EXMD504 lecture 2

What was found in chickens?

• they would take lysates → filter → still caused tumours in other chickens

• determined that it was this filterable agent (virus) that caused the tumours

• named: Rous sarcoma virus → contains this oncogene called VSARC that causes cancer

What are viruses?

• obligate parasites

• want to make more copies of themselves

How do viruses cause cancer?

cancer not part of their normal replication cycle → normally it reticulates, spreads and then goes away but in rare accidental events, they encode powerful oncogenes which get left behind and then cause cell growth = cancer

What is the direct mechanism?

• viral oncogene drives transformation

• p53 and Rb are inhibited

• increased proliferation and genomic instability

• DNA viruses

virus infects → viral oncoproteins made → p53 and Rb inactivated → uncontrolled growth → cancer

What is the indirect mechanism?

• persistant infection

• causes chronic inflammation

• oxidative stress and DNA damage by ROS

• tissue repair and regeneration

• don’t encode oncogenes

• + strand RNA viruses

When looking at virus associated cancer how must you break things down?

must break things down in terms of gender → different cancers affect the two genders differently

• females highest proportion of cancers are cervical (driven by papilloma virus)

• males highest proportion of cancers are liver

What are the categories in the Baltimore classification of viruses?

based on composition of genome:

DNA

• dsDNA

• ssDNA

• dsDNA (RT)

RNA

• dsRNA

• ssRNA (-)

• ssRNA (+)

Which viruses are referred to as the DNA tumour viruses?

• Herpesviridae

• Papillomaviridae

• Adenoviridae

• Polyomaviridae

because they have been shown to cause cancer in humans

In general, how do DNA viruses replicate?

they usually infect epithelial cells which are not actively dividing = not a great environment for a DNA virus

• they require oncogenic factors in this environment to replicate their genome

  • needs nucleotides and translation before it can replicate

• forces the epithelial cells back into S phase to make its DNA → eventually causing cancer

• leave some of their DNA behind = problem

What is the odds ratio?

probability that someone develops cancer with a factor divided by probability that someone develops cancer without that same factor

What is the most common viral mediated cancer?

cervical cancer

What does the herpes virus cause?

• Burkitts lymphoma

• Epstein Barr

• Kaposi’s sarcoma

What are the general characteristics of the Adenovirus?

• DNA tumour virus

• non-enveloped

• icosahedral

• 90nm in diameter

• have powerful oncogenes

• has a protein capsid

• Genome: dsDNA, 35-45kb, linear

what does the adenovirus cause?

• upper respiratory infections

• doesn’t cause cancer in humans but does in rodents

• a human virus

• doesn’t replicate in mice → machinery of mice not compatible with the human virus so it stalls → delivers it DNA containing oncogenes to the mouse cells → causes cancer

  • whereas in humans it just replicates and leaves → doesn’t lead to cancer

How many genes does the adenovirus encode?

40 genes → lots of splicing

• where splicing was discovered

What is the difference between early and late genes?

• early genes set up the cell to help the virus replicate

• the late genes encode all of the capsid parts to put together new virus particles

what are the genes of the adenovirus that are sufficient to cause cancer?

• region called E1A and E1B

• they are early genes, expressed first to set the groundwork to make the cell permissive for viral replication

What does E1A do?

it is a transcriptional activator → activates all of the other early genes

• an on switch

• also bind TBP (an initiation factor) → recruits potent transactivation domain that turns on the rest of the early genes and other transcription factors like ATF

also binds to Rb family of proteins

*comes in two variants ( 289R and 243R) → only differ by conserved region 3

What is the Rb protein?

• very important tumour suppressor

• it inhibits cell cycle continuation

• binds to another family of transcription factors called E2F which turn on S phase genes

normally Cdk will phosphorylate Rb and E2F gets released and can activate S phase

with an adenovirus infection E1A binds to Rb and E2F is free to continuously activate S phase → continued proliferation

What is important about the G1-S checkpoint in the cell cycle?

its the GO/ NO GO site → once it passes this boundary it will be committed to go through the cell cycle

• on button

• requires growth factors or a viral infection to get past the checkpoint

Why isn’t E1A alone enough to transform the cell?

because cells have this prideful protective mechanism that they turn on using another tumour suppressor gene called p14 ARF is E2F is constitutively on

normally: MDM2 (oncogene) which is bound to p53 (tumour suppressor) → causes p53 to be degraded

in cells that express too much EF2: p14 binds to MDM2 - p53 complex → inhibits degradation of p53 → cells undergo apoptosis to prevent too many cells from replicating

What is p53?

• protects body from cancer

• it itself is a transcription factor

• universal constant that must be lost/mutated to produce a tumour

• also prevents angiogenesis

what are similar to getting rid of p53?

• MDM2 over expression

• losing p14 ARF

• expression of viral oncogenes

What is p21?

• one of the strongest p53 induced genes

• Cdk inhibitor → stops cell cycle

What else helps E1A to transform the cell?

