L2 - Human Papilloma Virus (HPV) - Luca

Question 1

What are papillomaviruses?

Answer 1

Papillomaviruses are a large group of small DNA viruses (~55 nm virion diamter) that infect a variety of higher vertebrates, including humans.
They typically cause mild symptoms like warts, but in some cases can lead to serious diseases such as cancer.

Question 2

What is the evolutionary origin of papillomaviruses and how does it relate to their virulence?

Answer 2

🦎🧓🔄 Papillomaviruses are evolutionarily ancient, thought to have evolved alongside the epithelium of reptiles, dating back ~350 million years. Because of this long co-evolution with hosts, they typically cause mild or no symptoms (although certain strains have developed oncogenic potential)

Question 3

what is the general structure of papillomaviruses envelope and capsid?

Answer 3

They are non-enveloped, meaning they lack a lipid envelope, making them resistant to many disinfectants that rely on disrupting this structure.

Their genome is enclosed in a capsid made of 72 pentameric capsomer subunits.

Question 4

How were papillomaviruses first linked to cancer?

Answer 4

🐇🔍🧪 The first link between a virus and cancer was made in 1933, when researchers described cottontail rabbit papillomatosis, a disease caused by a papillomavirus that led to cancerous growths. (this was a landmark in virology ad helped launch the field of viral oncology)

Question 5

Which HPV types are linked to cervical cancer?

Answer 5

HPV types 16 and 18 are responsible for the majority of cervical cancer cases. These are high risk alpha papillomaviruses and because of their oncogenic potential they are the focus of vaccination and screening

Question 6

What is the genetic structure of papillomaviruses

Answer 6

The HPV genome includes:

1. A Long Control Region LCR) – non-coding but essential for replication regulation.
2. Eight genes total which includes 6 early genes (E1–E7): Involved in replication, regulation, and immune evasion.
   and
   2 late genes (L1, L2): Code for structural proteins forming the viral capsid.
   *** Due to mRNA splicing, these 8 genes produce a larger variety of gene products.

Question 7

How are papillomaviruses classified into genera?

Answer 7

Classification is based on genomic sequence alignments.

Question 8

What are the main differences between papilomavirus generas?

Answer 8

• the size and position of the major ORF can vary e.g. Beta HPV types lack an E5 ORF
• different life cycle characteristics / epithelial tropisms
• disease associations

Question 9

how many papillomavirus generas are there?

Answer 9

There are currently 29 genera of papillomaviruses (PVs) within the Papillomaviridae family each adapted to infect different types of epithelium. ( Human PVs: 5 genera. Mammalian PVs: 20 genera.
Avian PVs: 3 genera.
Reptile PVs:1 genus.)

Question 10

What type of epithelium do Alpha, Beta and Mu genus infect>

Answer 10

🧬 Alpha: Infect mucosal tissues; includes oncogenic HPV-16 and HPV-18.
🧬 Beta: Infect cutaneous skin often asymptomatic, but can contribute to non-melanoma skin cancers.
Often lack the E5 gene .
🧬 Mu: Infect cutaneous skin, usually associated with common warts (e.g., HPV-1).

Question 11

which type of human papillomavirus is the most heavily studied

Answer 11

the Alpha-papillomavirus

Question 12

what does the Alpha-papillomavirus include and cause

Answer 12

1. the low risk mucosal types that cause genital warts
2. the high risk mucosal types that can cause cervical neoplasia’s and cancer

Question 13

What is the purpose of the long control region (LCR) in a virus?

Answer 13

The LCR contains binding sites for cellular transcription factors e.g. SP1, AP1 and viral proteins e.g. E1 and E2 to control viral replication and gene expression

Question 14

Is the long control region (LCR) translated into proteins?

Answer 14

No

Question 15

What type of proteins are E1 and E2?

Answer 15

E1: a DNA helicase - like enzyme (hence required for viral DNA replication)
E2 : a sequence-specific DNA-binding protein which helps regulate transcription and genome partitioning during cell division

Question 16

what do HPV proteins E1 and E2 do?

Answer 16

both viral proteins interact with the host cell DNA replication machinery (required for exploitation - a parasite) to initiate viral DNA replication

Question 17

In which epithelial cell layers are viral E1 and E2 proteins expressed, and what is their function?

Answer 17

in the basal layers

Question 18

What are the roles of E4 and E5 proteins?

Answer 18

they are both expressed in the upper layers of the epithelium:
E4 : involved in genome amplification and may disrupt keratin networks
E5 : Modulates cellular signalling supporting immune evasion and potentially enhancing viral genome amplification

Question 19

at what point of the cell cycle are the viral E4 proteins typically expressed?

