HPV Flashcards
What is the baltimore class of HPV
baltimore class 1:
non-enveloped, dsdna with an icosahedral capsid
What is the family of HPV
Papillomaviridae previously papovaviridae which was with the polyomaviruses
What do HPV infect?
it infects:
-Infect keratinocytes within differentiating epithelia
of the skin and mucosa.
-Viral life cycle depends on the differentiation program that keratinocytes undergo in these epithelia
-this is why it is hard to culture it in labs, gotta have differentiated cell lines
what can HPV cause?
-Cause hyper-proliferative benign and malignant lesions
of the skin and mucosa, notably cervical cancer in women
-most don’t cause cancer but warts
True or false: HPV has tropism for certain anatomical locations
true
-200 types each with a skin tropism
How did scientists discover that warts are cause by a virus?
o Induction of cutaneous warts in volunteers with extracts prepared from
common warts (Jadassohn, 1896)
o A cell-free filtrate of common warts can transfer infection (Ciuffo, 1907)
how did scientists discover that HPV could be an oncogenic agent?
-they looked at skin lesions on:
o Cottontail Rabbit Papillomavirus (Shope, 1933)
o Induction of epithelial malignancy in domestic
rabbits by CRPV (Rous, 1935)
who associated hpv with cancer
o Harald zur Hausen, 2008 Nobel prize
in physiology or medicine
-aka 16 and 18
-we kinda already knew that cancer could be cause by viruses, we just did not know which one
rue or false: HPV has been with us for a long ass time
true
what does the genus alpha of hpv infect?
-it infects cutaneous and mucosal tissues
-some mucosal alpha hpvs are oncogenic aka the high risk type
what are the other genus of HPVs
-genus beta: cutaneous
-genus beta: cutaneous
how are the HPV types defined?
HPV types are defined based on the nucleotide sequence of L1 ORF
-L1 is the major capsid protein
* Types: L1 ORF is >10% different than other known types
* Variant: L1 ORF is <10% different than other known types
what are the mucosal aka sexually transmitted disease you can get with hpv
HPV16, 18, other high-risk types
* Cervical and anogenital cancers
(anal, vulvar, vaginal, penile)
* Oropharyngeal cancers
that type of cancer is getting less popular from people who smoke and is more caused by sexually active people
HPV6, 11, other low-risk types
* Genital warts
* Laryngeal papillomas
what are the cutaneous stuff that you can get from HPV
-planter warts: hpv1
-common warts: hpv2,4,29
-flat warts: hpv3,10,28 and 49
what is EV?
-Epidermodysplasia verruciformis (EV)
* Very rare genetic disease characterized
by a higher risk of developing skin carcinoma on sun-exposed skin
* Abnormal susceptibility to HPV5 and HPV8,
and other EV types
* EV types are β-HPVs that are present in the skin
of most people without symptoms
* EV is caused by mutations in the EVER1, EVER2 and CIB1 genes.
* It was recently suggested that EVER1, EVER2 and CIB1 proteins form a
complex involved in keratinocyte-intrinsic immune response to β-HPVs
true or flase: the hpv vaccine protects and does not cure
true
HPV infections of the anogenital mucosa
-more alpha type
-One of the most common sexually transmitted infections
* Both men and woman can be infected
* 50% to 70 % of women are infected during their lifetime
* Average length of infection: 8 to 12 months
* Most infections are not diagnosed !
how can an infection of HPV persist
it is when your immune system is not able to clear the infection cuz it is too weak or smth then you could get cancer
what is the % of people before the vaccine that got cervical dysplasia
4%
4% Cervical dysplasia (HPV16, 18, 31, 33, 45, 52, 58)
what is the purpose of screening programs for HPV
The purpose of screening programs is to identify cervical
lesions that could progress or have progressed to cancer
pap test
- Dr. George Papanicolaou (1883-1962)
- Cytological test
- Detection of abnormal keratinocytes
-take cells from the cervix
Molecular detection of HPV:
-if you have a sus pap test
-you use PCR
-is there a high risk HPV?
