1. DNA Tumour Viruses Flashcards
what type of viruses cause cancer
DNA viruses
how do viruses infect cells to cause cancer?
infect non-dividing cells and forces them into cell cycle to provide machinery for viral replication
how are RNA viruses associated with cancer?
RNA viruses associated with cancer via inflammation
what is the top cancer for men? top cancer for women?
men: liver cancer
women: cervical cancer
3 characteristics of adenovirus?
- non-enveloped
- icosahedral
- 90nm diameter
3 characteristics of adenovirus genome
- dsDNA
- 35-45kb
- linear
who are at high risk for adenovirus? why
military recruits –> high stress environment + lots of exercise + tight conditions = become immunocompromised
what are 8 diseases that adenoviruses cause? what’s the most prominent one?
- acute respiratory issues
- pharyngitis
- gastroenteritis
- conjunctivitis
- pneumonia
- keratoconjunctivitis
- acute hemorrhagic cystitis
- hepatitis
upper respiratory illness is main one
describe adenovirus as a model for cancer biology
when first isolated, found it caused tumours in rodents
but we know now that injecting RODENTS with HUMAN adenovirus means the virus cannot replicate well in rodents but virus produces oncogenes to cause cancer
in humans, human adenovirus replicates very well to kill cells but we also have good immune response against it
what type of cells does adenovirus infect?
epithelial cells
what does adenovirus produce in the nucleus? what does this lead to?
creates inclusion bodies with crystal lattices of the virus in the nucleus to lead to lysis
how can we purify adenovirus?
CsCl gradient
how many proteins does adenovirus genome encode?
dsDNA genome encodes for 40 proteins
what is the role of early adenovirus genes?
sets up the cell so virus can replicate
what is the role of late adenovirus genes?
structural components, etc. for function
how long is the adenovirus life cycle?
12-14h
what did we major discovery came from adenovirus studies? how did we confirm this?
SPLICING –> mRNAs looked different than genome
hybridized viral RNA to genomic DNA and found introns looped out aka spliced out
which gene causes cancer from adenovirus? what type of gene is this?
first gene produced = E1A –> oncogene
what is the role of E1A?
transcription factor –> turns gene transcription ON
what happens if there is no E1A?
no E1A = no replication = dead virus
what are the functional domains of E1A?
- Nterminus
- CR1
- CR2
- CR3
what are the 2 roles of CR3?
- Binds TATA Box Binding Protein (TBP) which binds TATA at promoters to turn on gene expression
- Binds Activating Transcription Factor (ATF) at many promoters for genes required by adenovirus
what are the 2 types of E1A? what is the difference? how does this change the cancer outcome? what does this mean?
289R and 243R
243R does not have CR3 but can still cause cancer –> therefore not just transcription causes cancer
which functional domains of E1A are most important?
CR1, CR2, and N
what protein does E1A interact with to cause cancer?
Rb
describe E1A’s activity at Rb
E1A ACTIVATES E2F TRANSCRIPTION
- Normally, Rb binds E2F to block it from activating S phase genes
- but E1A sequesters Rb so E2F is able to activate S phase and E2 genes
how do cells normally activate E2F?
growth factors activate CDK to phosphorylate Rb so E2F can leave and activate genes
why is it important that E1A blocks Rb to cause cancer?
Rb acts as the guardian of G1-S checkpoint –> once it passes this boundary, it must commit to cancer
what happens once E2F is activated?
p14 ARF gene is active and sequesters MDM, releasing and stabilizing p53 to cause apoptosis
how is apoptosis inhibited? (2)
- E1B-55K inhibits p53 so the cell can survive
- E1B-19K inhibits apoptosis
what is p53?
a transcription factor that is a tumour suppressor –> most mutated gene in cancer bc essential to prevent cancer
what are the 3 domains of p53?