Answer 19

S or G2 phases

Question 20

Why are E6 and E7 proteins critical in high risk HPV types? ⚠️🧬🔥

Answer 20

these are the oncoproteins of high risk HPV ( e.g. 16 and 18). Both cooperate to immortalise cells and induce genomic instability through different mechanisms.
E6 : binds and degrades p53 tumour suppressor proteins preventing apoptosis
E7 : inactivates Rb protein, pushing the cell into uncontrolled cell division

Question 21

what are L1 and L2 proteins and when are they expressed?

Answer 21

they are both structural proteins that form the capsid (outer shell). Because of this they are expressed late in the replication cycle - in differentiated upper epithelial cells.

Question 22

which viral protein is targeted in HPV vaccines and why?

Answer 22

L1 and L2 proteins (particularly L1 which is the most abundant protein in the HPV capsid making yo 80% of the total virion protein and being highly immunogenic with the ability of inducing a high titre of neutralising antibodies) used as virus-like particles

Question 23

what does the LCR contain

Answer 23

most of the regulatory DNA sequences needed for proper replication of the viral genome ( origin of DNA replication) and for the expression of viral genes (promoter regions)

Question 24

what are E3 and E8 viral genes

Answer 24

putative genes found only in a few papillomaviruses

Question 25

HPV replication step by step general

Answer 25

1. Attachment & Entry – HPV binds to basal epithelial cells via receptors (e.g., heparan sulfate). 2. Uncoating & Genome Entry – Capsid breaks down; DNA enters cytoplasm, then nucleus. 3. Replication & Transcription – Viral genome replicated, early proteins expressed. 4.Gene Expression & Capsid Formation – L1/L2 produced in upper layers, capsids form. 5. Assembly & Release – Virions assemble in nucleus, released by cell lysis. 6. Infection of Adjacent Cells – Virions infect nearby epithelial cells.

Question 26

how does HPV enter the body

Answer 26

Via microwounds in multi layered stratified epithelium ( this allows the virion to access the basal lamina)

Question 27

What happens during viral attachment and entry

Answer 27

The HPV virion (L1 capsid proteins) attaches to the basal cells of the epithelium ( where the stem cells are) through interactions with cell surface receptors for entry

Question 28

what happens during uncoating and genome entry?

Answer 28

After entry, the viral capsid is uncoated, and the viral genome ( circular double stranded DNA) is released into the host cell cytoplasm.

Question 29

what happens during replication and transcription

Answer 29

The viral genome enters the cell nucleus, where it replicates and transcribes early genes using host cellular machinery. The early proteins regulate viral DNA replication and control host cell functions resulting in synthesis of the viral genome as the host cell undergoes keratinocyte differentiation

Question 30

what orchestrates the pattern of viral gene expression

Answer 30

Keratinocyte differentiation ( makes sure there is a productive infection)

Question 31

what happens in late gene expression and capsid formation?

Answer 31

Late genes are expressed which leads to the production of structural proteins for the viral capsid which is then assembles in the host cell nucleus

Question 32

What happens during assembly and release

Answer 32

New virions are assembled within the nucleus and accumulate in the cytoplasm. the mature virion is then released from the host cell as a result of cell lysis or other mechanism

Question 33

What happens during infection of adjacent cells?

Answer 33

The released virions can infect adjacent epithelial cells starting the cycle anew. Notably, some HPV types can establish persistent infections and certain high-risk types are associated with an increased risk of developing cervical and other cancers

Question 34

what plays a crucial role in determining the outcome of HPV infection?

Answer 34

the interplay between the virus and the host's immune response as well as whether the HPV genome integrates itself into the host genome ( this disrupts viral regulation balance - with less capsid protein expression and increaed E6/E7 oncogene expression --> strong predictor of cancer development especially cervical cancer)

Question 35

what makes high risk mucosal HPV types more pathogenic

Answer 35

they infect mucosal epithelium e.g. cervix, anus or oropharynx where they are more likely to persist, integrate and evade immune responses

Question 36

Where does productive HPV replication primarily occur?

Answer 36

In the multi-layered stratified epithelium.

Question 37

What is thought to be required for initial HPV infection to reach the basal lamina?

Answer 37

The presence of a microwound that allows the infectious virions to access the basal lamina.

Question 38

How does the HPV genome exist in the basal and parabasal cells?

Answer 38

As a low copy number episome (maintained as separate circular DNA).

Question 39

In which epithelial cell layers do proteins necessary for genome amplification become elevated?

