Lecture 15-16-17

Question 1

Structure of HPV

Answer 1

Non-enveloped, dsDNA with icosahedral capsid

Question 2

Family of HPV

Answer 2

Papillomaviridae (previously papovaviridae with polyoma since similar structure)

Question 3

HPV Baltimore classification

Answer 3

Class I

Question 4

What cells are infected by HPV?

Answer 4

Within keratinocytes differentiating in epithelia of the skin and mucosa, viral life cycle depnds on the differentiation program that keratinocytes undergo in epithelia

Question 5

Pathology caused by HPV

Answer 5

Cause hyper-proliferative benign (most cause being warts) and malignant lesions of the skin and mucosa, notably cervical cancer in women

Question 6

How many types of HPV?

Answer 6

Over 200 types, each with a preferece (why preference is still a mystery) (I.e. tropism) for certain anatomical locations
16,18 => cervical cancer (can be in other mucosal tissue, but never skin)

Question 7

Historical perspective

Answer 7

Warts are caused by a virus
- cutaneous warts induced in volunteers prepared with extracts from common warts (Jadassohn, 1896)
- a cell-free filtrate (so caused by a virus) of common warts can transfer infections (cuff, 1907)
Oncogenic potential of HPV
- cottontail rabbit papillomavirus (shoppen, 1933)
- Induction of epithelial malignancy in domestic rabbits by CRPV
Association of HPV with cervical cancer
- Haral zur hausen, 2008 Nobel Prize in phgy or medicine (not all infections will cause cancer)

Question 8

The Jackalope: myth or reality?

Answer 8

Bestie, it is just CRPV-caused tumors

Question 9

Phylogeny and classification of papillomaviruses

Answer 9

• shows evolution
• Genus alpha => cutaneous and mucosal
  some mucosal alpha HPVs are oncogenic (aka high-risk types)
  All HPVs that cause cancer are in this red section
• Genus Beta => cutaneous
• Genus Gamma => cutaneous
• Other genera => cutaneous
  HPV defined based on NT sequence of L1 (major capsid protein component of vaccine) ORF (also can be classified by tropism)
  Dif types if L1 ORF is more than 10% different than other known types
  Dif variant if L1 ORF is less than 10% dif than other known types
  1st isolate of any type is known as a prototype

Question 10

Disease caused by HPV

Answer 10

Mucosal (STD): cervical and anogenital cancer (anal, vulvar, vaginal and penile) and oropharyngeal cancers (50% of them are caused by HPVs) => HPV16,18, other high-risk types / genital warts and laryngeal papillomas (larynx of newborns passed by infected mothers => so much surgery eventually voice loss) (HPV 6, 11, other low-risk types)
Cutaneous (transmitted by contact): plantar warts (HPV1), common warts (HPV2, 4, 29), Flat warts (HPV3, 10, 28, 49)
Persistence of infection => highest risk factor for cancer

Question 11

Explain epidermodysplasia verruciformis (EV)

Answer 11

• only few 100 cases documented in literature
• very rare genetic disease characterized by a higher risk of developing skin carcinoma on sun-exposed skin
• abnormal susceptibility to HPV5 and HPV8, (BETA genus but become cancerous in these patients 1000 fold more likely esp if in sun with help of UV radiation) and other EV types
  EV types are beta HPVs that are present in the skin of most people without symptoms
  EV is caused by mutations in the EVER1, EVER2 and CIB1 genes, these proteins forma complex involved in keratinocyte-intrinsic immune response to beta-HPVs (innate immune response)

Question 12

HPV infection of the anogenital mucosa clinical progression and statistics

Answer 12

• one of the most common STIs
• Both men and women can be infected, 50-70% of women are infected during their lifetime (often in early sexual experiences, so important to vaccinate early)
• average length of infection: 8 - 12 months
• Most infections are not diagnosed
  inoculated => incubation 1-8 months => first lesion proliferation => immune response host containment (6-8 months) => remission for most people or infection persists which is a risk factor for cancer
• 4% cervical dysplasia (HPV 16, 18,31, 33, 45, 52, 58)
• 1% condylomas (HPV6 and 11)
• 10% presence of viral nucleic acids (DNA or RNA)
• 60% antibodies to capsid proteins
• 25% no sign of prior or active infection

Question 13

What is the purpose of screening programs with HPV?

