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MIMM 324 > HPV > Flashcards

HPV Flashcards

1
Q

What is the baltimore class of HPV

A

baltimore class 1:
non-enveloped, dsdna with an icosahedral capsid

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2
Q

What is the family of HPV

A

Papillomaviridae previously papovaviridae which was with the polyomaviruses

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3
Q

What do HPV infect?

A

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

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4
Q

what can HPV cause?

A

-Cause hyper-proliferative benign and malignant lesions
of the skin and mucosa, notably cervical cancer in women
-most don’t cause cancer but warts

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5
Q

True or false: HPV has tropism for certain anatomical locations

A

true
-200 types each with a skin tropism

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6
Q

How did scientists discover that warts are cause by a virus?

A

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)

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7
Q

how did scientists discover that HPV could be an oncogenic agent?

A

-they looked at skin lesions on:
o Cottontail Rabbit Papillomavirus (Shope, 1933)
o Induction of epithelial malignancy in domestic
rabbits by CRPV (Rous, 1935)

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8
Q

who associated hpv with cancer

A

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

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9
Q

rue or false: HPV has been with us for a long ass time

A

true

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10
Q

what does the genus alpha of hpv infect?

A

-it infects cutaneous and mucosal tissues
-some mucosal alpha hpvs are oncogenic aka the high risk type

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11
Q

what are the other genus of HPVs

A

-genus beta: cutaneous
-genus beta: cutaneous

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12
Q

how are the HPV types defined?

A

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

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13
Q

what are the mucosal aka sexually transmitted disease you can get with hpv

A

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

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14
Q

what are the cutaneous stuff that you can get from HPV

A

-planter warts: hpv1
-common warts: hpv2,4,29
-flat warts: hpv3,10,28 and 49

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15
Q

what is EV?

A

-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

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16
Q

true or flase: the hpv vaccine protects and does not cure

A

true

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17
Q

HPV infections of the anogenital mucosa

A

-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 !

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18
Q

how can an infection of HPV persist

A

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

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19
Q

what is the % of people before the vaccine that got cervical dysplasia

A

4%
4% Cervical dysplasia (HPV16, 18, 31, 33, 45, 52, 58)

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20
Q

what is the purpose of screening programs for HPV

A

The purpose of screening programs is to identify cervical
lesions that could progress or have progressed to cancer

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21
Q

pap test

A
  • Dr. George Papanicolaou (1883-1962)
  • Cytological test
  • Detection of abnormal keratinocytes
    -take cells from the cervix
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22
Q

Molecular detection of HPV:

A

-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

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23
Q

colposcopy

A

-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

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24
Q

Treatment for HPV

A
  • 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
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25
Q

Prophylactic vaccines for HPV

A
  • 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.
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26
Q

On what does the HPV cycle rely on?

A

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

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27
Q

true or false: virus infects undifferenciated cell but produce new virions in differentiated cells

A

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

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28
Q

how does the HPV virus infects the cells?

A

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

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29
Q

Infection of basal cells (HPV)

A

genome is established as an episome in the nucleus and replicated by E1 and E2 to 50=100 copies/nucleus

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30
Q

true or false: it is during the genome amplification that the viral capsid is expressed

A

true

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31
Q

oncogene-induced cell proliferation: HPV

A

-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

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32
Q

Papillomavirus genome

A
  • ~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
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33
Q

What is the role of E1 and E2 in HPV

A

-E1: DNA helicase, the HPV enzyme
-E2:
Helicase loader
transcription repressor
segregation factor

Both work for the viral DNA replication, gene expression and segragation

34
Q

What is the role of E4 and E5 in HPV

A

-E4: disrupts cytokeratin network
-E5: recycles growth factor repressor

both work for genome amplification

35
Q

true or false: in HPV E6 and E7 are oncogene

A

true

36
Q

what is the role of L1 and L2 in HPV

A

-L1: major capsid protein
-L2: minor capsid protein
it contains a highly conserved region

37
Q

why do primary cells are kinda sucky

A

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

38
Q

what is the main hallmark of cancer

A

uncontroled cell growth

39
Q

How can you immortalize a cell

A

-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

40
Q

how do you have a fully transformed cell to study HPV

A

-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

41
Q

what is the def of senescence

A

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.

