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Papillomaviruses
Chris Buck
Email: [email protected]
National Cancer Institute, Bethesda, Maryland
Papillomaviruses
National Cancer Institute*, Bethesda, Maryland
*The views expressed in this talk are my own
and do not necessarily represent the views of
the NCI
*I receive royalties from inventions related to
HPV vaccines
Baltimore Classification Scheme
Baltimore Classification Scheme
No integration into host cell
DNA. Viral genome only exists
as an “episome”
Papillomavirus Virion
•Non-enveloped icosahedral
shell formed by 72 pentamers of
a single protein, L1 (basis of
current HPV vaccine)
•60 nanometer diameter
•Many features in common with
polyomaviridae (e.g., SV40).
Most notably: the viral genome
is a small circular dsDNA
molecule decorated with host
cell histones (chromatin)
Human papillomavirus type 16
Taxonomy
•“Crown Group”
hypothesis: the last
common ancestor
of placental
mammals already
had several distinct
types of
papillomavirus.
Each ancestral virus
has co-speciated
with its host animal.
Little or no evidence
of papillomaviruses
transmission
between distantly
related animals
Taxonomy
Genus: Mu
Species: 1
Type: HPV1
causes warts
on the soles of
the feet
Taxonomy
Genus: Alpha
Species: 4
Type: HPV2
causes warts
on the palms
of the hand
Taxonomy
Genus: Gamma
Species: 1
Type: HPV4
Caused a wart
on the palm of
my hand. Grrr!
Taxonomy
Genus: Alpha
Species: 10
Type: HPV6
causes genital
warts. Does
NOT cause
cervical cancer
Taxonomy
Genus: Alpha
Species: 9
Type: HPV16
Generally
asymptomatic, but
causes cervical
cancer in a small
minority of infected
individuals
Genome
•8 kilobase
circular dsDNA.
Persists as
episome (doesn’t
integrate into
cellular DNA)
L1
E6
E7
7904/1
1000
7000
2000
6000
5000
L2
3000
4000
E5
E4
E2
E1
Genome
•8 kilobase
circular dsDNA.
Persists as
episome (doesn’t
integrate into
cellular DNA)
L1
E6
7904/1
1000
7000
2000
6000
5000
L2
•One coding strand.
Genome is divided
into Late and Early
regions
E7
3000
4000
E5
E4
E2
E1
Capsid Genes
•virion formation
•cell association
L1
7904/1
1000
7000
2000
6000
5000
L2
E7
E6
3000
4000
E5
E4
E2
E1
Capsid Genes
E7
E6
7904/1
L1
1000
7000
2000
6000
5000
L2
•genome encapsidation
•membrane penetration
•post-entry trafficking
3000
4000
E5
E4
E2
E1
Capsid Genes
E7
E6
7904/1
L1
1000
7000
2000
6000
5000
L2
•genome encapsidation
•membrane penetration
•post-entry trafficking
3000
4000
E5
E4
E2
E1
Oncogenes
•Together the
viral oncogenes
immortalize the
cell and prime it
for viral DNA
replication by
disrupting the
cell cycle.
L2
E6
E7
7904/1
L1
1000
7000
2000
6000
5000
3000
4000
E4
E2
E5
E1
Oncogenes
•Together the
viral oncogenes
immortalize the
cell and prime it
for viral DNA
replication by
disrupting the
cell cycle.
L2
E6
E7
7904/1
L1
1000
7000
2000
6000
5000
E1
3000
4000
E4
Small genome! No
E2
room for fancy stuff
like DNA polymerase,
E5
DNA primase, ribonucleotide reductase, etc. The host cell
must be forced to provide these factors.
Cell Cycle Disruption
Adenovirus E1B
SV40 T Ag
Adenovirus E1A
•The E6 and E7 genes of
cancer-causing HPVs
mediate destruction of the
tumor-supressor genes
p53 and pRB
(respectively)
•Other types of DNA virus
also disrupt p53 and pRB.
This promotes entry into
the S phase of the cell
cycle, allowing replication
of the viral DNA
E6 & E7 - Other Functions
E6: Activates telomerase.
Interferes with about a dozen other known cellular
targets, resulting in p53-independent effects.
E7: Triggers chromosomal instability
Interferes with about a dozen other known cellular
targets, resulting in pRB-independent effects.
Ongoing E6 and E7 expression is crucial for
maintenance of HPV-transformed cells. For
example, the famous HeLa cell line dies if E6
and/or E7 expression is knocked down.