Answer 13

identify cervical lesions that could progress or have progressed to cancer

Question 14

PAP test

Answer 14

Dr George Papanicolaou (1883-1962)
Cytological test
Detection of abnormal keratinocytes (huge nuclei and crunchy looking bunched up cells)

Question 15

Molecular detection

Answer 15

Viral nucleic acids (DNA/RNA)
Primarily of high-risk (Hr) HPV types
Can be performed on PAP sample

Question 16

Colposcopy

Answer 16

Can be done in anus throat
Direct visualization of lesions
Biopsies for histology
Often couple with treatment

Question 17

Name three screening programs

Answer 17

PAP test
Molecular detection
Colposcopy

Question 18

What are some treatments for HPV?

Answer 18

• Physical ablation
• Cytotoxic agents
• Immunomodulation (Imiquimod, for genital warts) cream used topically => just stimulates immune system (more production of TNF alpha and IFN alpha, etc.)

Question 19

About prophylactic vaccines

Answer 19

• Virus-like particles (VLPs) madeby expression of L1 in yeast or insect cells
• Often mixtures of VLPs from prevalent HPV types (necessary for broader protection) Cervarix (HPV 16,18) Gardasil (HOV 6, 11, 16, 18) Gardasil 9 (HPV 6,11 16, 18, 31, 33, 45, 52, 58)
• Provide high levels of antibodies and protection against cervical cancer and condylomas (expected to protect agains other HPV-associated cancers like throat and anus)
• More unvaccinated, more chance of cancer

Question 20

Rules of HPV vaccination in Quebec

Answer 20

Since sept 2020: Gardasil 9 in grade 4 and Cervarix in Secondary 3 (slightly better immune response against 16, 18 and economic reasons)

Question 21

Global burden of HPV-associated cancers in 2018

Answer 21

80% of all cases of HPV-associated cancers are cervical cancers (incidence of cervical cancer is inversely correlated with access to cervical screening programs
- incidence of oropharyngeal cancers is increasing in high-income countries, but the total number of cases remains small
- barriers to vaccination are greater in countries with the highest need

Question 22

What is the WHO’s plan to eliminate cervical cancer?

Answer 22

-2030 cervical cancer elimination targets
- 90% of girls fully vexed against HPV by 15
- 70% of women screened with a high-performance test by 35 and 45 years of age
- 90% of women identified with cervical disease receive treatment for precancerous lesions or invasive cancer
vax + screen + treat (precancerous lesions) + diagnosis and treatment (cancer) = eliminate cervical cancer as a public health problem and make it a disease of the past

Question 23

HPV replication cycle where and how?

Answer 23

HPV replication cycle depends on differentiation of keratinocytes in stratifies epithelia / virus infects undifferentiated basal cells (bottom Strat so only if deep cut) => genome established as an episome in the nucleus and replicated by E1 and E2 (50-100 copies/nucleus) in early phase of infection, acts as a reservoir so important when you have wart to cut deep in so wart doesn’t come back but produce new virions in differentiated cells
- oncogene-induced cell proliferation, E7 forces differentiated cells to enter S-phase, E6 prevents death by apoptosis, cells express host DNA replication factors (needs machinery of host to replicate but Dif cells don’t proliferate so must simulate S phase)
- Genome amplification: viral episomes are replicated by E1 and E2 to more than 1000 copies/nucleus
- Late gene/capsid protein expression
- Packaging and release of virions in higher cells cuz easier to evade immune response up there than closer to blood
(as move down list of steps move up Strat of skin from basal to cornified)

Question 24

keratinocyte differentiation from bottom to top

Answer 24

dermis, basal, spinous, granular and cornified

Question 25

Papillomavirus genome

Answer 25

- 8 kbp ds DNA circle - Encodes less than 10 proteins - small genomes, typically rely heavily on host proteins, must be able to interact with them - Three functional regions: early genes, late genes, regulatory region named LCR (long control region) or URR (upstream regulatory region) this region is the regulatory area for Transcription, DNA replication, Segregation of the genome has origin of replication (right before E6/E7)

Question 26

Main HPV proteins (hint: 8 of them)

