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MCB132 > LECTURE 3 + 4: Tumor Viruses > Flashcards

LECTURE 3 + 4: Tumor Viruses Flashcards

1
Q

Are all carcinogens mutagens? Are all mutagens carcinogens?

A

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

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2
Q

Raus Sarcoma Virus

A

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

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3
Q

RNA tumor virus

A

virus with an RNA genome

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4
Q

Retrovirus

A

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

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5
Q

Retrovirus Virion
(label diagram)

A

pic

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6
Q

Sections of Retrovirus RNA genome

A
  • 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
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7
Q

Retrovirus life cycle

A
  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

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8
Q

regulatory regions at end of viral RNA

A

LTR/Long Terminal Repeat

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9
Q

Retrovirus RNA genome (w LTR)

A

*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′

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10
Q

LTR composition

A

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

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11
Q

Why was the discovery of reverse transcriptase so transformative?

A
  • New understanding of evolutionary origin of DNA
  • New tool for cloning genes
  • Revealed how RNA viruses can cause cancer
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12
Q

Most RNA viruses __are/are not__ cytolytic

A

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

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13
Q

How do RNA viruses impact cells they infect?

A

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

transforming - permanently change the cell, typically cancerous

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14
Q

Temin & Ruben (1958)

A

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

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15
Q

GS Martin (1970) - How does RSV work?

What did the experiment show?

A

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

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16
Q

GS Martin (1970) - How does RSV work?

Explain the temperature specific mechanism

A

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

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17
Q

Temperature-specific mutations

A

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

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18
Q

Temperature specific mutations of RSV

A

37C - permissive temp
41C - non permissive temp

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19
Q

Slide 27 i don’t get it tara help

A

tara will help

20
Q

Southern Blot

A

Detect specific DNA sequence in a sample

21
Q

Northern Blot

A

Detect specific **RNA **sequence in a sample

22
Q

Western Blot

A

Detect specific **protein **sequence in a sample

23
Q

Northern vs Southern vs Western Blot

A

RNA, DNA, Protein
more differences

24
Q

Varmus-Bishop (1975)
What did they show?

A

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

25
Q

proto-oncogene vs oncogene

A

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

26
Q

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

A

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

27
Q

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

A

** 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

28
Q

Varmus-Bishop (1975)
observations of the src probe

A
  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
29
Q

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

A

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)

30
Q

RSV RNA genome

A

– gag pol env src –

31
Q

What type of protein is (c)src?

A

a tyrosine kinase

32
Q

What is a tyrosine kinase

A

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

33
Q

Protein structure of (c)src

A

SH3
Kinase
C-terminal
SH2

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

34
Q

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

A

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

35
Q

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

A

dephosphorylation of the tyrosine reside activates c-src

36
Q

How is the protein structure of v-src different?

A

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

37
Q

why are transduced proto-oncogenes transforming?

A
  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
38
Q

Lecture 4 transition - no question

A

take a little break woohoooo

39
Q

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

A

tara please help

40
Q

Inject chicken with RSV
Inject chicken with ALV

What do you observe?

A

RSV
-> tumor in 1-2 weeks
-> all infected cells transformed

ALV
-> leukemia in ~6 months
-> tumor development initiated in very few cells

41
Q

leukemogenesis in ALV

A

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

42
Q

Insertional mutagenesis

A

provirus integration adjacent to a proto-oncogene

leads to increased expression by downstream promotion or enhancement

43
Q

Insertional mutagenesis: downstream promotion

A

integration of provirus directly upstream of proto-oncogene

drives transcription of proto-oncogene (readthrough)

44
Q

Insertional mutagenesis: enhancement

A

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

45
Q

Insertional mutagenesis can activate many
proto-oncogenes
examples

A

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

46
Q

Viruses as causes of human cancer
examples

A

RNA viruses rarely cause human cancer
DNA viruses cause ~15%
table

MCB132 (7 decks)

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