typically cancerous
Temin & Ruben (1958)
Were working to understand how RSV infects cells to make them cancerous
Developed the focus formation assay
- infected chicken fibroblast cells with RSV and observed the formation of foci (overcame contact inhibition)
First demo of how virus could induce cancer
GS Martin (1970) - How does RSV work?
What did the experiment show?
Discovered SRC, showed that it was essential to the transformation process and not growth
Showed
* viral transformation was separate from replication
* src was needed to maintain the transformation state
* not a hit and run
GS Martin (1970) - How does RSV work?
Explain the temperature specific mechanism
Infected with RSV ts (temperature sensitive) mutant
at 37 - transformed morphology
at 41 - normal morphology
at 37 - again transformed morphology
virus replicates at either temperature
Activity, independent of viral replication, is required for viral transformation
Activity is also reversible
Temperature-specific mutations
Higher temps cause alteration of protein stability and function, unfolds the protein
Temperature specific mutations of RSV
37C - permissive temp
41C - non permissive temp
Slide 27 i don’t get it tara help
tara will help
Southern Blot
Detect specific DNA sequence in a sample
Northern Blot
Detect specific **RNA **sequence in a sample
Western Blot
Detect specific **protein **sequence in a sample
Northern vs Southern vs Western Blot
RNA, DNA, Protein
more differences
Varmus-Bishop (1975)
What did they show?
Showed that genes that cause cancer were normal genes hijacked by viruses - cancer came from within
proto-oncogene vs oncogene
proto-oncogene is a precursor to an oncogene
has the potential to cause transformations in the cell and cause cancer
Varmus-Bishop (1975)
Making of the src DNA probe: Idea
Make single-stranded src-specific complementary DNA (cDNA) probe.
Follow v-src DNA after infection.
Varmus-Bishop (1975)
Making of the src DNA probe: Procedure
** wildtype RSV **
- all parts of viral RNA genome intact, including src
- can replicate and transform
mutant RSV
- lost the src sequence
- can only replicate, could **not transform **
took RNA from the wildtype RSV
reverse transcriptase to make single stranded cDNA with radiolabeled deoxyribonucleoside triphosphates
wildtype RNA destroyed with alkali
sscDNA hybridised to viral RNA genome of mutant RSV => DNA-RNA hybrid
most of the DNA annealed except for the part encoding for c-SRC
hybrids were discards
ssDNA fragments left –> used as a src-specific probe
Varmus-Bishop (1975)
observations of the src probe
- v-src probe hybridizes to
cellular gene (c-src)! - c-src conserved to sponges
- Normal cellular gene (introns,
exons) - Unlinked to endogenous viral
genes
Varmus-Bishop (1975)
v-src vs c-src
the viral src gene (v-src) was a captured cellular src gene (c-src)
c-src exists in segments in the cellular genome, has introns (proto-oncogene)
v-src in the virus is continuous (oncogene)
RSV RNA genome
– gag pol env src –
What type of protein is (c)src?
a tyrosine kinase
What is a tyrosine kinase
Enzyme that transfers a phosphate group from ATP to the tyrosine residues of specific proteins inside a cell
Acts as an on/off switch in cells
Protein structure of (c)src
SH3
Kinase
C-terminal
SH2
Regulatory (SH3, SH2) and Catalytic domain (Kinase)
C-terminal tail contains a tyrosine residue
Protein structure of c-src: how is it kept inactive?
Phosphorylated Y at C-terminus keeps c-src inactive until cellular tyrosine signals remove this pY and activate Src
Protein structure of c-src:
how does it activate?
dephosphorylation of the tyrosine reside activates c-src
How is the protein structure of v-src different?
Contains mutations and C-terminal deletions that prevent autoinhibition
Mutations prevent phosphorylation, keeping it permanently active
why are transduced proto-oncogenes transforming?
- overexpression - viral oncogene expressed from strong viral promoter and enhancer
- protein activation - normal protein switches on and off (autoinhibition). truncated protein is always on
Lecture 4 transition - no question
take a little break woohoooo
How would you determine if a RSV-like RNA
tumor virus contains an oncogene that is active
due to the truncation of a proto-oncogene?
tara please help
tara please help
Inject chicken with RSV
Inject chicken with ALV
What do you observe?
RSV
-> tumor in 1-2 weeks
-> all infected cells transformed
ALV
-> leukemia in ~6 months
-> tumor development initiated in very few cells
leukemogenesis in ALV
leukemogenesis in ALV requires provirus integration in specific sites
tumor development initiated when provirus integrates in a certain site only
all tumor cells have provirus integrated at the same site - shown by southern blot
Insertional mutagenesis
provirus integration adjacent to a proto-oncogene
leads to increased expression by downstream promotion or enhancement
Insertional mutagenesis: downstream promotion
integration of provirus directly upstream of proto-oncogene
drives transcription of proto-oncogene (readthrough)
Insertional mutagenesis: enhancement
proto-oncogene independently transcribed, but its expression is enhanced by the U3
Insertional mutagenesis can activate many
proto-oncogenes
examples
ALV (chicken, myc gene, leukosis)
MLV (mouse, pim-1, t-cell lymphoma)
MMTV (mouse, int-1, mammary carcinoma)
FeLV (cat, myc, t-cell lymphoma)
Viruses as causes of human cancer
examples
RNA viruses rarely cause human cancer
DNA viruses cause ~15%
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