Iahi: 3.4 - Human Papillomaviruses (Hpv)

Front 1

How can viruses indirectly cause cancer?

Back 1

- Chronic infection (e.g., Hepatitis B and C) cause cirrhosis, inflammation, tissue damage, high levels of cell division
- Cycle vastly increases probability that hepatocytes develop mutations and chromosomal aberrations that derail their "growth control"

Front 2

How can viruses directly cause cancer?

Back 2

- E.g., Papillomavirus
- Upon infection, some DNA viruses stimulate cells to enter S phase of cell cycle and ready themselves for DNA synthesis
- Virus needs this for replication
- If virus does not complete lifecycle and kill infected cell then the same viral proteins can continue to direct cell to override normal controls on cell growth
- Inappropriate division

Front 3

What kind of cancer are Hepatitis B and C associated with? What kind of viruses are they?

Back 3

- Hepatocellular Carcinoma
- Hep B = hepadnavirus
- Hep C = flavivirus

Front 4

What kind of cancer are herpesvirus associated with? What viruses specifically?

Back 4

- Lymphoma; nasopharyngeal carcinoma (EBV)
- Kaposi's sarcoma KSHV (HHV8)

Front 5

What kind of cancer is lentivirus associated with? What virus specifically?

Back 5

- Adult T cell leukemia
- HTLV1

Front 6

What kind of cancer are papillomavirus associated with? What viruses specifically?

Back 6

- Cervical cancer
- Head and neck cancer
- HPV

Front 7

What kind of cancer is Merkel cell polyoma virus (MCPyV) associated with?

Back 7

Merkel Cell Carcinoma - aggressive skin cancer (in elderly and immunosuppressed patients)

Front 8

How was it discovered that viruses were associated with cancer?

Back 8

Observation that a particular cancer is more common in immunosuppressed individuals

Front 9

Human Papillomavirus (HPV) is a member of what viral family?

Back 9

Papovavirus

Front 10

What do Papillomaviruses infect?

Back 10

- Cutaneous and mucosal epithelia
- Hands, feet (warts)
- Anogenital tract

Front 11

What is the genome of Papillomaviruses?

Back 11

Small, circular, dsDNA

Front 12

How many subtypes of Papillomaviruses are there? Notable group of subtypes?

Back 12

~100
1/3 infect genital tract - sexually transmitted

Front 13

What genes do the Papillomaviruses encode? Functions?

Back 13

- Early (E) and Late (L) genes:
- E1 and E2 - mediate replication and transcription of viral DNA
- E4 - disrupts cytokeratins to facilitate virus exit
- E5 - stimulates constitutive growth factor receptor signaling
- E6 and E7 - neutralize major "brakes" that regulate cell cycle (p53 and Rb), uncoupling cell division from regulatory controls
- L1 and L2 - capsid proteins

Front 14

Which Papillomavirus genes mediate replication and transcription of viral DNA?

Back 14

E1 and E2

Front 15

Which Papillomavirus gene disrupts cytokeratins to facilitate virus exit?

Back 15

E4

Front 16

Which Papillomavirus gene stimulates constitutive growth factor receptor signaling?

Back 16

E5

Front 17

Which Papillomavirus genes neutralize major "brakes" that regulate cell cycle (p53 and Rb), uncoupling cell division from regulatory controls?

Back 17

E6 and E7

Front 18

Which Papillomavirus genes form capsid proteins?

Back 18

L1 and L2

Front 19

Which Papillomavirus genes intersect with key elements of cellular growth control, and are associated with oncogenesis?

Back 19

E5, E6, and E7
- E5 - stimulates constitutive growth factor receptor signaling
- E6 and E7 - neutralize major "brakes" that regulate cell cycle (p53 and Rb), uncoupling cell division from regulatory controls

Front 20

What is the course of a Papillomavirus infection?

Back 20

Papillomavirus -->
inoculation of epithelium (hand, foot, throat, or cervix) -->
local multiplication -->
wart -->
resolution (latency) or cell transformation

Front 21

How does Papillomavirus inoculate the epithelium?

