Papillomaviruses
Intro
Human papillomaviruses (HPVs) are a major public health problem. Some HPV types
cause benign skin warts, known as papillomas (hence the family name). Other HPV
types infect mucosal sites, such as the genital mucosa and are transmitted sexually. Many
sexually-transmitted HPV infections are not associated with overt symptoms, but in a
minority of infected women the infection can lead to cancer of the uterine cervix. Nearly
all cases of cervical cancer are caused by HPV. HPV vaccines are poised to effect
dramatic (but not total) reductions in the incidence of HPV-induced cervical cancer and
genital warts.
A)
Medical Significance
1) It’s likely that all humans become infected with multiple cutaneous (skin-tropic)
HPV types. Some infections are transient (e.g., childhood warts). Other
infections may be asymptomatic and lifelong.
2) Sexually-transmitted mucosal types are also ubiquitous (>75% lifetime risk of
infection with one or more cancer-associated HPV types).
a) Infection with cancer-associated HPV types is generally asymptomatic.
b) A minority (~1%) of infected women progress to cervical cancer.
3) Cervical cytology screening (Papanicolaou test = “Pap smear”) has dramatically
reduced the incidence of cervical cancer in the developed world.
a) Testing costs $billions/year
b) Some women still fall through the cracks
c) Diagnosis with cervical pre-cancer can be psychologically traumatic
4) Recently-approved HPV vaccines: based on antibody responses to recombinant
L1-only capsids
a) Phase III clinical efficacy trials recently demonstrated protection against
HPV16 in vaccinated women. Gained FDA and CDC approval this past
summer.
b) Protection primarily due to large HPV-neutralizing antibody response
c) Probably only prophylactic (not effective for women who are already
infected)
d) Probably HPV type-specific (only protect against certain HPV types)
5) Condoms are at most 70% effective for preventing genital HPV transmission.
This may be due to the fact that genital HPVs can infect skin surfaces that
condoms don’t cover.
B)
Physical Characteristics and Genetic Organization
1) non-enveloped icosahedral capsid formed by 72 pentamers of one protein, L1.
a) Medium size capsid (60 nanometer diameter).
b) Minor capsid protein present at variable copy number – up to one L2 per L1
pentamer.
c) Although papillomaviruses share many common features with polyomaviruses
(e.g., simian virus 40), it is thought that the two virus families (formerly
joined as family papovaviridae) actually never shared a common ancestor.
2) Circular 8,000 basepair dsDNA genome
a) genes all encoded on one strand of DNA
3) Early genes
a) Oncogenes E6 and E7 inactivate cellular p53 and pRb tumor suppressor
proteins.
b) E1: viral helicase. Binds to the viral origin of replication (Ori) in the long
control region (LCR). Helps recruit cellular DNA replication machinery.
Note that high-fidelity replication by cellular DNA polymerases means
papillomaviruses have low mutation rates.
c) E2: master transcriptional regulator. Also facilitates binding of E1 to the
viral Ori. In most cervical cancer, loss of E2 leads to up-regulation of E6 and
E7 expression.
4) Late genes – L1 and L2 (see above)
C)
Life Cycle: Maintenance and Oncogenesis
1) Exquisite tissue tropism: all PVs replicate exclusively epithelial tissues (skin or
mucosal surfaces). Most types have preference for a particular body surface.
a) Many HPV types are essentially commensal (little detriment to host)
b) Palms of hands and soles of feet are cutaneous hotspots.
c) Susceptible mucosal surfaces include vagina, cervix, foreskin, anus, oral and
respiratory surfaces.
2) Viral life cycle is intimately linked to differentiation of stratified squamous
epithelia
a) Unique feature of HPVs: may be nature’s only non-lytic non-enveloped virus.
Reliant on “desquamation” (spontaneous disintegration of cells at the skin
surface) for virion release.
3) Tissue architecture / immune evasion strategy. Late gene expression is limited to
the topmost layers of the epithelium. The early genes needed for maintenance of
the viral genome in basal layer stem cells are expressed at extremely low levels.
