Clinical Science (2006) 110, 525–541 (Printed in Great Britain) doi:10.

1042/CS20050369 525

R E V I E W

Molecular biology of human papillomavirus

infection and cervical cancer

John DOORBAR
Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.

A B S T R A C T

HPVs (human papillomaviruses) infect epithelial cells and cause a variety of lesions ranging from
common warts/verrucas to cervical neoplasia and cancer. Over 100 different HPV types have
been identified so far, with a subset of these being classified as high risk. High-risk HPV DNA is
found in almost all cervical cancers (> 99.7 %), with HPV16 being the most prevalent type in both
low-grade disease and cervical neoplasia. Productive infection by high-risk HPV types is manifest
as cervical flat warts or condyloma that shed infectious virions from their surface. Viral genomes
are maintained as episomes in the basal layer, with viral gene expression being tightly controlled
as the infected cells move towards the epithelial surface. The pattern of viral gene expression in
low-grade cervical lesions resembles that seen in productive warts caused by other HPV types.
High-grade neoplasia represents an abortive infection in which viral gene expression becomes
deregulated, and the normal life cycle of the virus cannot be completed. Most cervical cancers
arise within the cervical transformation zone at the squamous/columnar junction, and it has
been suggested that this is a site where productive infection may be inefficiently supported. The
high-risk E6 and E7 proteins drive cell proliferation through their association with PDZ domain
proteins and Rb (retinoblastoma), and contribute to neoplastic progression, whereas E6-mediated
p53 degradation prevents the normal repair of chance mutations in the cellular genome. Cancers
usually arise in individuals who fail to resolve their infection and who retain oncogene expression
for years or decades. In most individuals, immune regression eventually leads to clearance of the
virus, or to its maintenance in a latent or asymptomatic state in the basal cells.

HPVs (HUMAN PAPILLOMAVIRUSES)
AND DISEASE
HPVs cause a diverse range of epithelial lesions. Over 100
different HPV types have been identified based on DNA
sequence analysis [1], with each being associated with
infection at specific epithelial sites [2]. At an evolutionary
level, HPVs fall into a number of distinct groups or
genera (Figure 1), and the lesions they cause have different
characteristics.
The two main HPV genera are the Alpha and Beta
papillomaviruses, with approx. 90 % of currently char-
acterized HPVs belonging to one or other of these
groups. Beta papillomaviruses are typically associated
with inapparent cutaneous infections in humans but,
in immunocompromised individuals and in patients

Key words: cervical cancer, epithelial cell, human papillomavirus, immunity, infection, neoplasia.
Abbreviations: ARF, ADP-ribosylation factor; BPV, bovine papillomavirus; CIN, cervical intraepithelial neoplasia; COPV, canine
oral papillomavirus; DAPI, 4,6-diamidino-2-phenylindole; Dlg, the Drosophila discs large protein; E6AP, E6-associated protein;
EV, epidermodysplasia cerruciformis; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial neoplasia lesions;
Hsp, heat-shock protein; hTERT, human telomerase reverse transcriptase; LSIL, low-grade squamous intraepithelial neoplasia
lesions; MCM, multicopy maintenance; ORF, open reading frame; PCNA, proliferating-cell nuclear antigen; PML, promyelocytic
leukaemia; Rb, retinoblastoma; pRb, Rb protein; ROPV, rabbit oral papillomavirus; SCC, squamous cell carcinoma; URR, upstream
regulatory region.
Correspondence: Dr John Doorbar (email jdoorba@nimr.mrc.ac.uk).

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526 J. Doorbar
Figure 1 HPVs and their association with cervical disease
(A) HPVs are contained within five evolutionary groups. HPV types that infect
the cervix come from the Alpha group which contains over 60 members. HPV
types from the Beta, Gamma, Mu and Nu groups primarily infect cutaneous sites.
(B) The Alpha papillomaviruses can be subdivided into three categories (high risk,
low risk and cutaneous), depending on their prevalence in the general population
and on the frequency with which they cause cervical cancer (shown in the right-
most column). High-risk types come from the Alpha 5, 6, 7, 9 and 11 groups. The
frequency with which the different HPV types are found in cervical cancers (squam-
ous cell carcinoma and adenocarcinoma/adenosquamous carcinoma) is shown in
the central columns (based on information contained in [170]). Where no percent-
age is listed, the HPV type is not generally associated with cervical cancer.
suffering from the inherited disease EV (epidermodys-
plasia verruciformis), these viruses can spread unchecked
and become associated with the development of non-


C 2006 The Biochemical Society
melanoma skin cancer [3,4]. EV patients carry mutations
in their TMC6 (previously known as EVER1) or TMC8
(previously known as EVER2) genes, which renders them
susceptible to these viruses [5,6].
The largest group of HPVs comprise the Alpha papi-
llomaviruses, and it is this group that contains the genital/
mucosal HPV types (Figure 1). The Alpha papilloma-
viruses also include cutaneous viruses such as HPV2,
which cause common warts, and which are only very
rarely associated with cancers [7]. More than 30 different
HPV types are known to infect cervical epithelium, with
a subset of these being associated with lesions that can
progress to cancer. These cancer-associated HPVs are
classified as high-risk HPV types. HPV16 is the most
prevalent high-risk HPV in the general population, and
is responsible for approx. 50 % of all cervical cancers. The
remaining mucosal types are classified as intermediate or
low risk depending on the frequency with which they are
found in cancers. Low-risk HPV types, such as HPV11,
are associated with cervical cancer only very rarely,
but are still medically important because they cause
genital warts. Genital warts are a major sexually trans-
mitted disease in many countries and can affect 1–2 % of
young adults [8].
The remaining HPVs come from three genera
(Gamma, Mu and Nu) and generally cause cutaneous
papillomas and verrucas that do not progress to cancer
(but see [9,10]). The relationship between the different
genera of HPVs, and in particular the high-risk HPV
types associated with cervical cancer, is shown in Figure 1.
NORMAL PRODUCTIVE INFECTION
Many HPV types produce only productive lesions fol-
lowing infection and are not associated with human
cancers. In such lesions, the expression of viral gene prod-
ucts is carefully regulated, with viral proteins being
produced at defined times and at regulated levels as the
infected cell migrates towards the epithelial surface.
The events that lead to virus synthesis in the upper
epithelial layers appear common to both the low- and
high-risk HPV types, with protein expression patterns
in low-grade CIN (cervical intraepithelial neoplasia) 1
resembling the patterns found in benign warts caused
by other papillomaviruses. Productive infection can be
divided into distinct phases, with the different viral
proteins playing specific roles.
Establishment of HPV infection
All papillomaviruses share a number of characteristics
and contain double-stranded circular DNA within an
icosahedral capsid (Figure 2). Although the viral genome
can vary slightly in size between different HPV types,
it typically contains around 8000 bp [7904 bp for HPV16
(GenBank® accession number NC 001526)], and encodes