DNA Handling Genes
LCR/
Ori
E6
E7
7904/1
L1
1000
7000
2000
6000
5000
L2
•Helicase,
recruits
cellular DNA
polymerase
E1
3000
4000
E4
E2
E5
•Transcriptional
regulation
•recruit E1 to Ori,
•tethers viral DNA to
chromosomes
during cell division
Viral Lifestyles
•“Live fast, die young” - high-level viremia. Virus
attempts to outrun the adaptive immune response.
Examples: Ebola, influenza
•“Tiptoe past the graveyard” - virus can linger and lie
low in a safehouse cell type (neurons, stem cells).
Latently infected cells express few or no viral proteins.
Example: HPVs, polyomaviruses, herpesviruses.
Immunosuppression can lead to emergence from
latency and high level virion production
•Second strategy is somewhat more common among
DNA viruses (RNA is hard to preserve in a latent state?)
Life Cycle - Initial Infection (Skin)
micro-trauma
virions
Epidermis
(Keratinocytes)
Basement
Membrane
Basal
Cells
Dermis
Slide courtesy M. Kast
Gene Expression (Wart)
Progeny virions
Viral Proteins
Capsid &
Early
Epidermis
Early only
Basement
Membrane
Dermis
None
Immune Evasion
Epidermis
Langerhans
cells
Basement
Membrane
Dermis
Dendritic
cells
Draining Lymphatics
Immune Evasion
Epidermis
Langerhans
cells
Basement
Membrane
Dermis
Dendritic
cells
In traditional cell culture monolayers, the virus
thinks it’s in a basal keratinocyte, where
hiding from the immune system is top priority.
The only way to trick the virus into late phase
Draining Lymphatics(virion production) in vitro is through the use
of keratinocyte raft cultures
HPV Reporter Vectors
(Pseudoviruses)
GFP
codon-modified
L1+L2
(helper plasmid)
GFP
reporter
(8 kb)
SV40 Ori
transfect
transduce
T Ag
“293TT”
HPV Reporter Vectors
(Pseudoviruses)
GFP
codon-modified
L1+L2
(helper plasmid)
GFP
reporter
(8 kb)
Neutralizing
antibody
SV40 Ori
transfect
transduce
T Ag
“293TT”
HPV-Based Gene Transfer Vectors
•L1 and L2 can package non-viral DNA and deliver
packaged DNA to cells with high efficiency. HPVderived DNA sequences are not required for efficient
packaging (though some types of non-viral DNAs are
packaged better than others)
•HPV vectors may become useful for genetic delivery
of vaccine immunogens (e.g., vaccines capable of
eliciting cytotoxic T cells against HIV or other viruses)
Cryo Therapy, Wounding
Epidermis
Langerhans
cells
Basement
Membrane
Dermis
Dendritic
cells
Draining Lymphatics
DC & LC Activation =
HPV Specific Immunity
Vaccination
Epidermis
Langerhans
cells
Basement
Membrane
Dermis
Draining Lymphatics
Dendritic
cells
DC Activation =
HPV-specific Response
Progression to Cancer is Accompanied by
Deregulation of Viral Gene Expression
CIN 1
CIN 2
CIN 3
Doorbar, J Clin Virol 32:7-15, 2005
Common molecular events:
•Viral genome integration into cellular DNA
•Loss of E2 leads to increased E6/E7 expression
•Loss of L1, L2 expression. Therefore, current
vaccine can’t clear pre-cancerous lesions.
Therapeutic Vaccines
CIN 1
CIN 2
CIN 3
Doorbar, J Clin Virol 32:7-15, 2005
•Goal: recruit T cells against HPV E6 and/or E6
•Goal: eradicate transformed cells (including precancerous lesions)
Gene Expression (Wart)
Progeny virions
Viral Proteins
Capsid &
Early
Epidermis
Early only
Basement
Membrane
Dermis
Bad news: stably infected keratinocyte stem
cells don’t produce L1. Therefore, the L1
vaccine can’t possibly clear existing
infections. This is why you must vaccinate
kids before they’re exposed.
