Papillomavirus Flashcards
Does papillomaviruses have an envelope?
No
Describe papillomaviruses
Double stranded DNA
Icosahedral capsid
What family are papillomaviruses part of?
Papillomaviridae
Papillomaviruses are part of which Baltimore class?
Class I
What do papillomaviruses infect?
Keratinocytes within differentiating epithelia of skin and mucosa
What do papillomaviruses cause?
Hyper-proliferation benign and malignant lesions of the skin and mucosa
Notably cervical cancer in women
How is HPV defined?
Types: L1 ORF is more than 10% different than known types
Variant: less than 10%
What are the diseases caused by HPV?
Mucosal:
- Cervical and anogenital cancer
- Oropharyngeal cancer
- Genital warts
- Laryngeal papillomas
Cutaneous:
- Plantar warts
- Common warts
- Flat warts
What is EV?
Epidermodysplasia verruciformis
Rare genetic disease with a higher risk of developing skin carcinoma on sun-exposed skin
Abnormal susceptibility to HPV 5 and HPV 8, and other EV types
EV types are beta-HPVs that are present in the skin of most people without symptoms
What is the cause of EV?
Mutations in EVER1, EVER2, and CIB1 genes
Average length of HPV infection is…
8-12 months
What are the screening programs and treatments for HPV?
PAP test: detect abnormal keratinocytes
Molecular detection: viral nucleic acids, performed on PAP sample
Colposcopy: direct visualization of lesions, biopsies for histology
Treatments: physical ablation, cytotoxic agents, immunomodulation
What are prophylactic vaccines?
Virus-like particles made by expression of L1 in yeast or insect cells
Mixture of VLPs from prevalent HPV types
Provide high levels of antibodies and protection against cervical cancer and condylomas
Expected to protect against other HPV-associated cancers
Describe papillomavirus capsid.
One viral genome wrapped in his tones
Icosahedral symmetry with atypical T=7 arrangement
Made of 72 pentamers of L1, which interact through long tails for stability
- no hexamers
- 60 of the 72 L1 pentamers are surrounded by 6 neighbouring pentamers arranged along a 6-fold symmetry axis
- capsid is made of 360 subunits instead of 420
12 copies of minor capsid protein L2 are found in the inside of the capsid and may help with packaging the genome
80% of all cases of HPV-associated cancers are ___
Cervical cancers
The incidence of cervical cancer is inversely correlated with access to ____
Cervical screening programs
HPV replication cycle depends on ____
Differentiation of keratinocytes in stratified epithelia
HPV infects ______ but produces new visions in ____
Undifferentiated basal cells
Differentiated cells
How does HPV infect basal cells?
L1 binding to heparin surface proteoglycans triggers clathrin-dependent endocytosis
Genome is established as an episome in the nucleus and replicated by E1 and E2 to about 50-100 copies/nucleus
What does HPV do in differentiated cells?
Packaging and release of virions
Late gene/capsid protein expression
Genome amplification
- viral episodes are replicated by E1 and E2 to 1000 copies/nucleus
Oncogene-induced cell proliferation
- E7 forces differentiated cells to enter S-phase
- E6 prevents their death by apoptosis
- Cells express host DNA replication factors
Describe papillomavirus genome
8kbp double stranded DNA circle
Encodes less than 10 proteins
Three functional regions
- Early genes
- Late genes
- Regulatory region named LCR or URR
- LCR = Long control region
- URR = Upstream regulatory region
LCR/URR is the regulatory area for
- transcription
- DNA replication
- Segregation of the genome
What are the main proteins in HPV?
E1: DNA helicase, the only HPV enzyme
E2: Helicase loader, transcriptional repressor, segregation factor
E1 and E2 are in charge of viral DNA replication, gene expression, and segregation
E4: Disrupts cytokeratin network
E5: Recycles growth factor receptors
E4 and E5 are in charge of genome amplification
E6: Binds multiple targets including p53
E7: Binds multiple targets including pRb
E6 and E7 are viral oncogenes
L1: Major capsid protein
L2: Minor capsid protein
How does HPV transform a cell?
Telomerase activation
Inhibition of p53, pRb tumor suppressors = immortalization
Ras V12 activated oncogenes = transformation
What causes the immortalization of HPV?
HPV E6 and E7
Ad E1A and E1B
SV40 large T antigen
What is senescence?
When primary cells are put in culture in vitro, they only divide a limited number of times and eventually die because of the erosion of their telomeres
What is immortalization?
When primary keratinocytes are made to express the two HPV oncogenes E6 and E7, they become immortal and will divide indefinitely in cell culture
What is transformation of a cell?
When immortalized cells acquire additional mutations, such as mutations that activate the Ras oncogene, they become transformed. Cells that are transformed have additional growth properties, including the capability of forming tumours in animals
What kind of assay do you use to study HPV cycle in vitro?
Southern blot to examine viral DNA
Episomal shown in WT. Not shown in mutant genomes: mutations in viral genome that suppress DNA replication
What is the difference between epithelial tissue that are normal and have HPV?