Answer 26

E1 (DNA helices, the only HPV enzyme) + E2 (helices loader, transcriptional repressor, segregation factor) => viral DNA replication, gene expression, and segregation E4 (disrupts cytokeratin network) + E5 (recycles growth factor receptors) => genome amplification (accessory proteins, don't know exactly how they work) E6 (binds multiple targets including p53) + E7 (binds multiple targets including pRb) => viral oncogenes L1 (major capsid protein / need mixture of VLPs to offer cross-protection) + L2 (minor capsid protein / can offer cross-protection, has conserved regions) => capsid

Question 27

What is the conceptual framework of cellular transformation?

Answer 27

- Primary cell (keratinocyte like from circucision) => telomerase activation by E6 to prevent telomere shortening which would lead to senescence aka programmed cell death (RT type enzyme not typically expressed in primary cells) => telomere maintenance => inhibition of p53, pRb tumour suppressors by E6, E7 so no more breaks in cell division, cells is immortal but not transformed yet (both copies of genes must be KO to work) => immortalization => Ras V12 (render Ras constitutively active => constant growth despite what may happen around cells => so even in absence of GFs) activated oncogenes => transformation No plaque assay with HPV, uncontrolled proliferation, hallmark of cancer

Question 28

Immortalizing cells by DNA viruses

Answer 28

E6/E7 => HPV E1A/E1B => adenovirus Large T antigen => SV40 All target p53/Rb

Question 29

Senescence definition

Answer 29

When primary cells, such as keratinocytes, are put in culture in vitro, they only died a limited number of times and eventually die by senes fence, triggered by erosion of their telomeres

Question 30

Immortalization definition

Answer 30

When primary keratinocytes are made to express the two HPV oncogenes E6 and E7, they become immortal and will divide indefinitely in cell culture

Question 31

Transformation definition

Answer 31

When immortalized cells acquire additional mutations, such as mutations that activate the Rat oncogene, they become transformed. Cells that are transformed have additional growth properties, including the capability of forming tutors in animals.

Question 32

Describe an in vitro model to study HPV life cycle

Answer 32

Introduce high-risk HPV in primary human keratinocytes (PHK) which become immortalized due to E6 and E7 oncogenes. Viral genome is replicated and maintained as episomes by E1 and E2 and a suitable assay can be performed for reverse genetic experiments. Can use southern blot to examine viral DNA and see that mutated genome is integrated or multi Eric rather than linear or supercooled episomes like in WT

Question 33

We can perform reverse genetics but what else can we do experimentally to study HPV?

Answer 33

- keratinocytes put at the interface between the cell culture medium and air differentiate into a stratified epithelium, with PHK only basal cells have nuclei / HPV-immortalized PHK have a thicke epithelium and most cells retain their nucleus in order to replicate, DNA synthesis is seen with fluorescence and even goes up near top epithelial layers when immortalized

Question 34

Does E6E7 always target p53 and Rb?

Answer 34

Not in non-oncogenes HPV types => targets other pathways to stimulate proliferation without immortalizing

Question 35

High-risk HPV types are associated with…

Answer 35

- Almost all cervical cancers - 40% of vaginak and vulvar cancers - 50% of oropharyngeal cancers - 40-50% of penile cancers - 80-90% of anal cancers - Rare event relative to the large number of high-risk HPV infections - infection is necessary but not sufficient for the development of cancer => need additional mutations which can take 20-30 years after initial infection

Question 36

How does viral genome integration help to develop cancer?

Answer 36

- maintained in episomes in normal cycle - integrated in most high-grade lesions and in cancers, aberration not normal part of cycle - integration is a bio marker of cancer progression - results in disruption of E2 gene (no longer made in cancerous cells) which normally represses transcription (is TF) of E6 and E7 oncogenes, so higher expression of E6 and E7

Question 37

Why do we say that Cervical carcinomas are addicted to viral oncogenes?