eight or nine ORFs (open reading frames) (Figure 2). The
virus shell is made up of two coat proteins. The L1 protein
is the primary structural element, with infectious virions
containing 360 copies of the protein organized into 72
capsomeres [11]. L2 is a minor virion component, and
it is thought that a single L2 molecule may be present
in the centre of the pentavalent capsomeres at the virion
vertices [11,12]. Both proteins play an important role in
mediating efficient virus infectivity.
Infection by papillomaviruses requires that virus part-
icles gain access to the epithelial basal layer and enter the
dividing basal cells. At present there is some controversy
as to the precise nature of the receptor for virus entry
[13], but it is thought that heparan sulphate proteoglycans
may play a role in initial binding and/or virus uptake
[13–15]. As with other viruses [16,17], it seems that HPV
infection requires the presence of secondary receptors for
efficient infection, and it has been suggested that this role
may be played by the α6 integrin [18–20]. Papillomavirus
particles are taken into the cell relatively slowly following
binding [21] and, for HPV16, this occurs by clathrin-
coated endocytosis [22]. This mode of entry may not be
conserved among all HPV types, however, and it has been
suggested that HPV31 may gain entry via caveolae [23].
Papillomavirus particles disassemble in late endosomes
and/or lysosomes, with the transfer of viral DNA to
the nucleus being facilitated by the minor capsid protein
L2 [22,24]. In experimental systems, viral transcripts can
be detected as early as 12 h post-infection, with mRNA
levels increasing over the course of several days [24].
Infection leads to the establishment of the viral genome
as a stable episome (without integration into the host cell
genome) in cells of the basal layer, and this is thought to
require expression of the viral replication proteins, E1 and
E2. The E2 protein plays several roles during productive
infection and, in basal cells, is required for the initiation
of viral DNA replication and genome segregation. E2 is
a DNA-binding protein that recognizes a palindromic
motif [AACCg(N4 )cGGTT] in the non-coding region of
the viral genome ([25], and Figure 2). HPV16 has four
such motifs, one of which lies adjacent to the viral origin
of replication. E2 binding is necessary for the recruit-
ment of the E1 helicase to the viral origin, which binds
in turn to cellular proteins necessary for DNA repli-
cation, including RPA (replication protein A) and DNA
polymerase α primase [26–29]. The E2 protein sub-
sequently dissociates from the viral origin, allowing the
assembly of E1 into a double hexameric ring that func-
tionally resembles the hexameric ring structures that
assemble at cellular replication origins and which are
built from the cellular MCM (multicopy maintenance)
proteins.
In the basal cells, it appears that the viral genome
replicates with the cellular DNA during S-phase, with
the replicated genomes being partitioned equally during
cell division. The role of E2 in anchoring viral episomes
Papillomavirus molecular biology 527
to mitotic chromosomes is critical for correct segregation
[30] and, in some papillomavirus types, involves the
cellular Brd4 protein, which directly associates through
its C-terminus with the viral E2 protein [30,31]. For
the high-risk HPV types that are associated with human
cancers, association appears to be via the spindle, with
additional cellular proteins being involved. In addition
to its role in replication and genome segregation, E2 can
also act as a transcription factor and can regulate the viral
early promoter (p97 in HPV16; p99 in HPV31) and con-
trol expression of the viral oncogenes (E6 and E7). At
low levels, E2 acts as a transcriptional activator, whereas
at high levels E2 represses oncogene expression by dis-
placing SP1 transcriptional activator from a site adjacent
to the early promoter (Figure 2). It is unclear whether
E2-mediated transcriptional regulation is important in
basal epithelial cells, where the nucleosomal structure
and/or methylation status of the viral DNA may not
be compatible with efficient activation of the early pro-
moter [32,33]. The analysis of genome maintenance in
BPV (bovine papillomavirus)-transformed keratinocytes
and cell lines derived from cervical lesions [34,35] has
suggested that viral episomes may be maintained at 10–
200 copies in basal cells, although, as yet, this has not been
adequately demonstrated in lesions. Knockout mutants in
the viral genome have suggested that both E1 and E2 are
required for viral genome maintenance in the basal layer
[36,37].
Stimulation of cell proliferation
A number of model systems have been used to examine
the papillomavirus productive cycle during in vivo infec-
tion. Following experimental inoculation of mucosal
epithelial tissue by ROPV (rabbit oral papillomavirus)
or COPV (canine oral papillomavirus), an increase in
cell proliferation in the basal and suprabasal cellular
compartments is readily apparent, with mature warts
becoming visible by 4 weeks post-infection [38,39]. The
time between initial infection and the appearance of pro-
ductive papillomas can vary depending on the titre of
the infecting virus and possibly also on the nature of the
infecting papillomavirus type, and it has been suggested
that latency may result when inoculating titres are low
[40,41]. In cervical lesions caused by HPVs, the increased
proliferation of suprabasal epithelial cells is attributed
to the expression of the viral oncogenes, E6 and E7.
During natural infection, the activity of these genes
allows the small number of infected cells to expand, in-
creasing the number of cells that subsequently go on to
produce infectious virions. The ability of E6 and E7
to drive cells into S-phase is also necessary, along with
E1 and E2, for the replication of viral episomes above
the basal layer. Suprabasal cells normally exit the cell
cycle and begin the process of terminal differentiation in
order to produce the protective barrier that is normally
provided by the skin [42]. In HPV-infected keratinocytes,