* Viral nucleic acids (DNA/RNA)
* Primarily of high-risk (Hr) HPV types
* Can be performed on PAP sample
colposcopy
-if you have high risk HPV
-hpv lesions turn white when you add acid
* Direct visualization of lesions
* Biopsies for histology
* Often coupled with treatment
Treatment for HPV
- Physical ablation
- Cytotoxic agents
- Immunomodulation (Imiquimod; for genital warts)
it is a cream that you apply on the skin
-stimulates immune system to make for INF alpha
-antiviral drug not available
Prophylactic vaccines for HPV
- Virus-like particles (VLPs) made by expression of L1 in yeast or insect cells
L1 self assembles and the vaccine makes like a fake capsid=pseudo virus - Mixture of VLPs from prevalent HPV types
❑ Cervarix (HPV 16, 18)
❑ Gardasil (HPV 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 against other HPV-associated cancers.
On what does the HPV cycle rely on?
It depends on the differentiation of keratinocytes in stratified epithelia
-the virus needs to infect undifferentiated cells because it needs the host machinery to replicate aka it loves basal cells
-differenciated cells loose their nucleus and organelles
true or false: virus infects undifferenciated cell but produce new virions in differentiated cells
true
-the infected basal cells will replicate and the infected daughter cell will start to go through some sort of differentiation program and move upward
-making more progeny virions in these upper “differentiated” cells is good because the immune system has a harder time to clear them
-it is also there that the genome implication occurs
how does the HPV virus infects the cells?
it infects through small cuts
-som,e places like the cervix have stratified epithelium is juxtaposed to a granular epithelium which is only a 1 cell layer epithesiul=easy access to basal cells
Infection of basal cells (HPV)
genome is established as an episome in the nucleus and replicated by E1 and E2 to 50=100 copies/nucleus
true or false: it is during the genome amplification that the viral capsid is expressed
true
oncogene-induced cell proliferation: HPV
-E7 forces differenciated cells to enter S phase by binding to lots of targets including pRb
-E6 prevents their death by apoptosis by degrading the p53
-cells express host DNA replication factors
Papillomavirus genome
- ~8 kbp double stranded DNA circle
- Encodes less than 10 proteins
- Three functional regions
o Early genes
o Late genes
o Regulatory region named LCR or URR
-where the ORF is situated
LCR = Long Control Region
URR = Upstream Regulatory Region - LCR/URR is the regulatory area for:
o Transcription
o DNA replication
o Segregation of the genome
What is the role of E1 and E2 in HPV
-E1: DNA helicase, the HPV enzyme
-E2:
Helicase loader
transcription repressor
segregation factor
Both work for the viral DNA replication, gene expression and segragation
What is the role of E4 and E5 in HPV
-E4: disrupts cytokeratin network
-E5: recycles growth factor repressor
both work for genome amplification
true or false: in HPV E6 and E7 are oncogene
true
what is the role of L1 and L2 in HPV
-L1: major capsid protein
-L2: minor capsid protein
it contains a highly conserved region
why do primary cells are kinda sucky
because a primary cell can only replicate up to 20x because of telomere shortening
-everytime it replicates: the telomeres get shorter
-it leads to senescense when telomeres are too short and then the cell dies
what is the main hallmark of cancer
uncontroled cell growth
How can you immortalize a cell
-first, the telomerase has to be activated
-it is a reverse transcriptase that repairs the telomeres
it is the E6 that will activate that enzyme=hpv can’t die of senescence
-then you need to inhibit p53 and pRb aka tumor suppressors
-E6 and E7 will remove the breaks aka no apoptosis
-the cell is now immortalized: the cells can now replicate indefinitely
-the cells are not fully transformed yet: if we put them in immunodeficient cells they won’t form tumours
how do you have a fully transformed cell to study HPV
-follow the steps for immortalization
-then you need mutations in pathways that usually respond to TF to cell cells that it is time to proliferate. In cancer it is usually the RasV12. It renders a GTPase always active aka it always tells the cells that they need to proliferate
-the cells are now fully transformed
what is the def of senescence
Senescence
When primary cells, such as keratinocytes, are put in culture in vitro, they only divide a limited number of times and eventually die by senescence, triggered by erosion of their telomeres.
What is the def of immortalization
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.