E1B

it binds to p53 and inhibits it → apoptosis of cells cannot occur

What is the E1B-19k homolog?

• functional homolog of Bcl2 which is a cellular protein → unregulated in B cell lymphomas

• it inhibits cell death

Why can the virus selectively replicate in tumour cells?

without EB1, the cell wouldn’t be able to replicate but because tumour cells don’t have p53 → having just EA1 is enough to transform the cells and cause more cancer

What is polyomavirus?

• causes cancer in humans

• non-enveloped

• protein capsid

• icosahedral

• 45nm in diameter

• genome: dsDNA, 4.5-5.5kb, circular

What does the polyomavirus tied to?

• SV-40 → monkey virus

• Polio

when making the vaccine for polio, they knew that the virus replicated really well in monkey kidney cells → grew monkey cells → infect the cells with polio → take the virus and inactivate with formalin → basis for vaccine

• it worked to prevent polio, however you could still see residual replication which was the SV-40 → this was causing tumours

what do all the polyomaviruses have in common?

they all tend to affect people that are immunocompromised

• usually the immune system can keep the virus latent but in these individuals this can’t happen

What does polyomavirus do with histones?

it steals the histones from the hist cell and wraps its DNA around the histones

What is the SV40 LgT oncogene?

an oncogene that if put into rodent cells it will transform the cells into cancer cells

• has a p53 binding domain and Rb binding domain

• it binds to Rb → takes it out of the picture leaving E2F available to transcribe S phase genes → binds p53 as well

What is the papilloma virus?

• non-enveloped

• icosahedral

• 55nm in diameter

• diverse and present everywhere in the skin → don’t all cause cancer

• genome: dsDNA, 8kb, circular

how does % of cervical cancer change with low income countries?

it increases significantly

• there is no screening or vaccines, making it way more dangerous when they can be preventable

What are the pieces of papilloma virus that are left behind called?

viral DNA and viral gene expression in cervical cancers like HeLa cells

Which papilloma virus types cause warts?

• 4

• 65

• 37

• 2a

• 57

What does it mean if the virus types are low risk?

it means they cause genital warts but don’t usually cause cancer

What does it mean if the virus types are hight risk

• they cause cancer

• their DNA is left behind

What is the HPV vaccine?

• make virus like particles

• take capsid protein L-1 → express it → self assembles into empty capsids

• no DNA, but looks like the virus

Why is it important to be vaccinated early?

• if its not done early enough, virus will have already infected, DNA left behind → vaccine won’t be able to prevent the virus or cancer

describe the structure of the papillomavirus

5 of L1 monomer = 1 capsomere

72 capsomere = 1 VLP

how does papillomavirus infect?

must reproduce stratified epithelial cell layers that are seen in skin

• replication of the virus is tied to the differentiation process

• virus must get down to basal layer of cells

• early genes turned on and gets moved along with differentiation → late genes turned on → get assembled and released

How is cervical cancer currently screened?

PAP smear

• has 70-80% sensitivity

• involves examination of stained cervical cells for abnormalities

• annual examination

• looking for present of abnormal cells on the cervix (large nuclei)

How does HPV progress?

1. skin gets infected → thickening of basal layer

2. progresses to cervical neoplasia

3. majority of people → immune system takes care of it

4. in some people, there is a persistent infection → what you catch in the PAP smear

5. if not it can get worse and in several years progress to cancer

6. replication stops and viral DNA gets integrated

What are the pieces of the papilloma virus that causes cancer?

minimal piece retain in cancer cell is what’s present in the HeLa cell → piece of L1 and LCR that regulates expression of gene and two early genes E6 and E7

What does E6 do?

inhibits apoptosis of an infected cell (same as E1B)

What does E7 do?

Stimulates DNA synthesis in an infected cell (same as E1A)

What is the sequence motif that Rb binds to in papillomavirus?

LXEXE

• Sv40 LgT and E1A also have this conserved region

What is EBV?

• enveloped

• icosahedral capsid

• 200nm in diameter

• genome: dsDNA, 180kb, linear

what types of cancer do they cause?

cancers related to diseases of the immune system

What is Burkitt’s lymphoma?

hyperactivation of B cells due to malaria → with translocation and amplifications of myc you get this type of lymphoma

describe the genome of EBV

• expresses 70 different proteins (latent or lytic proteins)

• virus usually just exits in B cells (latent)

how does EBV infect?

infect via mouth epithelial cells → virus replicates and makes more virus only in the most → gets into blood stream → gets into B cells → proliferation starts → some get destroyed some continue to become memory B cells → virus activates and replicates more and gets released

how does EBV cause proliferation?

1. LMP1 (latent protein) can mimic the existing receptor on the B cell (CD40) → induces the sam signalling pathway without needing the helper T cell

2. LMP2 (latent protein) uses the PI3 kinase pathway (pro-growth genes) → downstream of the B cell receptor without needing the antigen