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528 J. Doorbar

Figure 2 Organization of the HPV genome and the virus life cycle

(A) The HPV16 genome (7904 bp) is shown as a black circle with the early (p97) and late (p670) promoters marked by arrows. The six early ORFs [E1, E2, E4 and
E5 (in green) and E6 and E7 (in red)] are expressed from either p97 or p670 at different stages during epithelial cell differentiation. The late ORFs [L1 and L2
(in yellow)] are also expressed from p670, following a change in splicing patterns, and a shift in polyadenylation site usage [from early polyadenylation site (PAE)
to late polyadenylation site (PAL)]. All the viral genes are encoded on one strand of the double-stranded circular DNA genome. The long control region (LCR from
7156–7184) is enlarged to allow visualization of the E2-binding sites and the TATA element of the p97 promoter. The location of the E1- and SP1-binding sites is
also shown. (B) The key events that occur following infection are shown diagrammatically on the left. The epidermis is shown in colour with the underlying dermis
being shown in grey. The different cell layers present in the epithelium are indicated on the left. Cells in the epidermis expressing cell cycle markers are shown with
red nuclei. The appearance of such cells above the basal layer is a consequence of virus infection, and in particular, the expression of the viral oncogenes, E6 and
E7. The expression of viral proteins necessary for genome replication occurs in cells expressing E6 and E7 following activation of p670 in the upper epithelial layers
(cells shown in green with red nuclei). The L1 and L2 genes (yellow) are expressed in a subset of the cells that contain amplified viral DNA in the upper epithelial

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 Papillomavirus molecular biology 529

however, the restraint on cell-cycle progression is lost and
normal terminal differentiation does not occur [43]. The
basic mechanism by which papillomaviruses stimulate
cell-cycle progression is well known and is similar to the
way that other tumour viruses deregulate cell growth.
E7 associates with pRb [Rb (retinoblastoma) protein]
and other members of the pocket protein family and
disrupts the association between pRb and the E2F family
of transcription factors, irrespective of the presence of
external growth factors (Figure 3). E2F subsequently
transactivates cellular proteins required for viral DNA
replication such as cyclins A and E. E7 also associates with
other proteins involved in cell proliferation, including
histone deacetylases [44], components of the AP1 tran-
scription complex [45] and the cyclin-dependent kinase
inhibitors p21 and p27 [46]. During natural infection,
however, the ability of E7 to drive cell proliferation is
inhibited in some cells, depending on the levels of the p21
and p27 cyclin-dependent kinase inhibitors (Figure 3).
High levels of p21 and p27 in differentiating keratinocytes
can lead to the formation of inactive complexes with E7
and cyclinE within the cell. It appears that the ability of
E7 to drive cells through mitosis in differentiating epi-
thelium may be limited to those cells which express p21
and p27 at low level or which express sufficient E7 to
overcome the block to cell-cycle progression [47]. This is
important given that deregulated expression of the viral
oncogenes is a predisposing factor in the development
of HPV-associated cancers (Figure 3). The function of
the viral E6 protein complements that of E7 and, in the
high-risk HPV types, the two proteins are expressed
together from a single polycistronic mRNA species [48].
A primary role of E6 is its association with p53 which, in
the case of the high-risk HPV types, mediates p53 ubi-
quitination and degradation. This is thought to prevent
growth arrest or apoptosis in response to E7-mediated
cell-cycle entry in the upper epithelial layers, which might
otherwise occur through activation of the ARF (ADP-
ribosylation factor) pathway (see Figure 3). The general
role of E6 as an anti-apoptotic protein is emphasized
further by the finding that it also associates with Bak [49]
and Bax [50]. This role of E6 is of key significance in the
development of cervical cancers (discussed in more detail
below), as it compromises the effectiveness of the cellular
DNA damage response and allows the accumulation of
secondary mutations to go unchecked. The E6 protein
of the high-risk HPV types also plays a role in mediating
cell proliferation independently of E7 through its C-
terminal PDZ ligand domain [the name PDZ is derived
from the first three proteins in which these domains
were found: PSD-95 (a 95 kDa protein involved in
signalling), Dlg (the Drosophila discs large protein), and
ZO1 (the zonula occludens 1 protein which is involved in
maintaining epithelial cell polarity)] [51]. E6 PDZ binding
can mediate suprabasal cell proliferation [52,53] and may
contribute to the development of metastatic tumours by
disrupting normal cell adhesion. In productive lesions,
cells are driven into cycle only in the lower epithelial
layers and extend towards the epithelial surface to varying
extents depending on lesion grade and the nature of the
infecting HPV type [54]. In benign warts caused by
HPV1, proliferating cells are restricted to the epithelial
basal layer, with genome amplification beginning as soon
as the infected cell enters the suprabasal cell layers. HPVs,
such as HPV6 and HPV16 (Alpha papillomaviruses),
typically have a region that may be many cell layers thick,
where cells are retained in cycle prior to the onset of
vegetative viral genome amplification [54]. In the case
of the high-risk HPV types that are associated with
cervical cancer, the relative thickness of these layers
increases with the grade of neoplasia, while the extent
of epithelial differentiation decreases.
Genome amplification
Although cell proliferation is required for lesion form-
ation and the maintenance of viral episomes, all papi-
llomaviruses must eventually amplify and package their
genomes if infectious virions are to be produced. What
triggers the onset of late events is not yet fully understood,
but appears to depend in part on changes in the cellular
environment as the infected cell moves towards the
epithelial surface. Critical for this is the up-regulation
of the differentiation-dependent promoter, which for
many HPV types is contained within the E7 ORF (P670
in HPV16; p742 in HPV31) [55,56]. The activation of the
differentiation-dependent promoter depends on changes
in cellular signalling, rather than on genome ampli-
fication [55,56], and leads to an increase in the level of
viral proteins necessary for replication (i.e. E1, E2, E4
and E5). Cells supporting productive infection can be
visualized using antibodies to E4 (Figure 4), whereas
those supporting genome amplification also contain E7
[57].