Epithelium is thicker, and cells still have their nucleus in everywhere
True or false: Infection by high-risk HPV type is necessary but not sufficient for the development of cervical cancer
True
During the normal viral life cycle, the HPV genome is maintained as …
Episomes
In most high-grade lesions and cancers, the viral genome is found ___ into the host chromosomes
Integrated
Integration is a bio marker of cancer progression
Integration of the HPV genome often results in ___
Disruption of the E2 gene
Because E2 normally represses transcription of the E6 and E7 oncogenes, disruption of E2 leads to higher expression of the E6 and E7 proteins
HPV: Re-expression of E2 ____ E6 and E7 and ___ p53 and pRb pathways
Represses
Reactivates
Cells cease to proliferate and die by senescence/apoptosis
Cervical carcinoma cells require continuous expression of ___ and ___
E6 and E7 are needed for growth and survival
What is the role of E7
Induces cellular proliferation
Separates (inhibits) E2F and pRB complex
What is the role of E6?
Prevents apoptosis that results from E7-induced proliferation
Inhibits p53
How does E7 induce cellular proliferation? (S phase entry)
Degrades pRb (induce its degradation by the protease by binding to it)
Stimulates Cln-Cdk2
Inhibits Cln-Cdk inhibitors (p21, p27)
Activate E2F: phosphorylation of pRb (E2F separates from pRb)
What is the structure of E7?
Small zinc-finger protein that contains a LxCxE Rb-binding motif
Structure of E7 LxCxE peptide bound to the pocket region of pRb. E7 peptide is an extended conformation
How does E6 induce degradation of p53?
E6 forms a complex with E6AP (E6-Associated Protein), a cellular E3-ubiquitin ligase
The E6-E6AP complex binds to p53 and promotes its polyubiquitination
Poly-ubiquitinated p53 is targeted to and degraded by the proteasome
E6AP is not implicated in p53 degradation in normal cells but is usurped by HPV E6
Describe the structure of E6 in HPV
Small protein with two zinc-fingers
E6AP peptide: E6 binds to an alpha-helical LxxLL motif in E6AP
The three leucines are on the same surface of the helix
How does E6 prevent telomere erosion in HPV?
Activates telomerase
- Ribonucleoprotein complex: TER RNA, TERT protein with reverse transcriptase activity
HPV E6 ___ TERT expression at the transcriptional level
Increases
HPV: genome replication is initiated at…
The viral origin (ori)
Why is it hard for drugs to target HPV?
DNA helicase is the only viral enzyme; the other proteins have no enzymatic activity
What is needed for DNA replication of HPV?
E1, E2, and host DNA replication machinery
What is the role of E1 in HPV?
Bind to replication origin
Helicase activity
Interact with cellular replication factors
HPV: The Ori contains 3 types of DNA elements:
E1 binding sites (E1BS)
E2 binding sites (E2BS)
An AT rich region
How is DNA replication initiated in HPV?
Ori
Assembly of E1-E2-ori complex
Assembly of the E1 double-hexamers
Replication-competent complex
How does the E1-E2-ori complex form?
E2 binds with high affinity and specificity to the origin. E2 also interacts with E1 to recruit it to the ori. E2 acts as a helicase loader
How is the E1 double-hexamers in HPV formed?
The binding and hydrolysis of ATP by E1 promotes its assembly into double hexamer
Each E1 hexamer encircles a DNA strand
HPV: How does the replication-competent complex work?
E1 unwinds DNA and interacts with host DNA replication factors like the ssDNA-binding protein RPA, polymerase alpha primase and topoisomerase I
Where does the DNA and E1 bind to in E2?
DNA-binding domain (in E2) binds to E2BS in the ori
E1-binding domain (in E2, also called TAD, Transactivation domain) binds to the E1 helicase domain
What are the functional domains of the E1 helicase?
N-terminal region: Contains motifs needed for E1 nuclear import and export
DNA binding domain (DBD): Binds to E1BS in the ori to facilitate assembly of double hexamer
HD (helicase domain):
- The enzymatic portion of the protein
- Has ATPase and DNA helicase/unwinding activity
- Sufficient for assembly into hexamers
- Interacts with E2 proteins and host DNA replication factors
How is DNA unwinding in HPV?
E1 assembles as a hexamer around ssDNA
DNA unwinding is largely the consequence of E1 trans locating along ssDNA (E1 is a translocase)
Translocation involves conformational changes in E1 induced by ATP-binding and hydrolysis
Six ssDNA-binding hairpins (beta-hairpins) interact with ssDNA in the central channel and change conformation upon ATP-binding and hydrolysis
HPV: Segregation of the viral genome is during…
Mitosis
How does HPV ensure equal partitioning of viral episomes to daughter cells during mitosis?
E2 tethers the viral genome to mitotic chromosomes
How does E2 tethers the viral genome to mitotic chromosomes?
By interacting with Brd4
What is Brd4?
Bromodomain-containing protein 4 is a chromatin reader that binds acetylated nucleosides and remains associated with chromatin during mitosis
E2 TAD interacts with the C-terminal 20 amino acids of Brd4
HPV viral gene transcription: Name the two promoters
Early gene transcription (pE) located in the LCR
Late gene transcription (pL) located in E6 gene. Activity of pL is dependent on keratinocyte differentiation
What does the keratinocyte-specific enhancer do in HPV?
Located in LCR
regulates both early and late promoters
How is viral gene transcription regulated in HPV?
Primarily by ubiquitous cellular transcription factors (AP1, SP1) that bind to the LCR
By E2 which functions as a repressor
Early and late mRNAs in HPV are ____
Alternatively spliced and poly-cistronic
