LECTURE 3 + 4: Tumor Viruses

Question 1

Are all carcinogens mutagens? Are all mutagens carcinogens?

Answer 1

All mutagens ARE carcinogens
All carcinogens are NOT mutagens (eg. immune cell activators, asbestos)

Question 2

Raus Sarcoma Virus

Answer 2

Chicken with sarcoma in breast tissue
removed, broken down, mixed with sand
filter
inject filtrate into young chicken
sarcoma develops

Question 3

RNA tumor virus

Answer 3

virus with an RNA genome

Question 4

Retrovirus

Answer 4

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

Question 5

Retrovirus Virion
(label diagram)

Answer 5

pic

Question 6

Sections of Retrovirus RNA genome

Answer 6

• 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

Question 7

Retrovirus life cycle

Answer 7

1. Reverse transcriptase (pol) synthesizes a complementary DNA strand using viral DNA as template (DNA-RNA hybrid)
2. RNA strand degraded (ssDNA)
3. Reverse transcriptase (pol) synthesizes a second DNA strand (Unintengrated dsDNA)
4. Integrase drops it in the DNA of the host cell (integrated DNA)

Viral DNA is now a provirus.
viral DNA -> viral RNA -> viral proteins

Question 8

regulatory regions at end of viral RNA

Answer 8

LTR/Long Terminal Repeat

Question 9

Retrovirus RNA genome (w LTR)

Answer 9

*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′

Question 10

LTR composition

Answer 10

U3 - Enhancer/promoter region
R - Repeating sequence of DNA
U5 - Initiation point for reverse transcriptase

Question 11

Why was the discovery of reverse transcriptase so transformative?

Answer 11

• New understanding of evolutionary origin of DNA
• New tool for cloning genes
• Revealed how RNA viruses can cause cancer

Question 12

Most RNA viruses __are/are not__ cytolytic

Answer 12

They are not cytolytic (DNA viruses are)
means they don’t kill the host cell they infect

Question 13

How do RNA viruses impact cells they infect?

Answer 13

Can be non-transforming (ALV) or transforming (RSV)

transforming - permanently change the cell, typically cancerous

Question 14

Temin & Ruben (1958)

Answer 14

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

Question 15

GS Martin (1970) - How does RSV work?

What did the experiment show?

Answer 15

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

Question 16

GS Martin (1970) - How does RSV work?

Explain the temperature specific mechanism

Answer 16

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

Question 17

Temperature-specific mutations

Answer 17

Higher temps cause alteration of protein stability and function, unfolds the protein

Question 18

Temperature specific mutations of RSV

Answer 18

37C - permissive temp
41C - non permissive temp

Question 19

Slide 27 i don’t get it tara help

Answer 19

tara will help

Question 20

Southern Blot

Answer 20

Detect specific DNA sequence in a sample

Question 21

Northern Blot

Answer 21

Detect specific **RNA **sequence in a sample

Question 22

Western Blot

Answer 22

Detect specific **protein **sequence in a sample

Question 23

Northern vs Southern vs Western Blot

Answer 23

RNA, DNA, Protein
more differences

Question 24

Varmus-Bishop (1975)
What did they show?

Answer 24

Showed that genes that cause cancer were normal genes hijacked by viruses - cancer came from within

Question 25

proto-oncogene vs oncogene

Answer 25

proto-oncogene is a precursor to an oncogene has the potential to cause transformations in the cell and cause cancer

Question 26

Varmus-Bishop (1975) Making of the src DNA probe: Idea

Answer 26

Make single-stranded src-specific complementary DNA (cDNA) probe. Follow v-src DNA after infection.

Question 27

Varmus-Bishop (1975) Making of the src DNA probe: Procedure

Answer 27

** 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

Question 28

Varmus-Bishop (1975) observations of the src probe

Answer 28

1. v-src probe hybridizes to cellular gene (c-src)! 2. c-src conserved to sponges 3. Normal cellular gene (introns, exons) 4. Unlinked to endogenous viral genes

Question 29

Varmus-Bishop (1975) v-src vs c-src

Answer 29

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**)

Question 30

RSV RNA genome

Answer 30

-- gag pol env src --

Question 31

What type of protein is (c)src?

Answer 31

a tyrosine kinase

Question 32

What is a tyrosine kinase

Answer 32

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

Question 33

Protein structure of (c)src

Answer 33

SH3 Kinase C-terminal SH2 Regulatory (SH3, SH2) and Catalytic domain (Kinase) C-terminal tail contains a tyrosine residue

Question 34

Protein structure of c-src: how is it kept inactive?

Answer 34

Phosphorylated Y at C-terminus keeps c-src inactive until cellular tyrosine signals remove this pY and activate Src

Question 35

Protein structure of c-src: how does it activate?

Answer 35

dephosphorylation of the tyrosine reside activates c-src

Question 36

How is the protein structure of v-src different?

Answer 36

Contains mutations and C-terminal deletions that prevent autoinhibition Mutations prevent phosphorylation, keeping it permanently active

Question 37

why are transduced proto-oncogenes transforming?

Answer 37

1. overexpression - viral oncogene expressed from strong viral promoter and enhancer 2. protein activation - normal protein switches on and off (autoinhibition). truncated protein is always on

Question 38

Lecture 4 transition - no question

Answer 38

take a little break woohoooo

Question 39

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

Answer 39

tara please help

Question 40

Inject chicken with RSV Inject chicken with ALV What do you observe?

Answer 40

RSV -> tumor in 1-2 weeks -> all infected cells transformed ALV -> leukemia in ~6 months -> tumor development initiated in very few cells

Question 41

leukemogenesis in ALV

Answer 41

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

Question 42

Insertional mutagenesis

Answer 42

provirus integration adjacent to a proto-oncogene leads to increased expression by downstream promotion or enhancement

Question 43

Insertional mutagenesis: downstream promotion

Answer 43

integration of provirus directly upstream of proto-oncogene drives transcription of proto-oncogene (readthrough)

Question 44

Insertional mutagenesis: enhancement

Answer 44

proto-oncogene independently transcribed, but its expression is enhanced by the U3

Question 45

Insertional mutagenesis can activate many proto-oncogenes examples

Answer 45

ALV (chicken, myc gene, leukosis) MLV (mouse, pim-1, t-cell lymphoma) MMTV (mouse, int-1, mammary carcinoma) FeLV (cat, myc, t-cell lymphoma)

Question 46

Viruses as causes of human cancer examples

Answer 46

RNA viruses rarely cause human cancer DNA viruses cause ~15% [table](https://imgur.com/a/Hme1jlR)