layers. Cells containing infectious particles are eventually shed from the epithelial surface (cells shown in green with yellow nuclei). In cutaneous tissue, this follows
nuclear degeneration and the formation of flattened squames. The timing and extent of expression of the various viral proteins are summarized using arrows at the
right of the Figure. The consequence of expressing viral gene products in this ordered way is shown on the far right. The expression of E6 and E7 in the presence of
low levels of E1, E2, E4 and E5 allows maintenance of the viral genome (genome maintenance/cell proliferation). Elevation in the levels of these replication proteins
facilitates viral genome amplification. The first appearance of L2 allows genome packaging to begin, with the expression of L1 allowing the formation of infectious
virions (virus assembly). The accumulation of E4 close to the epithelial surface may improve the efficiency of virus release.

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530 J. Doorbar

Figure 3 Stimulation of cell-cycle progression by high-risk HPV types

HPV infection leads to deregulation of the cell cycle. Regulation of protein expression in uninfected epithelium is shown in (A). In the presence of high-risk HPV
(B), the regulation of proteins necessary for cell proliferation is altered, allowing HPV to stimulate S-phase entry in the upper epithelial layers. (A) Uninfected epi-
thelium. The expression of proteins necessary for cell-cycle progression is controlled by pRB, which in non-cycling cells associates with members of the E2F transcription
factor family (centre). In the presence of growth factors, cyclinD/Cdk4/6 is activated, which leads to Rb phosphorylation and the release of the transcription factor
E2F, which drives the expression of proteins involved in S-phase progression. p16 regulates the levels of active cyclinD/Cdk in the cell, providing a feedback mechanism
that regulates the levels of MCM, PCNA (proliferating-cell nuclear antigen) and cyclinE. p14Arf , whose expression is directly linked to that of p16, regulates the activity
of the MDM (murine double minute) ubiquitin ligase, which maintains p53 at a level below that required for cell cycle arrest and/or apoptosis. (B) HPV-infected
epithelium. In cervical epithelium infected by high-risk HPV types, progression through the cell cycle is not dependent on external growth factors, but is stimulated
by the E7 protein, which binds and degrades pRB and facilitates E2F-mediated expression of cellular proteins necessary for S-phase entry. Although p16 levels rise,
normal feedback is by-passed, as HPV-mediated cell proliferation is not dependent on cyclinD/Cdk4/6. The rise in the level of p14Arf , which occurs in the absence
of p16-mediated feedback, leads to the inhibition of MDM function and an increase in the level of p53. This is countered by E6, which associates with the E6AP
ubiquitin ligase in order to stimulate the degradation of the p53 protein and prevent growth arrest and/or apoptosis. In low-grade cervical disease, where E7 levels
are carefully regulated, it is thought that E7-mediated cell proliferation can sometimes be inhibited as a result of association with p21 and cyclin E/Cdk. The high
levels of E7 found in cervical cancer cells are thought to overcome this block by binding and inactivating the Cdk inhibitor p21.

The role of E1 and E2 in viral genome amplification
is well established. E1 is highly conserved among papi-
llomaviruses and has a weak affinity for a consensus
motif (AACNAT) repeated 6 times in the viral origin.
During natural infection, E1 is expressed at very low
levels and requires the presence of E2 in order to be effi-
ciently targeted to its binding sites. E2 associates with
E1 primarily through its N-terminus and binds to DNA
as a dimer through its C-terminus [58,59] (Figure 2).
The formation of an E1–E2 complex at the origin of
replication induces localized distortion in the viral DNA,
which facilitates the recruitment of additional E1 mol-
ecules and the eventual displacement of E2 [60]. E1 can
also associate with cellular Hsps (heat-shock proteins),
in particular Hsp40 and Hsp70, and this contributes to
the formation of E1 dihexamers [61]. E1 and E2 also act
to regulate the viral early promoter (p97 in HPV16 and
p99 in HPV31), with high levels of E2 acting to down-
regulate the expression of E6 and E7 in experimental
systems. The ability of E2 to either repress or activate
early viral gene expression according to its abundance
is thought to result from differences in the affinity of
E2 for its various binding sites [62]. In HPV16, it is
thought that binding site 4 is the primary site that is occu-
pied when E2 is present at low levels and that binding to
this site and to binding site 3 (Figure 2) leads to pro-
moter activation [63]. As E2 increases in abundance,
occupancy of the remaining sites leads to the displacement