Introduction of high risk HPV genome in primary human keratinocytes (PHK)
- Cells become immortalized due to the action of the E6 and E6 oncogenes
- Viral genome is replicated and maintained as episomes by E1 and E2
- Suitable assay for reverse genetic experiments
-mutant genome is always shorter on southern blot
Introduction of high risk HPV genome in primary human keratinocytes (PHK)
- Cells become immortalized due to the action of the E6 and E6 oncogenes
- Viral genome is replicated and maintained as episomes by E1 and E2
- Suitable assay for reverse genetic experiments
-mutant genome is always shorter on southern blot
how do you make raft cultures
- Keratinocytes put at the interface between the cell culture
medium and air differentiate into a stratified epithelium
-the epithelium us thicker
-some cells differentiated and most cell retained their nucleus thanks to E6 and E6
=can still synthesize DNA
-while in normal cells only the basal cells are replicating
true or false: cervical cancer development is a common event relative
false it is a rare event relative to the large number of high risk hpv infections
takes 20-30 years to develop
-Infection by a high-risk HPV types is necessary but not sufficient for the development of cervical cancer
Viral genome integration HPV
-it is not a part of the viral life cycle: it is kinda like a mistake
* During the normal viral life cycle, the HPV genome is maintained as episomes.
* In most high-grade lesions and in cancers, the viral genome is found integrated into the host chromosomes.
* Integration is a biomarker of cancer progression.
* Integration 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.
Cervical carcinoma cells are addicted to?
- Cervical carcinoma cells require continuous expression of E6 and E7 for growth and survival. They are “addicted” to E6 and E7 !
-Validates E6 and E7 as targets for cancer therapy - Re-expression of E2 represses E6 and E7 and reactivates the p53 and pRb pathways. Cells cease to proliferate and die by senescence/apoptosis
true or false: E6 prevents apoptosis that result from E7 induced proliferation
true
E2F transcription factor family (HPV)
E2F transcription factor family
* Necessary for S-phase entry and progression.
* Kept inactive outside of S-phase by formation of a complex with pRb.
* Activated at the G1/S transition by Cyclin-Cdk2 phosphorylation of 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.
-it binds to lots of stuff
E6 induces the degradation of p53 by …..
a proteasome (don’t forget that ubiquitin targets the
* E6 forms a complex with E6AP (E6-Associated Protein), a cellular E3-ubiquitin ligase
* The E6-E6AP complex binds to p53 and promotes its poly-ubiquitination
* 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
Structure of E6
- Small protein with two zinc-fingers (it has a pocket that binds to E6Ap and this complex is enough to bind p53)
- E6AP peptide: E6 binds to an α-helical LxxLL
motif in E6AP. The three leucines, shown in green, are on the same surface of the helix - Crystal structures of E6 (green) in complex with LxxLL peptide from E6AP (red) and p53 (gray)
-Some labs have found compounds that bind top the pocket and prevents E6 from binding with E6Ap= E6 can’t degrade p53
E6 activated telomerase to…
sustain cellular proliferation
-expressed in most cancer cells
-made by the ribonucleoprotein complex
ter rna
tert protein with reverse-transcriptase activity when produced makes telomerase active
-solves the DNA replication “ends problem” to prevent telomere erosion
True or false: HPV E6 decreses TERT expression at the transcriptional level
false:
HPV E6 increases/reactivates TERT expression at the transcriptional level
What are the 3 main events for HPV to cause cancer?
-telomerase activation
-inhibition of p53 and prb tumor suppressors
-Ras V12 activated oncogenes
what are the 2 viral proteins required for the DNA replication of HPV
-E1 which is the helicase like DNAb
-E2; works as a TF for gene repression of E6 and E7, but for replication of the DNA it works as a helicase loader kinda like DNAa
-plus the host DNA replication machinery
rue or false: HPV DNA replication is bidirectional
true
Is E1 an inition protein?
yes
What are the 3 roles of initiator proteins in HPV
- They bind to the replication origin
(genetically defined as the replicator) - They have helicase activity that is used to melt
the origin and to unwind the DNA ahead of the
replication fork - They interact with cellular replication factors to orchestrate the replication of dna
What are the 4 steps for the initiation of papillomavirus DNA replication?
-the origin(Ori)
-Assembly of the E1-E2-ori complex
-Assembly of the E1 double hexamer
-replication-competent complex
true or false: E1 is the only viral protein at the replication fork
true
True or false: E1 does not need E2 to bind to the E1 binding sites to assemble
falseeee it does need it
What does the Ori for HPV contain?
The origin (Ori) contains 3 types of DNA elements:
* E1 binding sites (E1BS)
* E2 binding sites (E2BS)
* an AT-rich region (AT)
WHAT HAPPENS AT THE ASSEMBLY OF E1-E2-ORI COMPLEX
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”.