Answer 37

- re expression of E2 represses E6 and E7, reactivating p53 and pRb pathways so cells cease to proliferate and die by senescence or apoptosis - clerical carcinomas require continuous expression of E6 and E7 for growth and survival, are addicted - validates E6 and E7 as good targets for cancer therapy

Question 38

Mechanism of action by E6 and E7

Answer 38

E7 induces cellular proliferation, kicks cells into S phase since targets pRb, gatekeeper of S phase, E2F is released and activated S phase Usually p53 kicks in to induce apoptosis when sus proliferation, but E6 prevents it by inhibiting p53 by promoting its degradation ( no moutations in p53 like in non-virus induced cancers)

Question 39

Precise and detailed mechanism of E7

Answer 39

Binds pRb, induces its degradation by the proteasome (releasing E2F), binds and activates Cdk2 (activates E2F), inhibits p21 and p27 which usually inhibit cyclin-Cdk2 (inhibits the inhibitor) E2F is necessary for S-phase entry and progression, once you activate it, must commit all the way / kept inactive outside of S-phase by forming complex with pRb / activated at G1/S by cyclinxCdk2 phosphorylation of pRb

Question 40

Structure of E7

Answer 40

- small zinc-finger protein that contains LxCxE Rb binding motif (like in all viruses that bind Rb) - Structure of E7 LxCxE peptide bound to the pocket region of pRb / E7 peptide is in an extended conformation

Question 41

Is Rb the only E7 target?

Answer 41

No, many other targets all for proliferation, inhibition of apoptosis and genomic instability.

Question 42

How does E6 induce the degradation of p53 by the proteasome?

Answer 42

E6 forms a complex with E6AP (E6 associated protein, has E6 binding peptide and HECT domain), a cellular E3 ubiquitin ligase The complex binds to p53 and promotes poly ubiquitination (at least 4 moieties) by E1 and E2 ubi enzymes Poly U p53 targeted and degraded by proteasome E6APis not implicated innp53 degradation normally, it just usurped by HPV E6

Question 43

Structure of E6

Answer 43

- small protein with two zinc-fingers(pocket to bind E6AP) E6AP peptide: E6 binds to an alpha helical LxxLL motif in E6AP. the three leucines are on the same surface of the helix Perhaps a therapeutic agent can bind to this pocket to inhibit E6 binding to E6AP

Question 44

Besides p53 inhibition what other mechanism of action does E6 have to immortalize the cell?

Answer 44

- Activates telomerase to sustain cellular proliferation - expressed in most cancer cells - Ribonucleoprotein complex (TER RNA and TERT protein with reverse-transcriptase activity (not produced in normal cells) solves DNAreplication “ends problem” to prevent telomere erosion - HPV E6 increases TERT expression (mechanism not exactly known) at the transcriptional level

Question 45

Does E6 only target p53?

Answer 45

No, as seems also targets TERT for telomerase activation and other pathways for proliferation / immortalization / inhibit apoptosis

Question 46

After the cell is immortalized, is it cancerous?

Answer 46

Immortal and cancerous are not synonymous. The cell undergoes transformation to become cancerous through mutations in host genome like in Ras V12, activated oncogene. Aberration only 20-30 years later if the infection persists and HPV genome must also be integrated

Question 47

Give an overview of HPV DNA replication

Answer 47

- occurs in nucleus of infected keratinocytes - require two viral proteins, E1 (helices, only viral enzyme at replication fork, analogous to DnaB in bacteria) best helices ever lmao E2 (helices loader) and the host DNA replication machinery - Genome replication is initiated at the viral origin (ori) is bidirectional

Question 48

E1 as an initiator protein

Answer 48

- hexameric helicase Three main functions of initiator: - binds to the replication origin (genetically defined as the replicator) - has helicase activity that is used to melt the origin and to unwind (with ATP hydrolysis energy) ahead of the replication fork - interact with cellular replication factors to orchestrate DNA replication and their the same ones we use for our own replication

Question 49

How does replication initiate in HPV?

