Papillomaviruses (HPV)- pathogenesis

Question 1

HPV can cause a variety of diseases. Which can result from cutaneous infection and which can result from mucosal infection?

Answer 1

Cutaneous (skin)= warts/verucas (common) to ‘treeman’ (rare- this man also had immunodeficiency)

Mucosal (e.g genital)= genital warts (common) to cervical and oral cancer

Question 2

Transfection of HPV 16 and 18 genomes into primary keratinocytes permits immortalisation and transformation so to allow for the mapping of transforming factors from the virus. (HPV 6 and 11 do not allow this). What 3 proteins has this assay identified as oncogenic?

Answer 2

E5, E6 and E7

Question 3

Can both E6 and E7 induce tumours alone?

Answer 3

E7 can (in cell cultures and mice) but E6 cannot. E6 however significantly enhances E7 oncogenesis. (synergy)

Question 4

What is the main function of E6 and E7?

Answer 4

To keep undifferentiated and differentiated cells in the suprabasal region of the skin in the cell cycle. This is achieved through interaction with a growing number of cellular protein targets. There is no enzymatic activity, just rewiring function of proteins

Question 5

The oncogenes target certain pathways in the cell. E7 targets RB (retinoblastoma pathway). This is a family of proteins which includes pRB, p107 and p 130. What are these called and what do they do?

Answer 5

‘Pocket’ proteins. They are repressors of the cell cycle.

Question 6

How does E7 bind to proteins found in the RB family?

Answer 6

Through a highly conserved LXCXE motif found in all E7s across HPV types. This deregulates the activity of these pocket proteins

Question 7

What do the pocket proteins in the RB family actually regulate?

Answer 7

The RB pocket proteins control the G1-S phase transition by regulating the activity of the E2F family of transcription factors

Question 8

Where is the LXCXE motif found in the E7 protein?

Answer 8

In the amino terminus (near the end). This motif is also found in many other cancer causing viruses; an example of convergent evolution

Question 9

Apart from the LXCXE motif found in the E7 protein in HPV, what does the other half of the protein do?

Remember it is a very simple protein and has very few recognised motifs

Answer 9

Metal binding and dimerisation part which may enable the formation of complexes with multiple proteins

Question 10

Cell cycles are tightly regulated to prevent cancers developing. RB proteins are checkpoints in this process and interact with transcription factors known as E2F. How does this act as a break on cell cycle progression?

Answer 10

Converts this into a transcriptional repressor complex so basically this repressors genes necessary for the cell cycle to progress so acts as a break on cell cycle

Question 11

How does E7 then stop pRB from interacting with E2F complex and therefore stop pRB from stopping the cell cycle progress?

Answer 11

Interacts with it and couples it to the degradation machinery of the cell (ubiquitin mediated proteolysis). This removes the pRB from the cell and so E2F switches to a transcriptional activator by binding to promoters of genes necessary for cell progression

Question 12

The cell has mechanisms to tell whether pRB is being lost and compensates for this how?

Answer 12

By increasing levels of p53 which is also a tumour suppressor gene

Question 13

What is on the terminus of an E6 protein? What are there many of on an E6 protein?

Answer 13

PDZ domain. There are many cysteine residues on an E6 protein

Question 14

When do levels of p53 increase in a cell?

Answer 14

When a cell is stressed/insulted. E.g when levels of pRB are decreasing, levels and stability of p53 increase

Question 15

When levels of p53 are high, what does this induce in the cell?

Answer 15

production of pro-apoptotic factors (cell death) and p21 transcription which stops cell cycle

Question 16

In normal, uninfected cells how are p53 levels kept low?

Answer 16

hDM2 tag p53 with ubiquitin to degrade it

Question 17

In an infected cell, how does E6 keep levels of p53 low?

Answer 17

E6 forms a complex with p53 and E6AP which tags the p53 with ubiquitin and induces degradation of it

Question 18

In mutant viruses where E6 could not target p53, the virus was still able to transform cells, suggesting there was another pathway which was involved. Which one?

Answer 18

PDZ proteins

Question 19

PDZ proteins are involved in many roles including signalling enzymes and the cytoskeleton. What do they all share? What was the first PDZ protein shown to be a target for high-risk E6?

Answer 19

80-90 amino-acid motifs. The first PDZ protein shown to be targetted by E6 was Dlg, a tumour suppressor gene

Question 20

What did experiments with PDZ protein (in E6) mutants show?

Answer 20

Reduced growth rate, loss of viral episomes and frequent integration of viral genome into host chromosome. Transgenic mice did not show development of hyperplasia and tumours

Question 21

E6 and E7 can bind to many pathways. Give two examples of what each regulate (extra).

Answer 21

E7: apoptisis, proliferation, cell cycle progression, genomic instability

E6: apoptosis, p53, cytoskeleton

Question 22

Where is E5 found and what is it likely to be?

Answer 22

Has many transmembrane domains and is found a bit in the ER and Golgi (possibly nuclear envelope). It is an ion channel or viropore. Structure= hexamer

Question 23

What is E5 thought to be involved with?

Answer 23

Downregulation of MHC-1 which effectively ‘hides’ the infected cell from the immune system (cytotoxic/killler T-cells use these to identify infected cells). Also helps proliferation of the virus by switching on EGFR (epidermal growth factor); EGFR drives production of Cyclin B, this as seen earlier drives cell cycle

Question 24

HPV acts as an STD and is common. Do all cases lead to cervical cancer? What does this imply?

Answer 24

No! Very few lead to cancer. This suggests there is another factor contributing to developing cancer

Question 25

What happens to the viral genome as you progress from CIN 1 (first stage-mild displasia) through to CIN 3 (invasive carcinoma)?

Answer 25

Over expression of oncogenes and integration of the virus plasmid into the host chromosome. So basically there is something about the integration of HPV genome into the host chromosome which is linked to the development of cancer

Question 26

Why is integration of the viral genome into the genome actually bad news for the virus?

Answer 26

Linearalised genome (from its previous plasmid form) means it gets spliced and cut and so can no longer produce the virus!

Question 27

Why may integration of the viral genome actually be good for it?

Answer 27

E2 is lost and this acts as a repressor on oncogenes E6 and E7 so then it can transform the cells. This coupled with external or genetic factors can cause cancer