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Figure 4 Characterization of events during productive
papillomavirus infection by immunostaining
The pattern of viral protein expression is conserved in productive lesions caused by
papillomaviruses of diverse origins. Typical immunofluorescence staining patterns
are shown. (A) Productive papilloma showing the typical expression profile of PCNA
[a surrogate marker of E6/E7 gene expression (in red)] and E4 (in green), which
is thought to reflect the sites of expression of E1 and E2. Nuclei are counter-
stained with DAPI (4,6-diamidino-2-phenylindole; in blue). A small area of un-
infected tissue is apparent on either side of the papilloma. (B) Comparison of
the sites of viral genome amplification [in red; detected by FISH (fluorescence
in situ hybridization)] and E4 expression (in green) reveal that the two events cor-
relate very closely. Nuclei are counterstained with DAPI (blue). (C) The viral capsid
protein (L1; in red) is expressed in a subset of the cells that express E4 (in green)
in the upper epithelial layers. Nuclei are counterstained with DAPI (in blue).
of basal transcription factors, such as Sp1 and TBP
(TATA-box-binding protein), that are necessary for pro-
moter activation [64]. It appears that the increase in E2
Papillomavirus molecular biology 531
expression that is important in stimulating viral genome
amplification will lead eventually to the down-regulation
of E6/E7 expression and to the eventual loss of the
replicative environment necessary for viral DNA syn-
thesis. This curious link between replication and tran-
scription provides a mechanism by which the virus can
limit the timing and duration of genome amplification. In
cervical lesions caused by HPV16, cells supporting viral
genome amplification are often scarce and vary greatly
in their location within the lesion [57]. By contrast, in
verrucas caused by HPV1, cells supporting genome
amplification are relatively prevalent and are consistently
found immediately above the basal layer.
Although E1 and E2 are key players in viral genome
amplification, the viral E4 and E5 proteins also contribute
[65–69]. E5 mutant genomes of HPV16 and 31 exhibit
lower levels of genome amplification than wild type,
and it is thought that the ability of E5 to modulate cell
signalling is responsible for this. HPV E5 is a trans-
membrane protein that resides predominantly in the ER
(endoplasmic reticulum), but which can associate with
the vacuolar proton ATPase and delay the process of
endosomal acidification [70–72]. It is thought that this
affects the recycling of growth factor receptors on the cell
surface, leading to an increase in EGF (epidermal growth
factor)-mediated receptor signalling and the maintenance
of a replication competent environment in the upper
epithelial layers [73]. By contrast, the role of E4 in
genome amplification is not yet fully established. E4
accumulates in the cell at the time of viral genome ampli-
fication, and its loss has been shown to disrupt late events
in a number of experimental systems [67–69]. Part of
the effect may be related to the ability of certain HPV
types to associate with cyclinB/Cdk2 and to relocate
the complex to the cytoplasm [74,75]. This prevents the
nuclear accumulation of cyclinB/Cdk2, which is neces-
sary for progression through mitosis. The ability of E4
to cause cell-cycle arrest in G2 and to antagonize E7-
mediated cell proliferation is a common feature of the
E4 proteins encoded by several HPV types, including
HPV16, HPV11 [75], HPV18 [76] and HPV1 [77]. Inter-
estingly, recent work has suggested that the E4 proteins
of HPV16 and HPV18 can also associate with E2, which
suggests an additional mechanism by which E4 may act
[78].
Virus assembly and release
The final stage in the papillomavirus productive cycle
requires that the replicated genomes are packaged into
infectious particles. Capsid proteins (L1 and L2) accumu-
late after the onset of genome amplification, with L2
expression preceding the expression of L1 [79,80]. The
events that link genome amplification to the synthesis
of the capsid proteins are not yet fully understood, but
are dependent on changes in mRNA splicing and on
the generation of transcripts that terminate at the late


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532 J. Doorbar
(rather than the early) polyadenylation site (Figure 2).
During epithelial cell differentiation, the timing of capsid
synthesis is regulated both at the level of RNA processing
and at the level of protein synthesis [81–83]. Negative
regulatory elements that control RNA stability are
present in the coding regions and in the late untranslated
region of HPV16 [84,85], whereas a splicing silencer in
the HPV16 L1 gene leads to the preferential synthesis
of early transcripts in proliferating cells [86]. In addition
to this, the pattern of codon usage within the HPV16
L1 and L2 genes is distinct from the pattern usually
found in mammalian cells, which contributes further to
the inhibition of capsid expression in the lower epithelial
layers [82,87,88]. Similar regulatory mechanisms are also
thought to exist in HPV1 [89], HPV31 [90] and BPV [91]
and are likely to extend to other papillomavirus types.
The assembly of infectious virions in the upper epi-
thelial layers is thought to require E2 in addition to
the capsid proteins L1 and L2 [92,93], and it has been
suggested that E2 may improve the efficiency of genome
encapsidation during natural infection [93,94]. L2 local-
izes to the nucleus by virtue of nuclear localization
signals located at its N- and C-termini and, once there, it
associates with PML (promyelocytic leukaemia) bodies.
Although some papillomavirus L2 proteins can associate
directly with DNA [95], the specific recruitment of
viral genomes to PML bodies is thought to require E2,
which can associate with viral DNA through its specific
recognition sites. L1 assembles into capsomeres in the
cytoplasm prior to nuclear relocation and is recruited into
PML bodies only after L2 has bound and has displaced
the PML component sp100 [79]. The assembly of virus-
like particles in experimental systems requires the pres-
ence of the cellular protein Hsp70 [96], which is also
involved in recycling the viral E1 protein during repli-
cation [97], but does not appear to be strictly dependent
on PML localization [98]. Although papillomavirus part-
icles can assemble in the absence of L2, its presence con-
tributes to efficient packaging [99] and enhances virus
infectivity [100]. Loss of L2 in the context of the HPV31
genome results in a 10-fold reduction in packaging effi-
ciency and a 100-fold reduction in virus infectivity when
compared with wild-type HPV31 [101]. L2 associates
with L1 through a hydrophobic region near the C-ter-
minus of the protein that is thought to insert into the
central hole in the pentavalent L1 capsomeres [102].
The interaction between capsomeres requires the C-
terminus of the L1 protein [11] with virus maturation and
stabilization occurring as the infected cells approach the
epithelial surface as a result of disulphide cross-linking.
Although papillomaviruses are resistant to desiccation
[103], it is thought that their survival may be enhanced
by being shed from the epithelial surface as a cornified
squame [104]. The retention of papillomavirus antigens
until the infected cell reaches the epithelial surface is
thought to limit the ability of the immune system to