- DNA binding domain
- transactivation domain at N terminus
- regulatory domain at C terminus
what is the role of the DNA binding domain?
binds directly to specific DNA sequence at promoter of target gene to turn it on
what is the role of the transactivation domain at N terminus?
binds transcription factors
what is the role of the regulatory region at C terminus?
allows 4 p53 to bind DNA (bc p53 binds as tetramer)
why do cancers often mutate p53?
mutations prevent p53 from binding its normal genes so it cannot act as a tumour suppressor
when is p53 turned off? when is p53 turned on (3)?
normally off! usually no need for apoptosis
but turns on due to:
1. Ionizing radiation (UV, Xray)
2. DNA breaks that activate kinases
3. Oncogenes are expressed (ex. p14 ARF)
What is the relationship between MDM and p53?
negative feedback loop
describe the negative feedback loop btwn MDM and p53 (3 steps)
- p53 is activated and induces genes to prevent cancer
- p53 turns on MDM2
- MDM2 causes p53 destruction
describe the 5 types of genes that p53 turns on
- growth arrest
- DNA repair
- anti-angiogenesis
- apoptosis
- P21 (inhibits cell cycle kinases)
if we do a western blot for p53 under normal conditions will we see p53? what about under cancer conditions?
normal conditions: NO –> MDM is degrading it
cancer conditions: YES –> p53 is stabilized
describe the 1-2 punch of adenovirus cancer causing ability
- E1A sequesters Rb so E2F is active but this stabilizes p53
- E1B inhibits p53 –> cell survival
why is polyomavirus linked to non-cancer-causing virus poliovirus?
grew polio on monkey cells and used as a vaccine –> found there was still virus replicating which was SV40 –> injected in rodents and caused tumours
what are 3 general characteristics of polyomavirus?
- non-enveloped
- icosahedral
- 45 nm diameter
what is the most studied polyomavirus?
SV40
what are oncolytic viruses?
genetically alter the virus to be able to kill cancer cells
what are characteristics of polyomavirus genome?
- dsDNA
- 4.5-5.5 kb
- circular
what were the “firsts” of polyomavirus?
- first biological entity to have genome sequenced
- first transcription factor found
- first transcriptional enhancer found
- first nuclear localization sequence found
which polyomaviruses cause cancer in humans?
- JC virus –> brain disorder
- BK virus –> early childhood infection
- Merkel cell polyomavirus –> merkel cell carcinoma in immunosuppressed patients
what is the structure of polyomavirus DNA?
circular dsDNA wrapped in nucleosomes with histone octamer core
why did polyomavirus help us learn about human DNA synthesis?
DNA replication uses all the same enzymes as we use
what is the main protein in polyomavirus? is it early or late?
main protein = LgT
EARLY
is LgT an oncogene or tumour suppressor?
ONCOGENE –> causes cancer
what is the role of LgT and what does this indicate about its binding regions?
sequesters Rb for E2F activity AND inhibits p53 at the SAME TIME!!
therefore has Rb binding region AND p53 binding region
what happens if you put SV40 genome in test tube with LgT protein?
LgT forms hexamers and binds SV40 DNA and recruits all machinery for replication
what are 3 characteristics of papillomavirus?
- non-enveloped
- icosahedral
- 55nm in diameter
what are 3 characteristics of papillomavirus genome?
- dsDNA
- 8kb
- circular
which virus was papillomavirus previously grouped together with?
polyomavirus
what does papillomavirus lead to?
cervical cancer
incidence of cervical cancer in low vs high income countries
low incidence in high income countries bc preventable via screening
biggest mortality in low income countries
can HPV cause cancers other than cervical cancer?
yes, but at lower rates
is all cervical cancer caused by HPV?
YES!! no link is this strong –> 100% of the time we see viral DNA in the cell
what are HeLa cell?
cells stolen from cervical cancer patient which express oncogenes and viral DNA
what do all HPVs cause?
SKIN abnormality!
what is the difference btwn HPVs?
subtle sequence differences
what are 3 types of skin abnormalities that HPV causes?
- High risk
- Low risk
- Benign
what is benign HPV?
warts –> hyperproliferation of cells but doesn’t lead to cancer
which 2 HPVs are the highest risk for HPV cancers?
- HPV 16 and HPV 18
how is HPV vaccine made? (3)
- express recombinant capsid protein (L1) in eukaryotic cells
- L1 proteins self-assemble to form capsomeres which form VLPs without the DNA
- immune system can easily detect and fight virus
what are the 3 types of HPV vaccines?
- Bivalent (Gardacil) - only targets 16 and 18
- Quadrivalent
- 9-valent
why is it important for HPV vaccines to have high valency?
to target as many types of HPV as possible
why has the HPV vaccine taught us that we need diversity in population testing?