Back 21

Enters through a microabrasion / break in the skin

Front 22

What is the life cycle of Papillomavirus tied to?

Back 22

Differentiation state of the keratinocytes

Front 23

How does the gene expression / lifecycle of Papillomavirus relate to the keratinocytes?

Back 23

- Virus enters through break in skin to level of Dermis
- Begins early gene expression (E1, E2, E6, E7) in Stratum Basale and Spinosum
- Late gene expression and viral genome amplification in Stratum Spinosum and Granulosum
- Virion assembly and release in Stratum Granulosum and Corneum

Front 24

What does Human Papillomavirus (HPV) stimulate? Where? How does this differ from normal?

Back 24

- Stimulation of cell cycle
- Causes cells in statrum spinosum to replicate
- Normally only stratum basale cells replicate

Front 25

What are the stages of the cell cycle?

Back 25

- G1
- S (DNA replication)
- G2
- M

Front 26

What controls the movement through the stages of the cell cycle?

Back 26

- Cyclin dependent kinases (CDKs)
- Move cell cycle ahead by phosphorylating key substrates

Front 27

What regulates the cyclin dependent kinases (CDKs)?

Back 27

- Temporally regulated synthesis
- Proteasome-mediated degradation of cyclin subunits
- Stimulatory and inhibitory phosphorylation events
- Stoichiometric inhibitors

Front 28

What is the "brake" that blocks progression into S phase? How is it regulated?

Back 28

- Rb protein (Retinoblastoma)
- When sufficient Cdk/cyclin has accumulated in G1, Rb becomes phosphorylated and inactivated, cell can move into S phase

Front 29

What protein from Papillomavirus affects the Rb protein? How?

Back 29

E7 protein binds to Rb and prevents it from blocking the progression to S phase (removes a key regulator of cell cycle progression)

Front 30

When there is inappropriate entry into the cell cycle, what should happen?

Back 30

Activation of the p53 protein - "guardian of the genome"

Front 31

What is the role of p53?

Back 31

- Activated by inappropriate entry into the cell cycle
- "Guardian of the genome"
- Induces production of Cdk/cyclin inhibitors to stop cell cycle
- Major inducer of apoptosis
- Stops and weeds out cells that are dividing inappropriately

Front 32

What protein from Papillomavirus affects the p53 protein? How?

Back 32

E6 protein recruits a ubiquitin ligase that targets p53 for degradation, thereby preventing it from blocking the progression to S phase or inducing apoptosis; E6 removes a key regulator of cell "accuracy"

Front 33

What protein from Papillomavirus affects the telomeres? How?

Back 33

E6 protein induces the expression of telomerase, which enables cells to maintain their chromosomal telomeres and avoid senescence; E6 therefore induces a key component of cellular immortality

Front 34

What data supports the hypothesis that HPV causes cervical cancer?

Back 34

- Sexually active populations have a higher risk of cervical cancer
- Sexually inactive populations have an extremely low risk of cervical cancer
- Greater than 99% of all cervical cancer specimens show evidence of infection with "high risk" strains of HPV (16, 18, 6, and 11)
- Low risk strains are associated with warts
- Correlation between "high risk" strains and activity of E6 and E7 proteins --> immortalize keratinocytes

Front 35

What kind of proteins are p53 and Rb?

Back 35

"Tumor suppressor genes" - their loss or inactivation causes a predisposition to cancer formation

Front 36

What percentage of human cancers have a mutated p53?

Back 36

>50%

Front 37

What happens to the viral DNA from Papillomavirus in the earlier stages of acute and persistent infection?

Back 37

Maintained as an episome (genetic element that can replicate independently of the host and also in association with a chromosome with which it becomes integrated)

Front 38

What happens to the viral DNA from Papillomavirus during the development of carcionma?

Back 38

- Associated with the integration of the DNA into the host chromosome; E6 and E7 always retained in cancer cells
- Integration disrupts expression of E2 gene, which is a transcriptional repressor and allows increased levels of E6 and E7 expression
- Replication of integrated DNA triggers DNA amplifications and chromosomal abnormalities

Front 39

What is a key step towards oncogenesis of HPV infection?