None
Therapeutic Vaccines
Expert Opinion on Emerging Drugs, November 2012
•Good news: cervical
cancer cells depend
on ongoing E6/E7
expression
•Bad news: effective
T cell immunity is a
lot more difficult to
elicit than effective
antibody responses
History and Medical Significance
•1894-1924: Ciufo, Variot and others show that genital and
skin warts can be transmitted between individuals by a
filterable infectious agent
History and Medical Significance
•1894-1924: Ciufo, Variot and
others show that genital and
skin warts can be transmitted
between individuals by a
filterable infectious agent
•1933 (Shope) rabbit
papillomas have viral etiology
•1935 (Rous) papillomas can
progress to carcinoma
•Papillomaviruses found to
cause skin warts and other
tumors in vertebrates ranging
from birds to people
Infection with Shope cottontail
rabbit papillomavirus
History and Medical Significance
•1842: Rigoni-Stern reports that prostitutes have much
higher incidence of cervical cancer than nuns
History and Medical Significance
•1842: Rigoni-Stern reports that prostitutes have much
higher incidence of cervical cancer than nuns
•1928: Georgious Papanicolaou develops the “Pap smear”
technique for microscopic detection of cervical cancer and
pre-cancer
History and Medical Significance
•1842: Rigoni-Stern reports that prostitutes have much
higher incidence of cervical cancer than nuns
•1928: Georgious Papanicolaou develops the “Pap smear”
technique for microscopic detection of cervical cancer and
pre-cancer
•1951: George Otto Gey
establishes in vitro culture of
HeLa (Henrietta Lacks) cells
derived from a lethal cervical
cancer
History and Medical Significance
•1842: Rigoni-Stern reports that prostitutes have much
higher incidence of cervical cancer than nuns
•1928: Georgious Papanicolaou develops the “Pap smear”
technique for microscopic detection of cervical cancer and
pre-cancer
•1951: George Otto Gey establishes in vitro culture of HeLa
(Henrietta Lacks) cells derived from a lethal cervical cancer
•1983: Harald zur Hausen discovers
new HPV types (types 16 and 18) lurking
in HeLa cells and other cervical cancer
cells.
•2008: Harald zur Hausen wins Nobel
Prize for his work establishing a causal
link between HPVs and cervical cancer
Harald zur Hausen
HPV Vaccine: What Is It?
•Recombinant virus-like
particles based on HPV
capsid protein L1
•Produced in yeast (Merck) or
baculovirus (GSK)
•Elicits HPV-neutralizing
antibody responses that
protect against initial
infection
HPV Vaccine: Not Just for Girls Anymore
•Summer 2006: FDA and
CDC recommend new HPV
vaccine “Gardasil” (Merck)
for girls and women age 9 26
•October 2009: A second
vaccine, “Cervarix”
(GlaxoSmithKline)
approved by FDA
October 2009: Gardasil
approved for use in boys
and young men
HPV Vaccine: Highly Effective
Common Cancers in Women
“Pap” smear
Adapted from Parkin et al, Eur J Cancer 37:S4, 2001
Worldwide Incidence of
Cancers Attributable to HPV
Estimates from IARC
HPV and Non-Cervical Cancers
•About half of cancers of the upper throat (for example,
tonsils) are caused by HPV infection. A great majority of
HPV-associated throat cancers contain HPV type 16
(covered by the vaccine)
•Work by Maura Gillison and others has shown that oral
sex, particularly with multiple partners correlates with risk
of throat cancer (Michael Douglas: not crazy! actually kind
of heroic!)
•Most cases of anal cancer are caused by HPV. As with
throat cancer, HPV16 accounts for a great majority of
cases. No clear connection between anal sex and anal
cancer, though overall history of multiple sex partners is a
clear risk factor.
HPV Vaccine: Not Just for Girls Anymore
% of cervical cancers
Distribution of HPV Types in Cervical
Cancer by Geographical Region
100
90
80
70
60
50
40
30
20
10
0
HPV 45
HPV 31
HPV 18
HPV 16
Africa
Bosch et al, JNCI, 1995
Latin
South-East Europe
America
Asia
North
America
HPV Vaccine
Some Degree of Cross-Protection?
HPV31?
“high risk” types
partial
HPV16
HPV18
HPV45
no
yes (weak?),
Cervarix may
offer greater
cross-protection?
Harper (2006) Lancet 367:1247
HPV Vaccine
HPV Vaccine
HPV Vaccine
HPV Vaccine
HPV Vaccine
HPV Vaccine - Theory
HPV Vaccine - Theory
HPV Vaccine - Theory
•Other virologists should be trying to
figure out how to apply this magic to
their favorite virus. We’re looking at
you, HIV biologists!