Assembly of the E1 double hexamer
-The binding and hydrolysis of ATP by E1 promotes
its assembly into a double hexamer needed for bidirectional unwinding and replication. Each E1
hexamer encircles a DNA strand.
-2 hexamers that unwind DNA in 2 directions
what is the replication-competent complex in HPV
E1 unwinds DNA and interacts with host DNA
replication factors like the ssDNA-binding protein
RPA, Polymerase α primase, and Topoisomerase I.
Streuctures of E2 and E1-E2 complex
E2 binds DNA (ori) and E1 through separate domains
* The DNA-binding domain (DBD) binds to E2BS in the ori
* The E1-binding domain binds to the E1 helicase domain (HD)
* Note that the E1-binding domain is known as the “transactivation
domain (TAD)” referring to the role of E2 as a transcription factor
true or false: E2 is a dimer
true
what is the second role of the E1 binding domain/TAD related to E6 and E7
-important for the work fo E2 as a transcriptional repressor
-interacts with the hiosts transcription machinery to repress the expression of E6 and E7
What are the 2 domains of E2?
-E1 binding domain/TAD and the dna binding domain
What are the functional domains of E1?
The N-terminal region
▪ Contains motifs needed for E1 nuclear import and export
The DNA-binding domain (DBD)
▪ Also known as the origin-binding domain (OBD)
▪ Binds to the E1-binding sites (E1BS) in the ori to facilitate assembly of a double-hexamer.
The helicase domain (HD)
▪ The enzymatic portion of the protein; has ATPase and DNA helicase/unwinding activity
▪ Sufficient for assembly into hexamers
▪ Interacts with E2 protein and host DNA replication factors (e.g. polymerase α-primase)
how are the ATP binding domains of E1 formed?
-Six ATP-binding and hydrolysis sites are formed, at the interface of adjacent monomers
-basically they are between 2 monomers of the hexamers
-this means that they are able to “talk” to each other to relay conformational changes
Mechanisms of DNA unwinding HPV(1)
- E1 assembles as a hexamer around ssDNA
- DNA unwinding is largely the consequence of E1 translocating (i.e. moving) along ssDNA. E1 is a “translocase”
- Translocation involves conformational changes in E1 induced by ATP-binding and hydrolysis.
- Six ssDNA-binding hairpins (β-hairpins) interact with ssDNA in the central channel and change
conformation upon ATP-binding and hydrolysis
How does the translocase work with
E1 assembles ssdna through the power of atp binding and hydrolysis
-it will translocate this ssdna strans=helicase forces its way=complementary strand also gets displaced
-this translocating affects the beta hairpin
what are the beta hairpins in HIV
histif=dines that move ti grab DNA and the E1 move along
Segregation of the viral genome during mitosis HPV edition
- Mechanism for equal partitioning of the viral episomes to daughter cells during cell division (i.e. during mitosis nuclear membrane disappears).
- There could be loss of the viral episomes when the nuclear membrane is disrupted at mitosis=bad for virus.
- Papillomaviruses have evolved a way to tether their genome to the chromosomes of the host or to mitotic spindle.
What is the protein in HPV whose role is to tether the viral genome to mitotic chromosomes?
- E2 tethers the viral genome to mitotic chromosomes by interacting with Brd4
- Bromodomain-containing protein 4 (Brd4) is a chromatin reader that binds
acetylated nucleosomes and remains associated with chromatin during mitosis - The E2 TAD(E1 binding domain) interacts with the C-terminal 20 amino acids of Brd4 (Brd4-C) while being bonded to the c=viral episome via the DNA binding site
what are the 2 promotors of HPV
- One promoter for Early gene transcription (pE) is located in the LCR to make early mrna (has more than 1 orf=makes more than 1 protein)
- One promoter for Late gene transcription (pL) is Located in the E6 gene. The activity of pL is dependent on keratinocyte differentiation for capsid proteins L1 and L2
true or false: HPV has 2 keranocyte specific enhancers?
falseeee
One keratinocyte-specific enhancer
* The enhancer is located in the LCR and regulates both the Early and Late promoters
-made of ubiquitous TF sites which is sus since there is only one
What is the regualtion of viral gene transcription in HPV
- Primarily by ubiquitous cellular transcription factors (e.g. AP1, Sp1) that bind to the LCR
- By E2 which functions as a repressor
treur or false: there is lots of splicing in HPV
true
Several Early and Late mRNAs are synthesized
* Most are alternatively spliced and poly-cistronic