Answer 39

in the mid layers - this allows for genome amplification to occur in growth-arrested and differentiating competent cells

Question 40

What is the significance of HPV DNA being episomal vs. integrated?

Answer 40

Episomal HPV DNA leads to high viral load and long-term persistence but is not strongly associated with transformation. In contrast, integration of HPV DNA disrupts E2 protein expression, which normally regulates the viral oncogenes E6 and E7, allowing unchecked expression and promoting transformation. (integration of HPV DNA into host genome is a frequent event in cervical carcinogenesis)

Question 41

which two viral oncoproteins are the only ones expressed in cervical cancer as a consequence of viral integration

Answer 41

E6 and E7 oncoproteins

Question 42

what percentage of HPV types cause disease and cervical cancer ?

Answer 42

Of ~200 types, only ~30 cause disease; 3 types cause 75% of cervical cancers.

Question 43

What is the transformation zone in the cervix?

Answer 43

The area where the squamous epithelium meets the columnar epithelium, and a common site for HPV infection and subsequent dysplasia.

Question 44

What is a key characteristic of dysplasia in HPV infection?

Answer 44

Expansion of basal-like cells into the suprabasal layer and mitotic activity in these layers. One would be able to see nuclear abnormalities and failure for the cell to undergo normal differentiation

Question 45

what is a carcinoma in sity and invasive cancer in the context of HPV infection

Answer 45

- High-grade dysplasia where the abnormal cells occupy the full thickness of the epithelium but have not yet invaded the basement membrane. - Cancer where the dysplastic cells have penetrated the basement membrane and invaded the underlying stroma (connective tissue). there is a stepwise progression from normal epithelium to viral infection, dysplasia, carcinoma in situ, and finally, invasive cancer, highlighting the role of persistent viral infection and oncogene expression.

Question 46

what is the primary mode of transmission for HPV?

Answer 46

HPV is primarily transmitted through sexual contact including vaginal, anal and oral sex as well as skin-to-skin contact

Question 47

What factors increase the risk of HPV transmission

Answer 47

- Number of sexual partners - Early age of first sexual intercourse - Partner's sexual history

Question 48

Is the per-act transmission probability of HPV well known?

Answer 48

No. Unlike HIV, the per-act transmission probability for HPV is not well quantified, but transmission is highly efficient, especially in sexually active young adults.

Question 49

How has HPV screening changed in the UK since 2019?

Answer 49

The UK moved from lesion-based screening to HPV DNA testing, which detects high-risk HPV strains and provides: - Higher sensitivity - Fewer false negatives - Longer intervals between screenings

Question 50

What does a negative high risk HPV test imply?

Answer 50

It indicates a very low risk of developing cervical cancer in the next 5 years, allowing screening every 3 years instead of annually

Question 51

how frequent is the routine recall for ages 25 - 49 and for 50+ year olds in the UK

Answer 51

25 - 49yo = 3years 50+ = 5 years

Question 52

what two factors have dramatically reduced the rates of cervical cancer in the UK

Answer 52

screening and vaccination

Question 53

what type of HPVs are capable of inducing transient proliferation

Answer 53

all HPVs although only HPV 16 and 18 are capable of giving rise to immortalised cell lines

Question 54

Which HPV was found in Henrietta's cells

Answer 54

HPV-18 ( she had cervical cancer and then some of her cancer cells began to be used in research because of their unique ability to continuously grow and divide)

Question 55

can HPV 16 and 18 transform primary rat cells on their own

Answer 55

no. But they can co-operate with the "activated" ras oncogene to transform these cells

Question 56

which association with cervical cancer is stronger : HPV or cigarette smoking

Answer 56

HPV

Question 57

How many cases of HPV were there vs how many deaths were caused by cervical cancer globally in 2020?

Answer 57

Over 600,000 cases and over 300,000 deaths, mostly in low- and middle-income countries.

Question 58

Why is the burden of HPV-related disease higher in developing countries?

Answer 58

Limited access to vaccines and screening + Weaker healthcare infrastructure

Question 59

global burden of cervical cancer

Answer 59

- 84% of cancer occurs in less developed countries -90% of global burden occurs in low and middle income countries - It is the 3rd most common cancer in women - mortality incidence ratio is 52%

Question 60

what fold increased risk on cervical cancer does HPV16/18 cause

Answer 60

10-40 fold increase

Question 61

what are percentage of high grade cervical pre cancers are HPV16/18 responsible for

Answer 61

50%

Question 62

what is the latency perioid for HPV

Answer 62

20-40 years

Question 63

what percentage of healthy young vs women over 50 are infected with HPV

Answer 63

healthy young = 33% women over 50 = 3%

Question 64

what cofactors of cervical cancer and HPV likely to be

Answer 64

1. smoking 2. Age at first intercourse 3. Number of sexual partners

Question 65

why is the wait and see aproach implied when you see lesions caused by transient infections or initial neoplasia

Answer 65

because in most cases it will regress by itself / it will get cleared by the immune system (otherwise it can become an invasive cancer)

Question 66

how long does it typically take for patientients to clear infection?