C 2006 The Biochemical Society
detect infection. Ultimately, virus release requires effi-
cient escape from the cornified envelope at the cell surface,
which may be facilitated by the E4 protein. E4 can disrupt
the keratin network [105,106] and can affect the integrity
of the cornified envelope [104,107].
ABORTIVE INFECTION AS A PRECURSOR
TO CANCER
Virus-induced cancers often arise at sites where pro-
ductive infection cannot be properly supported. CRPV
(cottontail rabbit papillomavirus) induces productive
papillomas in its natural host (the cottontail rabbit),
but gives rise to lesions that cannot support virus syn-
thesis when inoculated into domestic rabbits. Such abor-
tive infections progress to cancer more frequently than
do infections in cottontails. A similar situation is seen
following the inoculation of SV40 (simian virus 40) and
adenovirus type 5 (which are also considered to be
DNA tumour viruses) into inappropriate hosts, such
as hamsters and rats, which support early, but not late,
viral gene expression. The papillomavirus types that
usually cause benign cutaneous warts in humans and are
considered low risk (e.g. HPV2 and 4) can be associated
occasionally with cancers at mucosal sites. The restricted
tissue tropism of the different HPV types, coupled with
their ability to infect non-optimal sites, may provide a
partial explanation as to why otherwise benign HPV
types are occasionally associated with human cancers.
The high-risk papillomavirus types that are associated
with human cancers more frequently come predomin-
antly from the Alpha 9 and Alpha 7 groups (see Figure 1),
with HPV16 and HPV18 being the most prevalent
types [108]. These viruses are found in women with no
cytological abnormalities, as well as in women with LSIL
and HSIL (low- and high-grade squamous intraepithelial
neoplasia lesions respectively) and/or cancer [109–111].
By PCR, HPV16 DNA is apparent in approx. 26 %
of LSIL [112], but can be seen in as many as 63 % of
SCCs (squamous cell carcinomas) of the cervix [113].
HPV18 is the second most common HPV type associated
with this disease (causing 10–14 % of all cases of
cervical SCCs), but is the primary HPV type associated
with cervical adenocarcinoma, causing 37–41 % (HPV16
causes 26–37 % of all such cases [113]). The prevalence of
high-risk HPV infection amongst young women (mean
age, 25 years) is typically approx. 20–40 % depending
on geographical location [111,114], with the incidence
declining with age as infections are either resolved or
brought under control by the host immune system. HPV
infections are very common in this age group, with
cumulative incidence of infection over 5 years being as
high as 60 % in some populations [110]. Most women
(80 %) infected with a specific HPV type will, however,
show no evidence of that type after 18 months, and it
 Papillomavirus molecular biology 533

Figure 5 Changes in expression patterns that accompany progression to cervical cancer

During cancer progression, the pattern of viral gene expression changes. In CIN1 (LSIL), the order of events is generally similar to that seen in productive lesions
(shown diagrammatically on the left). In CIN2 and CIN3, however, the onset of late events is retarded, and although the order of events remains the same, the
production of infectious virions becomes restricted to smaller and smaller areas close to the epithelial surface. Integration of HPV sequences into the host cell genome
can accompany these changes and can lead to further deregulation in the expression of E7 (and the loss of the E1 and E2 replication proteins). In cervical cancer
(shown on the right), the productive stages of the virus life cycle are no longer supported and viral episomes are usually lost.

Figure 6 Detection of viral proteins in cervical intraepithelial neoplasia

(A) The expression of surrogate markers of E7 (in this case MCM; in red) and E4 (in green) in HPV16-infected cervix resembles the pattern of expression seen in
productive papillomas caused by other papillomavirus types (see also Figure 4). Nuclei are counterstained with DAPI (in blue) and uninfected tissue is apparent either
side of the area of infection. (B) During cancer progression, the expression of surrogate markers of E7 (in this case MCM; in red) extends towards the epithelial surface
and the expression of E4 is restricted to isolated pockets close to the upper epithelial layers. CIN3 is shown on the left, whereas the pattern seen in many productive
CIN1 lesions is shown on the right. Nuclei are counterstained with DAPI (in blue).

is generally thought that re-infection by the same HPV
type is uncommon [115]. The development of high-
grade cervical neoplasia arises in women who cannot
resolve their infection and who maintain persistent
active infection for years or decades following initial
exposure. In such women, the continuous stimulation
of S-phase entry and cell proliferation by E7, coupled
with the loss of p53-mediated DNA repair pathways as
a result of E6 expression, allows the accumulation of
secondary point mutations in the cellular genome that
eventually lead to cancer. Previous work from a num-
ber of laboratories has suggested that not all cervical
cancers develop through this route, however, and that, in
some instances rapid onset of HSIL can arise after initial
infection [110,116]. Given the prevalence of genital HPV
types in the general population and the high life-time risk
of infection (estimated at approx. 80 %), the incidence
of cervical cancers is very low [typically approx. 0.03 %
in the absence of screening (0.018–0.044 %)] with most
infections being successfully resolved [117].
Low-grade cervical lesions (i.e. LSIL or grade 1 CIN)
caused by high- and low-risk HPV types are similar to
productive infections in the pattern of viral gene expres-
sion, and viral coat proteins can usually be detected in
cells at the epithelial surface [57] (Figure 5). High-grade
lesions (HSIL or grade 2 or 3 CIN) have a more extensive
proliferative phase, with the productive stages of the virus
life cycle being supported only poorly (Figures 5 and 6).
It has been estimated that approx. 20 % of CIN1 will
progress to CIN2, and that approx. 30 % of these lesions
will progress to more severe neoplasia if left untreated.
Approx. 40 % of CIN3 lesions can progress to cancer
[118], with cervical neoplasia generally arising within
the cervical transformation zone where the columnar
cells of the endocervix meet the stratified squamous
epithelial cells of the ectocervix. The extent of columnar