9-valent doesn’t cover high risk 35 bc wasn’t as frequent in western countries, but 10% of cases in Africa are caused by this
what is the function of long coding region with promoter, and origin of replication?
for regulation of viral gene expression and regulation
what are the 2 late genes and their functions. are they required?
- L1 –> required for capsid
- L2 –> acts like a glue inside the capsid but not required
what happens when you add papillomavirus to rodents?
no cancer!
how does papillomavirus infect?
- enters epithelium via microlesions and infects basal layers of skin where cells are slowly dividing and differentiating
- as cells differentiate up, virus progress up to top layer of skin to be released
why is papillomavirus infection hard to study?
need whole epithelium system to stimulate skin differentiation system
how does cervical cancer screening work?
- stain cervical cells
- cells with large nuclei = cancer
does HPV always cause cancer?
no, not part of the virus lifecycle –> rare accident
how common is HPV?
Most common STI
what are the 2 outcomes of HPV infection?
- immune system can usually get rid of it
- virus persists in 10-20% of cases (but we don’t know why)
what happens if HPV is left undetected?
can become invasive carcinoma , may take several years/decades to develop after intial infection
describe the HPV genome in early vs late stages
early = in episome
later = integrates into host DNA
what is the minimal piece of viral genome that is integrated
LCR, E6, E7, L1 –> minimal piece of DNA required to cause cervical cancer
which HPV proteins are always made in cancer?
E6 and E7
what is the role of E6?
inhibit apoptosis of infected cell
what is the role of E7?
stimulate DNA synthesis in infected cell
what is similar between E1A, LgT, and E7?
all have LxCxE motif in Rb binding domains to bind and displace Rb
describe the roles of E6 and E7 (5 steps)
- E7 sequesters Rb to release E2F
- E2F induces S phase genes and p14 ARF
- P14 ARF sequesters MDM to release p53
- E6-AP cellular protein binds P53
- E6 binds E6-AP to degrade p53
what is E6-AP?
cellular ubiquitin ligase that ubiquitinates p53 as a post-translational mechanism, targeting p53 for degradation
when does E6-AP target p53?
only if E6 is present
what are 3 characteristics of Epstein Barr virus?
- enveloped
- icosahedral capsid
- 200nm diameter
what are 3 characteristics of Epstein Barr virus genome?
- dsDNA
- 180kb (v large)
- linear
4 diseases that epstein barr viruses lead to?
- Lymphoproliferative disease (w immunodeficiency)
- Burkitt’s lymphoma (w malaria)
- Hodgkin’s lymphoma
- Nasopharangeal cancer (head and neck cancer)
what is the treatment for EBV? what is the cure rate?
treatment = cyclophosphamides
70-80% cure rate
what is different about the structure of EBV vs other cancer-causing viruses?
ENVELOPED! with viral proteins and receptors
why does EBV not usually cause cancer if it sits latent in most ppl?
- infects as linear genome, then becomes circular (episome) when latent
- cells divide and transmit the episome but still sits latent
- episome hangs around!
what does EBV express while it sits latent?
expresses EBNA and latent membrane proteins (LMP) genes
how is EBV spread?
by saliva (aka mono)
what cells does EBV infect? why?
enters EPITHELIAL CELLS in mouth bc it is the only place where there is a full replication system to produce viral particles that INFECT B CELLS
what happens if a person doesn’t have B cells?
no EBV infection!
why does EBV sit latent?
once it infects oral epithelium, there is big proliferation of cells (like WBC) with EBV –> immune system can control it and leaves memory B cells with latent EBV which may go back and infect oral epithelial cells
how is EBV associated with malaria?
malaria causes hyperactivation in genes that make antibodies –> causes increased mutational burden in immune cells which drive cancer
what happens if lymphoid and oral epithelial cells get mutations? example
can get EBV-driven cancer if mutated and infected
ex. smoking leads to epithelial cell mutations
does EBV also interact with Rb and P53?
EBV does sequester Rb and degrade p53
how does EBV cause cell proliferation?
viral proteins in B cells, LMP1 and LMP2A, mimic cellular proteins
what is the role of LMP1?
binds molecules and mimic CD40 to induce NF-KB pathway leading to cancer
what is the role of LMP2A?
mimics BCR