Answer 49

- the ori contains 3 types of DNA elements: E1 binding sites (4-6 of them), E2 binding sites (3-4 of them) and an AT rich region (easier to melt, only 2 H-bonds) - E2 binds with high affinity and specificity to the origin and also interacts with E1 to recruit it ti the ori since E1 doesn’t have AS high of affinity with its own binding site, E2 acts as a helicase loader - binding and hydrolysis of ATP in Eq promotes its assembly into a double hexamer needed for bidirectional unwinding and replication, each E1 hexamer encircle a DNA strand - E1 unwinds DNA and interacts with host DNA replication factors like the ssDNA binding protein RPA, polymerase alpha primase and topoisomerase I (yay replication-competent complex)

Question 50

E2 domains

Answer 50

E2 binds ori and E1 through separate domains (as a dimer) - DNA BINDING DOMAIN (DBD) binds to E2BS in ori ( c term) - E1 binding domain is known as trans activation domain (TAD) REFERRING TO THE ROLE OF e2 as a transcription factor (represses E6/E7) ( N term) Therapeutic molecules can be E2 ligand => E1 can’t bind anymore => no replication

Question 51

Functional domains of the E1 helicase

Answer 51

3 of them from N to C: - n terminal regionmotifs needed for E1 nuclear import and export (replication obvi in nucleus) - DNA-binding domain (DBD): aka origin-binding domain (OBD), binds to the E1-binding sites in the ori to facilitate assembly of a double hexamer - Helicase domain: enzymatic portion, ATPase and DNA helicase/unwinding activity Sufficient for assembly of (alternate monomers) around ssDNA interacts with E2 protein and host DNA replication factors (polymerase alpha primase)

Question 52

Where are the ATP binding/hydrolysis sites on E1?

Answer 52

- Six ATP-binding (hydrolysis important for unwinding energy)sites are formed at the interface (junction) of adjacent monomers (one monomer alone cannot hydrolyze ATP to do its thing Monomers communicate in this way, both know when ATP is hydrolyzed => relays conformational changes around the hexamer s during DNA unwinding cycle

Question 53

Mechanism of DNA unwinding

Answer 53

E1 assembles as a hexamer around ssDNA DNA unwinding is largely consequence of E1 translocating (moving using ATP hydrolysis) along ssDNA. E1 is a translocase Translocation involves conformational changes in E1 induced by ATPxbinding and hydrolysis and release of Pi) Six ssDNA BINDING HAIRPINS(BETA-hairpins) interact with ssDNA IN THE CENTRAL CHANNEL and change conformation (mediating translocation) (histidine residues that interact with phosphate backbone)

Question 54

How does translocation mediate unwinding?

Answer 54

- Second strand does not fit in the channel => is extruded base-pairing disrupted => ssDNA to be template - most helicase s function similarly

Question 55

Segregation of the viral genome during mitosis

Answer 55

- human genome segregated thanks to mitosis spindle - mechanism for equal partitioning of the viral episomes to daughter cells during cell division - there could be loss of the viral episomes when the nuclear membrane is disrupted at mitosis - Papillomaviruses have evolved to ensure they end up in the nucleus after the mitosis, no genome lost, a way to tether their genome to the chromosomes of the host, some viruses tether to the mitosis spindle

Question 56

What viral protein helps genome segregation in HPV?

Answer 56

E2 tethers the viral genome to mitosis chromosomes by interacting with Brd4 (bromodomain containing protein 4) which is a chromatin reader that binds acetylated nucleosomes and remains associated with chromatin during mitosis. The E2 TAD or E1 binding domaininteracts with the c term 20 amino acids of Brd4 (opposite side of TAD that binds E1)

Question 57

3 functions of E2

Answer 57

- helicase loader - depressor of E6/E7 - segregation of of chromosomes

Question 58

Describe viral gene transcription in HPV

Answer 58

Two promoters: one early pE located in the LCR, one late pL located in the E6 gene. The activity of pL depends on keratinocyte differentiation. pL same enhancer as pE but only active in more differentiated keratinocytes. LCRs early promoter, enhancer region, ori, many binding sites => 2 viral E1/E2, rest is host TFs binding sites One keratinocyte specific enhancer which is located in the LCR and regulates both pE and pL Transcript resembles bacteria, polycistronic mRNA more than one ORF and more than one viral protein/mRNA also alternative splicing Late transcripts => mRNA code for late genes capsid L1/L2 Regulation of viral gene transcription: Primarily by ubiquitous cellular transcription factors (AP1, Sp1) that bind to the LCR (promoter sequence to recruit RNA pol II) however keratinocytes have a special combo of these ubiquitous proteins and only that combo activates transcription, tissue selective / also regulated by E2 which functions as a repressor