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534 J. Doorbar
and stratified epithelium in this region changes markedly
during a woman’s life, and it is in this region of change that
most cervical neoplasias develop. It has been suggested
that the transformation zone may be a sub-optimal site
for completion of the productive life cycle of high-risk
HPV types such as HPV16. Interestingly, high-risk HPV
infections can be found at many sites in the anogenital
tract, including the vagina, vulva and penis, but, despite
this distribution, the incidence of cancer at these sites is
generally low (0.001 %). Only at the anus, in men who
have sex with men, does the incidence of HPV-associated
cancer rise to levels that are similar to those found at the
cervix (0.035 %) [119], and it is interesting that at both
these susceptible sites there is a transformation zone. In
the cervix, the reserve cells of the transformation zone
eventually form the basal cells of a stratified squamous
epithelium, and the higher frequency of cancers at such
sites may reflect the fact that the virus can access these
basal cells more readily than those that are protected
from infection by a permanent stratified epithelial layer.
Despite this possibility, it appears that the transformation
zone may in fact be an epithelial site where high-risk HPV
types cannot properly regulate their productive cycle,
and that variation both in the level and in the timing of
expression of viral proteins may underlie the development
of cancers at these sites.
MOLECULAR EVENTS DURING CANCER
PROGRESSION
The identification of cervical lesions as flat condyloma,
LSIL, HSIL or invasive cervical cancer reflects molecular
changes in the normal programme of epithelial cell differ-
entiation that occur following infection. Virus production
at the epithelial surface depends on the ordered and timely
expression of viral gene products (Figure 2) [54,57],
with the timing of such events becoming progressively
disturbed during neoplastic progression (Figures 5 and
6). In HSIL, viral genome amplification occurs closer
to the epithelial surface than in condylomas and LSIL,
and the expression of viral coat proteins is retarded
[57] (Figure 5). The molecular bases for these changes
are not fully understood, but may in some instances
reflect changes in the levels of E6 and E7 expression
that occur following integration of the viral genome
into the host cell chromosome. Integrated HPV DNA
is found in most invasive cancers and in a subset of
high-grade lesions [120,121], but can also be found in
some CIN1 lesions, and it has been suggested that
integration may be an early event in cancer progression
[122]. Indeed, p16INK4A expression, which is considered
a marker of elevated E7 expression, can be detected
in some CIN1 lesions, as well as in CIN2 and CIN3
lesions that show evidence of integration [123,124].
Integration of the HPV genome into the host cell


C 2006 The Biochemical Society
chromosome is a critical event in the development of most
cervical cancers and, although this can occur randomly
throughout the genome, several studies have indicated
a preference for integration at common fragile sites
and have suggested that changes in the expression of
genes at or near the integration site may participate
in cancer development [125]. It is clear, however, that
integration (which often results in the loss of E2) can
lead to the deregulation of E6/E7 expression, and that
this is critical for the enhanced growth characteristics
of cervical cancer cells. The requirement of these genes
for the maintenance of the cancer phenotype is shown
most dramatically by studies that have aimed to inhibit
viral oncogene activity in cervical cancer cells. Cell lines
such as HeLa and SiHa, which have been grown in the
laboratory for many decades, will undergo apoptotic
cell death in the presence of molecules that inhibit
E6 function [126–128]. Similar results are obtained
following the reintroduction of E2 into such cell lines,
which suppresses the expression of the viral oncogenes
by binding to the URR (upstream regulatory region),
preventing continued cell proliferation [129,130]. The
ubiquitous retention of the E6/E7 region of the viral
genome following integration into the host chromosome
is usually accompanied by the loss or disruption of viral
sequences encoding most of E1, as well as E2 and E4. The
viral E2 protein has a negative effect on cell proliferation
by regulating the viral URR and can also cause cell-cycle
arrest at the G2 phase of the cell cycle [131,132]. Similarly,
the E4 protein of HPV16 is able to inhibit mitosis
by preventing the nuclear localization of cyclinB/Cdk1,
and it is not surprising therefore that the expression of
these proteins is abrogated in HPV-associated cancers. In
addition to the loss of regulatory proteins, integration
also leads to the loss of sequences at the 3 -end of the
viral early transcripts that can suppress the production
of viral mRNA species encoding E6 and E7 [133–
135], contributing further to the deregulation of viral
oncogene expression. Although integrated HPV DNA
is found in the vast majority of cervical cancers, other
factors are likely to influence the development of the
precancerous changes seen in squamous intraepithelial
neoplasia. These include exposure to glucocorticoids and
progesterones, which can affect viral oncogene expression
[136–138], and the regulation of viral gene expression by
DNA methylation and chromatin organization [139].
Both factors can also regulate expression from integrated
sequences and, in instances where integration occurs as
concatamers, it is often only one copy of the viral genome
that is transcriptionally active [140].
Although many HPV types can infect the cervix, only
the high-risk HPV types are consistently associated with
cervical cancers because of the specific activity of their
oncogenes. HPV16 gene expression facilitates integration
of foreign DNA into the host cell chromosome, which
may be a consequence of the increased level of host