Therefore no “danger” signals are given to alert professional antigen presenting
cells such as Langerhans cells and dendritic cells.
a) Tearing away HPV’s veil: cryotherapy, imiquimod, vaccines. Tissue
disruption or the toll-like receptor-8 ligand imiquimod can reveal the infection
to professional antigen-presenting cells.
4) Genomic instability: oops, I caused cancer. A loss of HPV gene regulation leads
to over-expression of the viral oncogenes, E6 and E7, leading to cellular genetic
instability and ultimately cancer.
a) progression vs regression of HPV infection (cervical intra-epithelial
neoplasia)
D)
Life Cycle: Assembly
1) Papillomaviral vectors (a.k.a. pseudoviruses)
a) Use SV40 T antigen for DNA replication and “codon-modified” HPV L1 and
L2 genes to achieve intracellular assembly of an L1/L2 shell carrying a
reporter plasmid. Expression of wild-type L1 and L2 genes in normal cells is
very poor due to mRNA negative regulatory elements. Genes must be rebuilt
from scratch to incorporate numerous silent mutations (same protein encoded
by a different mRNA sequence). Packaged reporter plasmids can be taken on
a one-way trip into target cells. Very useful tool for a virus without a
convenient cell culture model.
2) Hypothetical “catch and release” assembly model to explain how L1 and L2 can
fish reporter plasmids away from cellular DNA without any apparent packaging
signals. L1 and L2 roam the nucleus grabbing DNA. If they find a small piece
of DNA (eg, reporter plasmid), they form a proto-capsid and stop shuttling.
Protocapsids containing reporter plasmid accumulate prior to cell lysis.
a) possible supporting evidence: PVs don’t pack their DNA very tightly.
3) Capsid maturation. After cell lysis, sulfhydryl groups slowly become oxidized
and form covalent cysteine crosslinks between neighboring L1 molecules.
Disulfide crosslinks lock the capsid into a more condensed configuration, with
greater stability.
E)
Life Cycle: Entry
1) Attachment – heparan sulfate proteoglycans. L1 binds avidly to surface proteins
decorated with sulfated polysaccharides (HSPGs).
2) More proteinaceous receptors. Everybody feels confident they exist but it’s very
unclear exactly which proteins are involved.
3) It is widely believed that clathrin-mediated endocytosis is required for infectivity.
However, more cell types / HPV types must be studied to be sure. Unknown
signaling events are probably involved. Process is very slow compared to other
viruses.
4) Furin protease (pathogens’ best friend!) must cut L2. Furin is involved in cutting
the envelope proteins of a variety of viruses (e.g, HIV, WNV, many others)
during assembly / trafficking through the Golgi. Furin cleavage is also needed to
activate various bacterial toxins (anthrax toxin, pseudomonas exotoxin, many
others) on the way into the cell.
5) Uncoating. L1 undergoes structural rearrangments to expose L2 and the viral
genome. Mechanism is not known.
a) melting of disulfide bonds?
b) other proteases?
6) Endosome disruption (L2). Membrane-destabilizing peptide in L2 mediates
release of the viral genome from endosomes. L2 accompanies the genome out of
the endosome, L1 probably gets left behind.
7) Genome trafficking / ND-10 sites. L2 drags the viral genome to a sub-nuclear site
known as nuclear domain-10 (ND-10). Many viruses interact with ND-10. Some
herpesviruses initiate transcription there as well, but later in the herpesvirus life
cycle viral genes disassemble ND-10.
F)
Future Directions
1) L2-based prophylactic vaccines
a) Antibody-based prophylactic vaccines. Ordinarily L2 is not very
immunogenic. However, antibodies to certain L2 epitopes are broadly crossneutralizing. i.e., antibodies to the L2 of one HPV type may block infection
of a diverse range of other types (in contrast to L1-only vaccines).
2) Therapeutic vaccines
a) Goal: elicit T cell response to oncogenes E6 and/or E7
b) E6 and E7 excellent tumor targets since they are required for maintenance of
transformation (example: HeLa die after treatment with E6/E7 siRNAs).
3) Topical microbicides – compounds that might be put on condoms to prevent HPV
transmission. Some promising candidates may already be on store shelves near
you.