L2 - Human Papilloma Virus

Question 1

Describe the link between papillomaviruses and cancer

Answer 1

Papillomaviruses typically cause no/mild symptoms e.g. warts but can sometimes lead to cancer. Cottontail rabbit papillomatosis was described in 1933 (first link between a virus and animal cancer).

HPV-16 and 18 are disproportionately responsible for the majority of cervical cancer cases. All HPVs are capable of inducing transient proliferation but only 16 and 18 give rise to immortalised cell lines e.g. HeLa. These types can’t cause cancer by themselves but are important co-factors in the development of human cancers.

HPV association with cervical cancer is stronger than that for cigarette smoking and lung cancer.

Question 2

Describe the categorisation of papillomaviruses.

Answer 2

By studying the genomes of papillomaviruses through sequence alignments, they have been categorised into 5 main genera (alpha, beta, gamma, nu and mu). They can be further subdivided based on their ability to infect different types of epithelia. Alpha-papillomaviruses are the most studied as these include those that cause serious disease in humans e.g. HPV-16 and HPV-18.

Question 3

Describe the structure of the HPV virion

Answer 3

Quite small (~55nm diameter), non-enveloped (lack outer lipid so resistant to some disinfection methods), capsid is comprised of 72 pentameric capsomer subunits.

Question 4

What do E1 and E2 do?

Answer 4

E1 and E2 are required for viral DNA replication, together with host cell DNA replication machinery.

Question 4

Describe the structure of the HPV genome

Answer 4

HPVs have DNA genomes which are typically comprised of an untranslated long control region (LCR) and 8 genes required for different stages of the virus life cycle (6 early and 2 late) which encode a larger number of gene products due to mRNA splicing.

All HPVs have a similar genome, but size and position of the major ORFs can vary e.g. beta-HPVs lack an E5 ORF

Question 5

What do E4 and E5 do?

Answer 5

E4 and E5 are required for amplification of the viral genome in the upper layers of the epithelium (spacial segregation).

Question 6

What do E6 and E7 do?

Answer 6

The E6 and E7 proteins of high-risk HPVs are oncogenic as they cooperate to immortalise cells and induce genomic instability.

Question 7

What do L1 and L2 do?

Answer 7

L1 and L2 are structural proteins that form the viral capsid and are expressed late in infection, in the upper layers of the epithelium.

Question 8

What does the LCR do?

Answer 8

The long control region contains most of the regulatory DNA sequences needed for proper replication of the viral genome (e.g. origin of DNA replication) and for the expression of the viral genes (promoter regions).

Question 9

Which 2 genes are putative and only expressed in a few papillomaviruses?

Answer 9

E3 and E8

Question 10

Describe the process of HPV replication

Answer 10

Attachment and entry - HPV virion attaches to basal cells of epithelium through interactions between capsid and cell surface receptors then virus enters host cell

Uncoating and genome entry - Viral capsid is uncoated and viral genome (circular dsDNA) released into host cell cytoplasm

Replication and transcription - Viral genome enters cell nucleus where it replicates and transcribes using host cell machinery, early genes expressed to produce proteins that regulate viral DNA replication and control host cell functions and new viral genomes are synthesised

— The following steps only happen in some cells ——-

Late gene expression and capsid formation - Late genes expressed, producing structural proteins for the viral capsid and viral capsids assemble in host cell nucleus

Assembly and release - New virions assembled within nucleus and accumulate in cytoplasm before mature virions released from host cell due to cell lysis or other mechanism

Infection of adjacent cells - Released virions can infect adjacent epithelial cells to restart the cycle

Note: Some HPVs can establish persistent infections and some high-risk types may lead to cancer

Question 11

How is HPV DNA replication regulated in epithelial cells?

Answer 11

HPV expression relies on host cell environment so a productive cycle can’t occur unless the epithelial cell is terminally differentiated. Therefore the virus will usually only complete its replication cycle in the upper/more differentiated cells.

In the basal cells, the viral genome is maintained as a low copy number episome. These cells express E6 and E7 and are stimulated to divide. Cells in the mid-layers express E4, undergo genome amplification and are typically in the S or G2 phase. In the upper layers, cells leave the cell cycle and in some E4+ cells, L1 and L2 are expressed to allow packaging of amplified genomes.