Answer 66

typically 3 years - hence why screening is done every 3-5 years

Question 67

in general which immune system is able to infiltrate and clear the HPV infected cell

Answer 67

lymphocytes

Question 68

what process does HPV ( along with HIV and Hep B ) undergo, to avoid lymphocyte / immune responses

Answer 68

viral latency

Question 69

what happens in viral latency

Answer 69

the situation where viral genes and proteins are expressed at low levels or not at all, despite the persistence of the viral genome, usually in the form of episomal DNA and integrated DNA in the basal cell layer. The main problem is that latency isn't absolute so can become reactivated and later cause persistent infection

Question 70

in what situations can HPV become reactivated after latency

Answer 70

a change in immune status e.g. individual immune system is weakened (after taking immunosuppressants)

Question 71

what can possibly induce viral latency?

Answer 71

Cytokine signalling from the infiltrating lymphocytes

Question 72

Which HPV proteins have roles in both immune evasion and cell cycle entry?

Answer 72

E5, E6, and E7.

Question 73

How do E5, E6, and E7 contribute to HPV persistence in infected epithelium?

Answer 73

Through their roles in immune evasion and promoting cell cycle entry.

Question 74

how does high risk HPV E6 act as an oncogene

Answer 74

1. interferes with p53 cell cycle regulation 2. activates telomerase

Question 75

p53 function

Answer 75

a tumour suppressor which gets activated during cellular stress / when there is DNA damage and induces cell arrest (p21) for repair or appoptosis (Puma or Bax) so the damage isn't passed down to daughter cells.

Question 76

how does high risk HPV E6 protein interfere with p53 and cell cycle regulation

Answer 76

High risk HPV E6 binds to p53 via E6-Ap (associated protein) and ubiquitinates it / targets it for degradation by the proteosome. This means no p21 or Puma / Bax is made and the cell cycle continues despite there being damage

Question 77

how does high risk HPV E7 act as an oncogene

Answer 77

1. modulates cell cycle progression 2. promotes telomer maintenance

Question 78

E2f function

Answer 78

heterodimeric proteins which stimulate transcription of cellular genes and allow passage through the cell cycle (G1 to S phase) when they are not complexed with Rb proteins

Question 79

How does E7 protein modulate cell cycle progression?

Answer 79

E7 binds to Rb (sequesting it) so it no longer binds to E2f, activating E2f-dependent transcription + increased synthesis of cellular and some viral replication proteins

Question 80

how do E6 and E7 promote telomere maintenance

Answer 80

E6 activates TERT (telomerase reverse transcriptase) through interaction with a variety of transcriotion factors, repressors and activators E7 then maintains the telomere length through the ALT pathway. this is what makes the host cell practically immortal

Question 81

what stages of cancer development are E6 and E7 important for

Answer 81

E6 is important for later stages whilst E7 is important for the early stages

Question 82

What are telomeres?

Answer 82

genome repeats of about 6-12kb on the ends of chromosomes that maintain their integrity by protecting them from NHEJ and HR events. They play an important role in aging as they shorten with each replication meaning that a typical cell has a limited number of divisions (~50-70)

Question 83

E6 and E7 tumour progression (from initiation to progression)

Answer 83

1. induction of the initial proliferation when E7 binds to RB and E2F is free to induce aberrant proliferation 2. E6 ubiquitinates p53 and induces extended proliferation 3. E6 and E7 act on telomeres so the cell has an indefinite life span 4. E6 and E7 cause genomic instability leading to cellular transformation and tumour progression

Question 84

What is a key overall functional difference of E6/E7 proteins between high-risk and low-risk HPVs, as indicated by the colored boxes?

Answer 84

High-risk HPV E6/E7 strongly stimulate cell cycle entry and cell proliferation, while low-risk HPV E6/E7 have a weaker effect on cell cycle entry.

Question 85

What is a characteristic of the E6 protein encoded by high-risk vs low-risk alpha HPVs?