Current HPV Vaccine - What It Will Do
•Protect against two HPV types that cause 70% of all
cervical cancer. Given the amazingly strong antibody
responses, protection will probably be life-long
•Gardasil (but not Cervarix) will protect against two HPV
types that cause 90% of all genital warts
•Protect against other HPV-induced cancers, such as
throat cancer and anal cancer
Current HPV Vaccine - What It Won’t Do
•Won’t be affordable for women in developing
countries
•Won’t protect against all of the dozen or so HPV
types that can cause cervical cancer
•Won’t protect against non-carcinogenic HPV types
that may still cause abnormal Pap smear
•Won’t protect women who are already infected (more
on why this is so coming up later in the lecture)
Life Cycle: Tissue Tropism
Life Cycle: Entry
Infectious Entry Pathway
Step 1: Attachment
+
+
+
-
+
-
-
cell membrane
heparan sulfate proteoglycans
(=surface proteins decorated with
sulfated polysaccharide side-chains)
Infectious Entry Pathway
Heparin,
carrageenan
-
-
-
Step 1: Attachment
Topical Microbicides
•Several brands of sex lube happen to use carrageenan
as the principal gelling agent
Topical Microbicides
•Recently-announced clinical trial results suggest
that a carrageenan gel protects women from HPV
infection when applied before sex
Infectious Entry Pathway
conformational
shift?
Step 2: L2 exposure
Infectious Entry Pathway
Step 3: Furin cuts L2
furin
protease
Infectious Entry Pathway
Step 3: Furin cuts L2
furin
protease
Note: many viruses use
furin during assembly.
•Some toxins (anthrax toxin) use furin during entry
Infectious Entry Pathway
conformational
shift?
Unidentified
second
receptor?
Infectious Entry Pathway
conformational
shift?
Receptor =
Annexin-A2?
Infectious Entry Pathway
conformational
shift?
nAb
Many hours
Receptor?
Recent Developments
•Infection of mouse female genital tract reveals that the virus
initially attaches to heparan sulfate on the basement membrane
furin
Day et al (2009) PNAS 106:20458
L2-Neutralization - Chop the Whole Tree?
Immunogen:
BPV1 L2 aa 1-88
cutaneous,
mostly asymptomatic
foot
warts
CRPV
BPV1
neutralization
HPV16, 31
hand
warts
genital
mucosa,
cancerassociated
HPV18
HPV6, 11
genital warts
Richard Roden
Diana Pastrana
(2005) Virol 337:365
Infectious Entry Pathway
Step 5: Endocytosis
PO4?
clathrin
2 to 8 hours?
Infectious Entry Pathway
Step 6: Endosome
H+
H+
protease(s)?
disulfide reduction?
H+
+
H+ H
Infectious Entry Pathway
Step 7: Membrane disruption
L2 carboxy-terminus
Syntaxin-18?
Sortin Nexin 17?
~8 to 20 hours?
Infectious Entry Pathway
Step 8: L2 drags viral
genome to nucleus
DNA
L2
Tubulin
Actin?
nuclear pore
Infectious Entry Pathway
Step 8: L2 drags viral
genome to ND-10
24 to 72 hours
mRNA
ND-10
Future Directions:
L2-Based Vaccines
•Antibody responses to certain portions of L2 can be
broadly cross-neutralizing. For example, antibody
response to HPV16 L2 can neutralize other genital and
skin-tropic HPV types. Maybe also true for conserved
portions of L1?
•Single immunogen might protect against all HPV types.
Would eliminate the need for expensive Pap testing
•Added bonus: protection against skin warts might
facilitate implementation of childhood vaccination
Future Directions:
Therapeutic Vaccines
•T cell responses to E6 and E7 might cure existing
cervical cancer and pre-cancerous lesions
•The fact that E6 and E7 are likely required for
maintenance of transformed phenotype in cervical
cancers makes them excellent cancer vaccine targets
Practicalities
•Unvaccinated college-age adults should consider getting the
HPV vaccine. This advice now applies to both men and women
•In contrast to other STIs, condoms provide limited (possibly
no) protection against HPV transmission. Large areas of
genital surfaces can be productively infected
•Papanicolaou (“Pap”) testing is highly effective for detecting
cervical pre-cancer
•Newer HPV DNA testing is also a highly effective screening
method
•Topical microbicides are in trials. Carrageenan, which is
already on store shelves, appears to be effective for protecting
women against HPV infection in an early clinical trial
the end