Question 12

Describe the persistent infection of HPV in epithelial cells

Answer 12

The genome can persist as an episome in the nucleus of basal cells but can sometimes get integrated which is a strong prognostic predictor of cancer. When this occurs, production of the virus itself decreases but expression of oncoproteins E6 and E7 increases and becomes permanent.

Question 13

Describe the link between HPV and disease

Answer 13

HPVs replicate in squamous epithelial cells - cutaneous and mucosal (most likely to cause disease)

There are over 200 HPVs but most are relatively harmless. ~30 HPVs are associated with genital warts/growths and ~15 HPVs are associated with cervical cancer (types 16, 18 and 33 account for 75%)

Question 14

How is HPV transmitted?

Answer 14

HPV is transmitted by sexual contact (sexual intercourse or other types but this is less common).

Transmission is determined by per partner transmission probability, infectiousness and average number of partnerships formed per unit time. However, there is little reliable epidemiological data on HPV transmission probability per sex-act or partnership unlike with other viruses e.g. HIV.

Question 15

How does HPV screening work?

Answer 15

Testing for high risk papillomaviruses has shown to be a better cancer predictor (less false negatives) than cytological tests (checking for lesions).

Women who test negative for high risk HPVs have very low chance of developing cancer in the next 5 years so screenings are less frequent.

Screening (along with vaccination) has drastically reduced rates of cervical cancer in the UK.

Question 16

Describe the epidemiology of cervical cancer across the world

Answer 16

• 604,000 annually cases globally (WHO 2020)
• 342,000 deaths annually (WHO 2020)
• Mortality : incidence ratio is 52%
• Third most common cancer in females
• Disease burden falls heavily on LMICs with strained healthcare systems (~90% cases, WHO 2020)

Question 17

Describe the statistics linking HPV and cervical cancer

Answer 17

• HPV 16/18 infection results in a 10-40 fold increase in cervical cancer risk and are responsible for ~50% of high-grade cervical pre-cancers
• HPV has a 20-40 year latency period
• HPV present in 33% of healthy young women but only 3% of women over 50 due to effective immune responses (1/300 of these women will have low grade CIN (cervical intraepithelial neoplasia))
• 7% of women with HPV-16 will develop cervical cancer within 45 years of infection
• Common cofactors are smoking, age at first intercourse and number of sexual partners

Question 18

Describe the major steps in development of cervical cancer

Answer 18

Transient infection:
Normal cervix —> HPV-infected cervix
(At this point the infection could be cleared)

HPV viral persistence:
HPV-infected cervix —progression—> precancerous lesion
(At this point the lesion could regress)
Precancerous lesion —invasion—> cancer

Question 19

Describe how HPV infection persists and becomes latent

Answer 19

E5, E6 an E7 proteins have roles in immune evasion, helping the virus persist in infected epithelium. Viral gene expression is also switched off in the presence of infiltrating lymphocytes (perhaps due to cytokine signalling) but viral episomes not effectively cleared from basal cell layer which leads to latency and allows the possibility of reactivation e.g. due to immunosuppression.

Question 20

Describe HPV DNA integration

Answer 20

Integration of HPV DNA into the host genome is a common event in cervical carcinogenesis. This integration disrupts E2 expression, leading to deregulated expression of early proteins. The E2 protein is thought to down-regulate the promoter that drives E6/E7 expression so its disruption can lead to only the 2 viral oncoproteins being expressed in cervical cancer.

Question 21

Describe the oncogenic mechanisms of E6

Answer 21

E6 interferes with p53 cell cycle regulation. p53 is upregulated due to viral infection/DNA damage/cell stress. Normally, high levels of p53 would induce cell cycle arrest. High risk HPV E6 binds p53 via the cellular ubiquitin protein ligase, E6-associated protein (E6-Ap), causing p53 to be ubiquinated and targeted for degradation by the proteasome. This brings p53 back down to normal levels, allowing cell cycle progression and therefore uncontrolled cell proliferation.