42
Q

What is the def of immortalization

A

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.

43
Q

Introduction of high risk HPV genome in primary human keratinocytes (PHK)

A
  • 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
44
Q

Introduction of high risk HPV genome in primary human keratinocytes (PHK)

A
  • 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
45
Q

how do you make raft cultures

A
  • 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
46
Q

true or false: cervical cancer development is a common event relative

A

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

47
Q

Viral genome integration HPV

A

-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.

48
Q

Cervical carcinoma cells are addicted to?

A
  • 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
49
Q

true or false: E6 prevents apoptosis that result from E7 induced proliferation

A

true

50
Q

E2F transcription factor family (HPV)

A

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

51
Q

What is the structure of E7

A

-* 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

52
Q

E6 induces the degradation of p53 by …..

A

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

53
Q

Structure of E6

A
  • 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
54
Q

E6 activated telomerase to…

A

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

55
Q

True or false: HPV E6 decreses TERT expression at the transcriptional level

A

false:
HPV E6 increases/reactivates TERT expression at the transcriptional level

56
Q

What are the 3 main events for HPV to cause cancer?

A

-telomerase activation
-inhibition of p53 and prb tumor suppressors
-Ras V12 activated oncogenes

57
Q

what are the 2 viral proteins required for the DNA replication of HPV

A

-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

58
Q

rue or false: HPV DNA replication is bidirectional

A

true

59
Q

Is E1 an inition protein?

A

yes

60
Q

What are the 3 roles of initiator proteins in HPV

A
  • 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
61
Q

What are the 4 steps for the initiation of papillomavirus DNA replication?

A

-the origin(Ori)
-Assembly of the E1-E2-ori complex
-Assembly of the E1 double hexamer
-replication-competent complex

62
Q

true or false: E1 is the only viral protein at the replication fork

A

true

63
Q

True or false: E1 does not need E2 to bind to the E1 binding sites to assemble

A

falseeee it does need it

64
Q

What does the Ori for HPV contain?

A

The origin (Ori) contains 3 types of DNA elements:
* E1 binding sites (E1BS)
* E2 binding sites (E2BS)
* an AT-rich region (AT)

65
Q

WHAT HAPPENS AT THE ASSEMBLY OF E1-E2-ORI COMPLEX

A

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”.

66
Q

Assembly of the E1 double hexamer

A

-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

67
Q

what is the replication-competent complex in HPV

A

E1 unwinds DNA and interacts with host DNA
replication factors like the ssDNA-binding protein
RPA, Polymerase α primase, and Topoisomerase I.

68
Q

Streuctures of E2 and E1-E2 complex

A

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

69
Q

true or false: E2 is a dimer

A

true

70
Q

what is the second role of the E1 binding domain/TAD related to E6 and E7

A

-important for the work fo E2 as a transcriptional repressor
-interacts with the hiosts transcription machinery to repress the expression of E6 and E7

71
Q

What are the 2 domains of E2?

A

-E1 binding domain/TAD and the dna binding domain

72
Q

What are the functional domains of E1?

A

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)

73
Q

how are the ATP binding domains of E1 formed?

A

-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

74
Q

Mechanisms of DNA unwinding HPV(1)

A
  • 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
75
Q

How does the translocase work with

A

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

76
Q

what are the beta hairpins in HIV

A

histif=dines that move ti grab DNA and the E1 move along

77
Q

Segregation of the viral genome during mitosis HPV edition

A
  • 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.
78
Q

What is the protein in HPV whose role is to tether the viral genome to mitotic chromosomes?

A
  • 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
79
Q

what are the 2 promotors of HPV

A
  • 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
80
Q

true or false: HPV has 2 keranocyte specific enhancers?

A

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

81
Q

What is the regualtion of viral gene transcription in HPV

A
  • Primarily by ubiquitous cellular transcription factors (e.g. AP1, Sp1) that bind to the LCR
  • By E2 which functions as a repressor
82
Q

treur or false: there is lots of splicing in HPV

A

true
Several Early and Late mRNAs are synthesized
* Most are alternatively spliced and poly-cistronic

MIMM 324 (26 decks)

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