Papillomaviruses

Question 1

structure

Answer 1

• Naked icosahedral capsid
• Circular double-stranded DNA
• DNA is packaged as a “minichromosome” with
  cellular nucleosomal histones

Question 2

genome

Answer 2

• circular, double-stranded DNA that contains 8-10 open reading frames (ORFs) – 6 early genes (E1, E2, E4, E5, E6, E7), 2 late genes (L1, L2)
• Only one of the DNA strands is used for mRNA transcription

Question 3

what are the viral mRNAs made from?

Answer 3

• two promoters and two polyadenylation signals
• Early promoter located upstream of E6 gene; late promoter located upstream of E1 gene
• Two poly(A) signals are located downstream of E5

Question 4

infectious cycle

Answer 4

• follows differentiation of epithelial cells
• infects the mucosal epithelium and have to reach non-differentiated basal cells
• attaches to heparin sulfate proteoglycans and α6-integrins and enters by endocytosis
• Viral DNA is maintained in the nucleus, replicating modestly until cell differentiates into a keratinocyte

Question 5

keratinocytes

Answer 5

• predominant cell type in the epidermis, the outermost layer of the skin
• originate in the basal layer of the epidermis and undergo differentiation as they migrate upward, eventually forming the skin’s tough outer layer

Question 6

what do the viral E1 and E2 proteins do?

Answer 6

bind to the origin of replication (ori, present in the long control region,
LCR) and direct initiation of DNA replication

Question 7

long control region (LCR)

Answer 7

• sometimes called URR – upstream regulatory region
• contains the origin of replication
• also contains enhancer sequence(s)
  (may be specific keratinocyte enhancer)

Question 8

E7 Protein

Answer 8

• small (~100 AA) protein that interacts with cell cycle regulatory proteins,
  particularly retinoblastoma (Rb) protein
• also induce the ubiquitin-mediated degradation of Rb
• leads to replication of
  viral DNA to high copy number

Question 9

retinoblastoma (Rb) protein

Answer 9

• tumor suppressor protein which represses the activity of E2F transcription factors under
  normal conditions
• blocks activation of cell-cycle genes (involved in G1 to S phase (DNA synthesis phase) transition
• blocks transcription of cell-cycle genes

Question 10

E2F protein

Answer 10

activates transcription of cell-cycle genes

Question 11

CDK/cyclin complex

Answer 11

• phosphorylates Rb
• results in dissociation from E2F
• allows expression of cell cycle genes
• P53 activates transcription of P21 that blocks this complex

Question 12

E6 protein

Answer 12

• controls the level of cellular p53 proteins, allows the virus sufficient
  time to replicate
• associates with cellular E6AP (ubiquitin-protein ligase, aka E3A), and the complex then stimulates
  the ubiquitination of p53 and its subsequent degradation by proteosome
• interacts with a number of
  cellular proteins involved in signal
  transduction, DNA replication,
  apoptosis, Ca2+ metabolism

Question 13

what does increased cell-cycle progression lead to?

Answer 13

• increase in p53 levels
• eventual apoptosis

Question 14

E6 protein from non-oncogenic HPVs

Answer 14

unable to mediate the degradation
of p53

Question 15

Diagnosis

Answer 15

Pap test is often combined with DNA test for HPV

Question 16

Pap Test

Answer 16

• recognizes morphologically abnormal cervical cells and is an effective means of reducing cervical cancer deaths
• Allows for identification of morphologically abnormal cervical cells
• Cervical warts and precancerous are routinely removed, prevents cervical cancer

Question 17

DNA tests

Answer 17

Self-sampling DNA tests may overcome the obstacles of a lack of trained pathologists (e.g. in
underdeveloped countries) and cultural restrictions (invasive procedure)

Question 18

Treatment

Answer 18

HPV vaccines (e.g. Gardasil, Cervarix) are commonly available and is part of free vaccination
programs in several countries (typical vaccination age ~11-13, but can be given to older
individuals (higher dose)

Question 19

Disease

Answer 19

• Oncogenic human papillomaviruses are a major cause of genital tract cancers and can cause genital warts (condylomas), neoplasias (uncontrolled growth of cells), and invasive squamous cell carcinomas
• Some types cause cervical carcinoma, a sexually transmitted disease and a major cause of cancer in women
• May cause other anogenital cancers (cancers of anus, genital organs) in both men and women, and cancers of the oral cavity, oropharynx and tonsils
• Benign warts at specific sites (skin, mucosa, larynx) depending on virus strain, transmitted by direct contact

Question 20

What is E1?

Answer 20

DNA helicase that locally unwinds the DNA

Question 21

What does E2 do?

Answer 21

E2 binds to specific sites near ori and forms a complex with E1, increases E1 affinity for ori

Question 22

what increases DNA replication?

Answer 22

• Late promoter is active in differentiated keratinocytes
• Some of the transcripts are polyadenylated (addition of poly (A) tail) at the late site
• produces abundant E1 and E2
  proteins
• increased level of E1 and E2 result in increased DNA replication, producing
  thousands of DNA molecules per cell

Question 23

what does inhibition of Rb lead to?

Answer 23

• uncontrolled proliferation (rapid reproduction) of the infected cell
• loss of p53’s ability to induce
  apoptosis
• oncogenic combination

Question 24

what is contained in cervical cell lines?

Answer 24

• copies of part of HPV genome integrated into different sites in cellular genome
• integration happens only in cancer cells

Question 25

what is the result of HPV genome integration?

Answer 25

• loss of all viral genes except E6 and E7
• expression of E6 and E7
  gene products (mRNA, proteins)

Question 26

E6 & E7 in cancer

Answer 26

• E7’s main function is inducing cell proliferation, leading to tumor formation
• E6’s has a cooperative role in increasing tumor size and progression of malignancy
• inhibiting these gene products is a potential target for therapeutics

Question 27

L2

Answer 27

• enhances capsid formation and is involved in encapsidation of viral
  genome
• Some evidence indicates it is
  involved in intracellular trafficking events; deposition of viral DNA
  into nucleus

Question 28

assembly and release

Answer 28

• Expression of L1 and L2 promotes the encapsidation of newly replicated genomes, resulting in virion release from the uppermost layers of the epithelium
• virus is released from the cell
  (lysis)

Question 29

what is viral capsid formed from?

Answer 29

72 capsomeres (aka capsomers; protein subunits that make up
capsid) which are formed as pentamers of L1

Question 30

vaccines and L1

Answer 30

Two different vaccines are made
by assembling L1 into virus-like
particles (VLP) that do not
contain the viral genome