Papillomaviruses

1
Papillomaviruses

• Numerous viruses that infect mammals and some
  birds
• Approximately 200 different human viruses
  identified
• Virions look like polyomaviruses but slightly
  bigger (55 nM diameter)
• Genome is circular dsDNA, 8,000 bp long
  (complexed with histones)
• Most papillomaviruses infect squamous epithelial
  cells, causing benign epithelial tumours (called
  papillomas or warts) eg. all human viruses (HPVs)
• A small group of papillomaviruses infect dermal
  fibroblasts causing benign fibroepithelial
  tumours (fibropapillomas) eg. bovine
  papillomavirus (BPV)

2
HPV Infection of Epithelium
Stages of infection of squamous epithelial cells
is tightly linked to the differentiation stage of
the cells. Initial infection can only occur in
cells capable of dividing (basal cells).
3
Genome Organization

• Genome forms nucleosomes with cellular histones
  as for polyomavirus
• Organized into early and late regions separated
  by regulatory sequences called LCR (long control
  region) or URR (upstream regulatory region)
• All genes transcribed from 1 DNA strand (same
  direction around genome)
• 1 early promoter in HPV several in BPV
• Final mRNA generated by alternative splicing

4
Early Gene Expression

• Early genes can only be expressed in certain cell
  types due to requirement for cellular factors
  that bind LCR enhancer elements
• Early genes
• E1 required for DNA replication
• E2 roles in transcription, DNA replication,
  viral DNA segregation
• E5, E6, E7 roles in cellular transformation
• E3, E8 functions unknown (not in HPV)

5
Transcription Regulation by E2

• LCR contains an enhancer that is activated by E2
• E2 activates the expression of all early genes by
  binding the enhancer
• E2 contains separate DNA binding and activation
  domains
• Transcription activation involves E2 recruiting
  cellular Brd4 protein to enhancer (E2 binds Brd4
  through activation domain)
• Truncated forms of E2 are also produced that bind
  DNA but lack the transactivation domain. These
  inhibit activation by full length E2 by competing
  for DNA binding sites. This results in down
  regulation of early gene expression

Full length E2
Truncated E2
6
Late Gene Expression

• Occurs only in differentiated keratinocytes for
  HPV
• Late proteins are L1, L2 and E4
• Transcribed from 1 promoter (PL) that is
  activated in differentiated cells of the wart
• L1 and L2 capsid proteins
• E4 in early region of genome but expressed late
  in infection
• - plays role in viral egress by disrupting
  keratin cytoskeleton

7
DNA Replication

• Two modes of DNA replication
• Plasmid replication
• Occurs in basal cells and lower portion of
  epidermus
• Viral genomes are initially amplified from low
  copy number to moderate copy number of 50-400
  copies/cell
• Moderate copy number is then stably maintained
  for many cell generations (maintenance phase)
• A copy number control mechanism ensures that the
  viral plasmids double in number every cell cycle.
  Some plasmids replicate once, some more than
  once and some not at all, such that on average
  the copy number remains the same.
• Maintenance phase replication is what occurs in
  tissue culture

8
DNA Replication

• 2) Vegetative DNA Replication
• Occurs in more differentiated epithelial cells of
  the wart
• Bursts of viral DNA synthesis occurs in the
  absence of cellular DNA synthesis
• Results in a great increase in viral copy number
• Generates genomes for packaging in the virion
• Not well understood

9
Maintenance Phase DNA Replication (BPV)

• Replication initiates from single origin in LCR
• Origin contains AT-rich region
• E1 binding site (dyad symmetry region)
• E2 binding sites

Replication requires 2 viral proteins, E1 and E2
(and many cellular proteins) E1 -
sequence-specific DNA binding protein but binds
DNA with low affinity - DNA helicase (as double
hexamer) - melts origin DNA (as double
trimer) E2- binds specific DNA sites with high
and interacts with E1 - facilitates E1
binding to the origin Replication proceeds by
theta structure mechanism using cellular DNA
polymerases and associated factors
10
Minimal origin
E1BS
AT-rich
E2BS
E2BS
E2
E1
E1
ATTGTTGTTAACAATAATTAACAACAATTGTTATTA
E2
E1
E1
ATTGTTGTTAACAATAATTAACAACAATTGTTATTA
E1
E1
Replicative helicase
E1
E1
11
Viral Plasmid Segregation (Partitioning)

• Papillomaviruses have a mechanism to ensure that
  viral genomes are not lost during cell division
  in the maintenance phase of replication
• Involves attachment of viral plasmids to the
  cellular mitotic chromosomes
• Requires a DNA sequences in the LCR that contains
  multiple binding sites for E2 the minichromosome
  maintenance element (MME)
• Also requires E2
• E2 binds MME then interacts with a cellular
  protein on the mitotic chromosome called Brd4
• the transactivation domain of E2 binds Brd4
• A recent paper shows a second cellular protein,
  ChlR1 (A DNA helicase) is also required for E2 to
  initially attach to mitotic chromosomes

12
BPV Segregation Mechanism
Cellular chromosome
Brd4
E2
BPV
BPV genome
E2
Mitotic Chromosome
Mother cell
Daughter cells
13
Cell Transformation by BPV (fibroblast
transformation)

• Assay BPV transforms rodent cell lines in tissue
  culture
• Cells acquire growth properties characteristic
  of cancer cells (loss of contact inhibition,
  anchorage independence, form tumours in nude
  mice)
• Viral DNA is maintained as a plasmid (not
  integrated)
• Involves E5, E6, E7
• E5 major transforming gene of BPV
• - responsible for proliferation of dermal
  fibroblasts in fibropapillomas
• - integral membrane protein
• - interacts with receptor for platelet-derived
  growth factor (PDGF)
• - PDGF receptor is activated by E5 leading to
  cell proliferation
• E6 and E7 required for full transformation of
  mouse cells
• - likely function by binding cellular
  proteins (mechanism not clear)
• - nuclear