Back 39

Genome integration

Front 40

What is the cytological progression of HPV-induced lesions?

Back 40

- Normal <-->
- Infection <-->
- LSIL (Low grade Squamous Intraepithelial Lesion) <-->
- HSIL (High grade Squamous Intraepithelial Lesion) -->
- Cervical Cancer
(doesn't always progress)

Front 41

What is the histological progression of HPV-induced lesions?

Back 41

CIN-1 --> CIN-2 --> CIN-3
(CIN = Cervical Intraepithelial Neoplasia)

Front 42

What kind of lesion occurs during a productive infection?

Back 42

LSIL - Low grade squamous intraepithelial lesion

Front 43

What kind of lesion occurs during cellular gene deregulation?

Back 43

HSIL - High grade squamous intraepithelial lesion

Front 44

What kind of lesion occurs with DNA integration and breaks in basal membrane?

Back 44

Cervical Cancer

Front 45

What immune, environmental, genetic, and life-style choices impact the outcome of HPV infection?

Back 45

- Host immune response involved in initial infection of HPV
- Smoking, Oral contraceptive use, viral-load, and coinfections determine conversion from Low grade to High grade squamous intraepithelial lesion
- Viral integration and methylation determines conversion from HSIL to cervical cancer

Front 46

What are some markers of the progression from a normal cervix --> HPV infection --> LGIL --> HGIL --> cervical cancer?

Back 46

- Pap test: (-) for HPV infection; (+) for LSIL, HSIL, and CC
- E6/E7 mRNA: (-) for HPV infection; (+) for LSIL, HSIL, and CC
- L1 protein: (+) for HPV infection and LSIL; (-) for HSIL and CC

Front 47

What diagnostic tests are used for CIN1, CIN2, and cervical cancer?

Back 47

- Pap Smear
- Hybrid Capture Assay for HPV DNA

Front 48

What are you looking for on a pap smear?

Back 48

- Cytological evidence of displasia or neoplasia; detection of koilocytotic cells (vacuolated cytoplasm) which are rounded and appear in clumps
- Odd looking cells (easy to miss and easy to get false positive)

Front 49

What is done if there is an abnormal pap smear?

Back 49

Hybrid Capture Assay - for detection, strain analysis, and quantification of HPV DNA

Front 50

What are the steps of the Hybrid Capture Assay for detection, strain analysis, and quantification of HPV DNA

Back 50

1. Release nucleic acids - by combination w/ base solution (releases target DNA)
2. Hybridize RNA probe w/ target DNA - creates RNA:DNA hybrids
3. Capture hybrids - onto solid phase coated w/ universal capture antibodies specific for RNA:DNA hybrids
4. Label for detection - conjugated to alkaline phosphatase; signal can be amplified >3000-fold
5. Detect, read, and interpret results - substrate produces light that is measured on luminometer in Relative Light Units (RLUs)

Front 51

What are the issues with making an effective vaccine that would prevent genital infection with high-risk strains of HPV?

Back 51

- Need for mucosal infection due to route of infection
- Need to recognize multiple high risk strains
- Administration of a vaccine that included "oncogenes" not possible
- Societal issues - is this to prevent cancer or STD?

Front 52

What protein from HPV is utilized in vaccines? How does it work?

Back 52

- Capsid protein L1 induces productive humoral immunity
- Recombinant L1 can be produced in culture (insect cells or yeast)
- L1 self-assembles into "VLPs - virus like particles" that resemble virions and induce appropriate immunological response

Front 53

What is the effect of the HPV vaccine that contains purified "VLPs - virus like proteins"?

Back 53

- Induces protective, humoral immunity
- Antibodies present at mucosal surface prevent incoming HPV from establishing infection

Front 54

When was HPV discovered? When was HPV proposed as a necessary cause of cervical cancer? When was the vaccine licensed?

Back 54

- Discovered in 1982
- HPV --> cervical cancer in 1999
- Vaccine (Gardasil) licensed in 1008

Front 55

What strains of HPV cause cervical cancer?

Back 55

Types 6, 11, 16, and 18