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Lecture 5

BIOL 4010 Lecture Notes - Lecture 5: Integrase, Avian Sarcoma Leukosis Virus, Colonoscopy


Department
Biology
Course Code
BIOL 4010
Professor
Anafi
Lecture
5

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How do retroviruses cause cancer?
Retrovirus genome:
3 genes: Gag (internal proteins), pol (RT and integrase), env (envelope) proteins (all are
important for replication of the virus). with LTRs (long terminal repeats) on both ends of the
genome = If the genome is responsible for the cancer something must be responsible for
replication of virus and transformation of cells
Replication and transformation are separate activities of RSV (found by Rous) which can
transform cells and replicate. However, naturally occurring relatives of RSV have the following
properties:
RAV: chicken normally has them as part of their ecology. replicates but does not transform cells
Bryen: strain of RSV transforms cells but does not replicate (the cells don’t make viruses)
Co-transfection of RAV and Bryen = Bryen becomes able to replicate with the help of RAV
machinery
Replication and transformation are separable activities of RSV. To prove this and figure out
activities of genes: mutate the viral genes and “hope” that one of the mutations would reduce
the protein stability at a certain temperature = temperature sensitive mutants.
Isolated of RSV mutant (T1 strain) that can replicate and transform the cells at 37 degrees. In 41-
42 degrees it loses the ability to transform cells, but it can still replicate the temperature
sensitive mutation is not related to the ability of the virus to replicate, but it is important for
transformation. replication and transformation are different activities and require different
genes
At 37 degrees, cells infected with mutant virus are transformed. At 41 degrees, the cells turn
normal. reduce temperature cells become transformed again. something in the
genome must explain that the replication and transformation are different activities

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RSV had a longer genome than RAV, it contains a new gene = src (usually viruses cant hold extra
genetic material because of their particle size) = RSV is a very unique virus
Src is responsible for transformation activity of the virus
RAV is transformation defective have a smaller genome than RSV
Sequencing was not available then most of this work was done by genetic analysis
How does RSV acquire a transforming gene? Where did it come from?
The discovery of protooncogenes
Mid 80s Bishop and Varmus
Showed that cancer doesn’t always start with a virus, but always starts with a They did a
southern blot: the use of short DNA probe (radioactive) in order to find its complementary
sequences. You can make a short probe to find your gene of interest. They were looking for Src.
Take DNA samples from cells (infected with RSV and uninfected= control)
They thought that uninfected cells would not contain Src and the infected cells would have Src.
They found that Src is also present in uninfected cells. Src is a normal gene that is not related
to transformation by retroviruses how is Src responsible for transformation in infected cells?
more analysis of the Src gene from infected and uninfected cells they found a point
mutation that activates Src from the virus the virus took the gene from normal cells. = they
started to understand Proto-oncogenes and oncogenes
Restriction enzyme cuts DNA, run on gel and transfer to membrane, specific DNA fingerprints,
src DNA from viral genome as a radioactive DNA probe detect matches = src is in infected and
uninfected cells more analysis of src infected cells and normal cells, they found that it is a
point mutation that activated the src from the Rous sarcoma virus = the virus took genes from
normal cells
Proto-oncogenes: the cell precursor of a retroviral transforming gene (oncogene)
Proto = non-mutated form (c-src)
They are usually important players in normally regulated signal transduction pathways (if you ko
Src in an animal it is lethal)
Oncogenes: mutated form (mutated c-src “when the endogenous proto-oncogene is mutated)”
or v-src)
Bishop and Varmus experiments others did similar experiments Oncogenes found in
retroviruses are oncogenes that were acquired by viruses in some way and turned to oncogenes
that contribute to cancer development
How do viruses turn proto-oncogenes to oncogenes?
RAV took normal src and turned to RSV = v-src
There are many ways that viruses can pick up genes from our genome:
Proto-onc gene in a normal cell infect with RAV enters the cell the virus was able to pick
up the gene and change it = v-src = RSV.
The virus transduces proto-onc as a result of an error during integration in/near a cellular gene,
(it may also pick up an mRNA from the cytoplasm and may include it in the viral genome during
reverse transcription) RSV that can infect other cells second round of infection: v-onc
(much stronger than the proto-onc) is integrated in the genome cell transformation
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Retroviruses don’t have proof reading machinery, they integrate randomly, they use at least 2
stages of copying the genome without proof reading (when the RT makes DNA from RNA and
when the virus is integrated in the DNA of the cell “proof reading is only in the nucleus”
made into RNA from DNA by the RNA pol 2 = no proof reading)
1 mutation in 5000 nts = 2 mutations in every virus produced (virus is 10 kbp) + they can take
parts of genes
Many of the oncogenes are truncated form of proto-onc = the regulation part is not there =
turns proto-oncogene to oncogene
Many viruses have done this
The study of Bishop and Varmus led many similar studies using different oncogenic retroviruses
that have mutated proto-oncogenes. (many are from chicken)
They found how to isolate the viruses from the cells and they found the oncogene in the virus
RSV has an extra gene, others have an oncogene instead of their regular genes (they don’t have
space to pack the extra gene) usually the genes are replaced (a gene is exchanged)
acquiring src was a rare event
Bryan strain has src instead of pol
Avian myelocytoma virus has myc instead of pol and gag
These two need a helper virus for replication because they don’t have the pol genes
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