14
Cell Transformation by HPVs (epithelial cell
transformation)

• Assay ability to transform primary rodent cells
  and primary human fibroblasts in tissue culture,
  resulting in immortalization of cells
• High risk HPVs associated with human
  malignancies (eg. HPV16, HPV18)
• - positive in above assay for cell
  transformation
• Low risk HPVs not associated with human
  malignancies (eg. HPV6, HPV11)
• - negative in above assay for cell
  transformation
• (all genital HPVs transiently induce cell
  proliferation but only high risk HPVs give rise
  to immortalized cells that are refractory to
  differentiation signals)

15
Role of HPV E5 in Cell Transformation

• Epithelial cells lack the PDGF receptor but
  express the epidermal growth factor (EGF)
  receptor
• Primary target of HPV E5 is the EGF receptor
• Results in increased EGF signalling
• Some transforming activity can be detected for E5
  from both high and low risk HPVs
• In most HPV-positive cancers, E5 is not expressed
• Suggests that growth stimulation by E5 is
  important in warts but not in cancer
• Alternatively E5 might participate in early steps
  of carcinogenesis

16
Role of HPV E6 in Cell Transformation

• Required for efficient immortalization of primary
  human cells
• Effect only seen with E6 from high risk HPVs
• 3 cellular proteins are bound by high risk (but
  not low risk) E6
• p53 high risk E6 inactivates p53 by inducing
  its degradation
• - involves cellular protein E6AP, a ubiquitin
  ligase
• - E6, E6AP and p53 form a complex
• - in presence of E6, E6AP ubiquitinates p53
  which targets it for degradation
• - p53 levels are 2-3 fold lower in
  E6-immortalized cells
• - enables cell proliferation
• 2) p300/CBP (histone acetyl transferases)
• - results in decreased transcription of cell
  cycle inhibitors and increased cell proliferation
• 3) Proteins with PDZ domains E6 causes
  ubiquitination of these proteins by E6AP,
  leading to their degradation
• Role of E6 in infection is likely to induce cells
  into S phase

17
Role of HPV E7 in Cell Transformation

• Part of E7 is similar to region of adenovirus E1A
  and SV40 T that binds Rb
• Accordingly, E7 found to bind Rb
• High risk E7s bind Rb 10-times better than low
  risk E7s (due to differences in Rb binding
  sequence)
• High risk but not low risk E7s induce cell
  transformation (could involve more than Rb
  binding)
• E7 binding to Rb causes increased expression of
  genes under E2F control (including cellular
  replication proteins)
• Role of E7 in viral infection (due to Rb binding)
• Induction of S phase
• Expression of cellular DNA replication proteins
• - would be particularly important for
  vegetative DNA synthesis which occurs in
  nondividing cells (replication proteins expressed
  in the absence of S phase)

18
A
. Adenovirus
E
1B
p
53
Cell transformation by adenovirus, SV40 and
papillomavirus all involve targeting of Rb, p53
and p300/CBP
E1A
B
. SV40
Large-T
p
53
Rb
C
. Papillomavirus
E6AP
E7
E6
p
53
E6
Rb
P53 degradation
19
Diseases associated with Papillomaviruses

• Most common manifestation is a wart
• Wart benign, self-limiting proliferative lesion
  that regresses after a period of time (months to
  years)
• - clonal in origin (develops from a single
  infected cell)
• - initial infection (basal cells) causes cells
  to proliferate
• - cells on outer surface gradually differentiate
  and stop dividing

20
Types of HPV

• 1) Cutaneous
• - Causes skin warts
• - infection occurs through an abrasion of the
  skin
• - HPV5 and 8 can cause carcinomas
• 2) Mucosal
• - reside mainly in the genital tract where they
  can produce genital warts
• - often genital HPV infections are without
  symptoms and transient
• - sexually transmitted
• - lifetime risk of a genital HPV infection is
  80
• - HPV6 and HPV11 are most common
• - Fifteen different HPV strains are considered
  high risk as they are associated with cervical
  cancer (HPV16 and 18 are the most common of
  these)

21
HPV and Cervical Cancer

• 450,000 cases of invasive cervical cancer per
  year worldwide (80 in developing countries
  without Pap screens). Results in death in about
  50 of cases.
• 99.7 of cervical squamous cell carcinomas (most
  common type of cervical cancer) contain HPV
• 80 of these contain HPV strains 16, 18, 31 or 45
• Requires a persistent infection with a high risk
  strain
• In most cervical cancer cells, the HPV genome is
  integrated into the cellular DNA
• Cancer cells have increased expression of E6 and
  E7
• Linearization/integration can result in increased
  expression of E6 and E7, which increases the
  immortalization capacity of the virus
• Cancer is a rare consequence of HPV infection and
  develops a long time (12-15 years) after
  infection, suggesting a requirement for
  additional genetic mutations in the infected cell
  prior to carcinogenesis

22
HPV and Genital Infections in Men

• Genital HVP infections in men range from no
  symptoms to squamous cell carcinomas on the penis
• HPV found in 42-80 of penile tumours
• Lower incidence of genital HPV infections in men
  as compared to women
• Vaccine
• Vaccine against HPV 6, 11,16 and 18 is available
  and proven to be effective in women for at least
  5 years.