Answer 85

High risk : encodes E6* products , binds strongly to p53, inhibits apoptosis, bypasses growth arrest following DNA damage, inhibits keratinocyte differentiation as well as interferon responses and activates the Akt, Wnt signalling pathway. It also activates telomerase. low risk : doesn't encode E6* products, weakly binds to p53, undergoes normal growth arrest following DNA damage and weakly inhibits the interferon response

Question 86

what is evidence for HPV 16 E5 proteins also being involved in tumorigenesis

Answer 86

- In transgenic mouse (genetically engineered to express the HPV 16 E5 protein) the presence of E5 alone has been shown to induce epithelial proliferation. This uncontrolled cell growth is a hallmark of early stages of cancer development, suggesting E5 can directly contribute to abnormal tissue growth. - When mice were treated with estrogen (a hormone known to influence cervical cell growth) and simultaneously expressed HPV 16 E5, E5 expression alone was sufficient to induce cervical cancers. This indicates that E5 can act synergistically with other factors, like hormonal signals, to drive the development of cancerous lesions in the cervix. - some human tumours contain viral epistomes as well as viral integrants. This means that E5 may alter the activity of the EGFR and reduce the surface levels of MHC class 1 proteins which alters the MAPK pathway and alters caveolin levels.

Question 87

what is evidence that immune responses towards papillomaviruses are good enough to obtain a vaccine-

Answer 87

- Warts are more common under immunouppression siggesting that the immune system normally controls HPV infections effectively (found that when immunosuppression stopped the warts regressed deepening this point) - CIN was linked to immunosuppression e.g. in AIDS patients, indicating that immune dysfunction allows HPV-induced lesions to develop and progress more readily - immune cell infiltrates are commonly observed in regression HPV lesions indicating an active immune response involved in clearance - Around 50% of cervical carcinoma patients have antibodies to E7, indicating the immune system recognises and responds to viral oncoproteins during cancer progression. - antibodies to L1 in individuals with genital warts showing it could be a key target

Question 88

conclusions towards the evidence that immune responses towards papillomaviruses are good enough to obtain a vaccine

Answer 88

that HPV is regulated effectively by the immune system most of the time and other factors allow the development of cancers. This suggested that both preventative and therapeutic vaccines are feasible

Question 89

what percentage of the total virion protein is made from the L1 protein

Answer 89

80% (making it a good target)

Question 90

Has the L1 vaccine been a success?

Answer 90

yes - been used against dog mucosal papilloma's and clinical trials suggest that prophylactic vaccines will be effective

Question 91

what are the three licensed HPV vaccines called?

Answer 91

1. Cervarix 2. Gardasil4 3. Gardasil9

Question 92

which is the best licensed HPV vaccine and why

Answer 92

Gardasil especially Gardasil 9 as it offers more protection against a wider variety of HPV types (6,11,16,18,31,33,45,52, 58) - this is the one which is routinely offered

Question 93

what age group are the doses given to?

Answer 93

Cervarix: 2 doses @ 9-14 and then 3 doses over 15 years Gardasil 4 : 2 doses @ 9-13 and then 3 doses over 14 years Gardasil 9: 2 doses @ 9-14 and then 3 doses over 15 years

Question 94

proof that Gardasil is effective according to data from New England Journal of Medicine looking at 1.7 million girls and women between 2006 and 2017

Answer 94

Great data is accumulating that among young women who were vaccinated prior to exposure to HPV, aprox -90% reduction in HPV infection by the strains of HPV vaccinated against - 90% reduction in genital wart infections - 45% reductio in low grade cervical cytological abnormalities - almost 85% reduction in high grade cytological abnormalities

Question 95

what sort of countries accounts for 80%+ of cervical cancer

Answer 95

developing countries

Question 96

provided that HPV primary screening is introduced by 2023, what is the estimated reduction of cervical cacer rates in ages 25-64

Answer 96

a 19% reduction

Question 97

how will the NHS make getting vaccinated against HPV more accessible

Answer 97

by improving access to online vaccination appointments nationally - this allows people to view their full vaccination records on the NHS app (see the ones they need)

Question 98

what are important questions to ask regarding new HPV 16/18 vaccines?

Answer 98

1. What is the duration of protection and the total effect on cancer incidence? 2. Do the vaccines provide cross-protection against a few related types, as previously suggested? 3. Does the vaccine prevent infection in men, and reduce the transmissibility of HPV from men to their partners? 4. Do these vaccines protect against other HPV-related cancers such as oropharyngeal and anal cancers? 5. In developing countries, where 80% or more of cervical cancer occurs, who can afford to get vaccinated, even with tiered pricing, in view of competing health priorities?

Question 99

when are boys vaccinated against HPV in the UK

Answer 99

between 12 and 13 yo

Question 100

what type of virus are Papillomaviruses

Answer 100

small, non-enveloped, DNA viruses