E6 also activates TERT (telomerase reverse transcription) to lengthen the telomeres and avoid the typical telomere shortening associated with cell proliferation. This process involves interaction with a variety of transcription factors, repressors and activators.

E6 is important in the later stages of carcinogenesis.

Question 22

Describe the oncogenic mechanisms of E7

Answer 22

E2f dimers (E2f + Dp) stimulate transcription of genes involved in replication and allow passage through the cell cycle. If Rb binds E2f, this transcription is repressed. E7 sequesters Rb, preventing it from binding E2f and therefore activating uncontrolled E2f-dependent transcription of cellular and viral replication proteins.

E7 is also involved in the alternative lengthening of telomeres (ALT) pathway which works to maintain telomere length despite proliferation.

E7 is important in the early stages of carcinogenesis.

Question 23

What are some of the key differences between high-risk alpha and low-risk alpha HPVs?

Answer 23

High-risk alpha HPVs stimulate cell cycle entry AND cell proliferation
- encode E6* (truncated E6 that modulates E6 function)
- E6 binds and degrades p53, inhibits apoptosis, inhibits IFN response, activates telomerase, activates c-myc
- Bypass of growth arrest following DNA damage
- E7 binds and degrades pRb, induces genomic instability and has functions involved in immortalisation and transformation

Low-risk alpha HPVs stimulate cell cycle entry
- does not encode E6*
- E6 weakly binds but does not degrade p53, more weakly inhibits IFN response, does not activate telomerase or c-myc
- Normal growth arrest following DNA damage
- E7 more weakly binds pRb, does not stimulate instability and does not have functions involved in immortalisation and transformation

Question 24

Describe the evidence of E5 involvement in disease caused by HPV-16

Answer 24

• In transgenic mouse models, HPV-16 E5 can induce epithelial proliferation
• In oestrogen-treated mice, E5 expression alone can induce cervical cancers
• Some human tumours contain viral episomes (contain E5) as well as viral integrants (no E5) - E5 may alter activity of EGFR and may reduce surface levels of MHC-I, modulate MAPK pathway and alter caveolin levels

Question 25

What evidence is there that the immune system can influence HPV infection?

Answer 25

• Warts are more common under immunosuppression
• CIN (cervical intraepithelial neoplasia) is much more common in AIDS patients
• Warts may regress when immunosuppression is lifted and immune cell infiltrates are found in regressing lesions
• The antibody to E7 is found in 50% of cervical carcinoma patients
• The antibody to L1 is found in those with genital warts

This evidence shows that HPV is usually regulated effectively by the immune system and therefore both preventative and therapeutic vaccines are feasible.

Question 26

Why is the L1 protein the logical target for HPV vaccines?

Answer 26

L1 is the major capsid protein and is 80% of the total virion protein - it can self-assemble in baculovirus and vaccinia expression systems. L2 is the minor capsid and is not required but is incorporated in self-assembly. Successfully targeting L1 would therefore prevent viral entry.

Question 27

Describe the HPV vaccine currently offered in the UK.

Answer 27

Gardasil9 is now offered routinely in the UK for those above 9 years old. It protects against HPV-16 and HPV-18 as well as 7 other types (broad protection).

Question 28

Describe the effect of HPV vaccination on young women who were vaccinated prior to HPV exposure at 9-12 years old

Answer 28

~90% reduction in HPV infection by the strains of HPV vaccinated against and ~90% reduction in genital warts

45% reduction in low grade cervical cytological abnormalities and almost 85% reduction in high grade cytological abnormalities.

Question 29

What questions are yet to be answered about the new HPV 16/18 vaccines?

Answer 29

What is the duration of protection and the total effect on cancer incidence?

How broad is the cross-protection of vaccines against a few related types?

Does the vaccine prevent infection in men, and reduce the transmissibility of HPV from men to their partners? (vaccine rollout recently started in boys)

Do these vaccines protect against other HPV-related cancers such as oropharyngeal and anal cancers?

In developing countries (>80% cervical cancer cases), who can afford to get vaccinated and is it cost-effective when competing with other health priorities?

Question 30

Describe the estimations of cervical cancer elimination

Answer 30

NHS aim is to eliminate cervical cancer in the UK by 2040.