genome instability in these cells [141]. The high-risk E7
proteins, but not the E7 proteins encoded by the low-
risk HPV types, induce centrosomal abnormalities in cell
culture and in transgenic animals [142,143], and it has
been suggested that high-risk E7 may act as a mitotic
mutator, which acts to increase the chance of errors
during each round of cell division [144]. Although the
molecular basis for E7-mediated genome instability is not
fully understood, it appears in part to be independent of
the well-characterized association of E7 with the pocket
proteins Rb, p107 and p130. The association of E7 with
these proteins does, however, contribute to the ability of
E7 to stimulate cell proliferation, with the high-risk E7
proteins binding Rb more efficiently than the E7 protein
of the low-risk HPV types [145,146]. The high-risk E7
proteins are also capable of mediating Rb degradation
through a proteosome-dependent mechanism [147,148],
which is important for E7-mediated cell transformation
[149]. As with E7, the E6 protein also differs in its
function between high- and low-risk HPV types. One
of the most important of these with regard to cancer
progression is the ability of high-risk E6 proteins to form
a tripartite complex with p53 and the cellular ubiquitin
ligase E6AP (E6-associated protein), which leads to
proteosome-mediated p53 degradation [150]. Low-risk
E6 proteins bind p53 with a lower affinity than the high-
risk types and have no significant ability to bind E6AP
and to stimulate p53 degradation [150,151]. The loss of the
p53-mediated DNA damage response in cells expressing
high-risk HPV E6 predisposes to the accumulation of
secondary changes in the host cell chromosome that
eventually lead to cancer. The high-risk E6 proteins also
differ from those encoded by the low-risk HPV types in
having a C-terminal PDZ-binding domain. High-risk E6
proteins bind and stimulate the degradation of several
cellular targets that contain PDZ motifs, such as hDlg
(human homologue of Dlg) and hSrib (human homologue
of the Drosophila scribble protein), which are thought
to be involved in the regulation of cell growth and
attachment [152]. PDZ binding is a conserved feature of
all high-risk E6 proteins, and it has been suggested that
the loss of cell–cell contacts mediated by tight junctions
may contribute to the loss of cell polarity seen in HPV-
associated cervical cancers [153]. PDZ binding, which is
distinct from the ability of E6 to bind and degrade p53,
is important for cell transformation [154] and has been
shown to be necessary for the stimulation of epithelial
hyperplasia in transgenic animals [53]. Another important
function for high-risk E6 is its ability to activate the
catalytic subunit of telomerase [hTERT (human telo-
merase reverse transcriptase)], which adds hexamer
repeats to the telomeric ends of chromosomes [155].
Telomerase activity is usually absent in somatic cells,
leading to the shortening of telomeres with successive cell
divisions and, eventually, to cell senescence. Although
the precise mechanism by which E6 mediates hTERT
Papillomavirus molecular biology 535
activation is not known, it is clear that such an ac-
tivity may predispose to long-term infection and the
development of cancer. Interestingly, recent studies have
shown that the viral oncogenes E6 and E7 can antagonize
BRCA-mediated inhibition of the hTERT promoter
[156].
Although aberrant expression of high-risk oncogenes
can predispose to the development of cervical cancer,
their expression alone is not considered sufficient, and
the viral proteins cannot fully transform human keratino-
cytes in culture [157]. It is generally accepted that papi-
llomavirus-mediated oncogenesis requires the accumu-
lation of additional genetic changes that occur over time
following initial infection. The average age of women
with invasive cervical cancer is approx. 50 years, whereas
the mean age of women with HSIL is approx. 28 years,
which suggests in most cases a long precancerous
state that allows the accumulation of secondary genetic
changes. Although secondary genetic changes may occur
randomly, the presence of tobacco metabolites in cervical
secretions is considered a risk factor in the develop-
ment of cervical cancer [158], with certain smoking car-
cinogens being found at significantly higher levels in the
cervical secretions of cigarette-smoking women [159].
Multiparity and the long-term use of oral contraceptives
are also associated with increased risk [160,161].
REGRESSION, LATENCY AND
ASYMPTOMATIC INFECTION
In contrast with our understanding of HPV-associated
cancers, our knowledge of the molecular events that
regulate the development of latent and/or asymptomatic
infections is only poorly developed. Experimental infec-
tions using animal papillomaviruses such as ROPV or
COPV lead to the development of lesions that persist
for months rather than years [38,162]. Lymphocyte
infiltration and lesion regression take place between 8
and 12 weeks post-infection and, by 16 weeks, infected
sites regain the appearance of uninfected epithelium [162].
A similar pattern of events occurs in cattle [163], and
regression of HPV-induced lesions in humans is also
thought to follow this path [164]. The immune system
is clearly important in controlling viral persistence, and
patients with immune defects can develop widespread
lesions that are refractory to treatment. Although the
immune responses involved in clearance of HPV in-
fections are considered in depth elsewhere [165,166],
it is worth noting that Alpha papillomaviruses (and
in particular those associated with cervical cancer) can
cause persistent infections and have evolved a number
of mechanisms to limit the chance of detection by the
immune system. Among these are the regulation of E-
cadherin expression and Langerhans cell density by E6
[167,168], the interference with MHC presentation by


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536 J. Doorbar
E5 [167] and interference with the function of inter-
feron response factor 3 by E7 [169]. Perhaps equally
important, however, is the fact that papillomaviruses
do not cause a lytic infection, with infectious particles
being produced only in the upper epithelial layers in
cells that are eventually lost from the epithelial surface
at the end of their life span. The viral early proteins
that mediate cell proliferation in the lower epithelial
layers are thought to be expressed at levels below those
required to reliably trigger an effective host immune
response. When it occurs, the stimulation of a cell-medi-
ated immune response that can clear infection appears
to depend on cross-priming of dendritic cells by viral
antigens expressed in keratinocytes. The frequent de-
tection of HPV DNA in cervical lesions in the absence
of any obvious disease may represent latent infection in
which viral genomes are maintained in the basal layer
in the absence of detectable productive infection. Immune
surveillance is thought to be important for this, as lesions
proliferate following immune supression, but this may
not be the only way in which viral latency is maintained.
Recent studies have suggested that the generation of
an asymptomatic infection in which viral DNA can
be detected in the absence of any abnormal pathology
may be a normal consequence of infection under some
circumstances, and may result from the silencing of viral
gene expression by methylation [139]. Latent infection is
thought to require the expression of the viral E1 and E2
proteins necessary for genome maintenance in the basal
layer, with the E6 and E7 genes not being required [40].
CONCLUSIONS
The consequence of papillomavirus infection depends
on the infecting HPV type and site of infection, as
well as on host factors that regulate virus persistence,
regression and latency. The characteristics of different
HPV types have been studied extensively and it is now
well known that high-risk types, such as HPV16, en-
code genes that can contribute to cancer progression
when aberrantly expressed. During productive infection,
however, these genes are carefully regulated and play
important roles in virus synthesis and in avoiding
detection by the host immune system. It seems that
papillomaviruses, like many other DNA tumour viruses,
cause cancers when their regulated pattern of gene
expression is disturbed. Viral oncoproteins such as E6
and E7, which are involved in cell proliferation and
the regulation of cell death, can be extremely dangerous
when inappropriately expressed. This can happen at the
cervical transformation zone, where most cervical cancers
originate, and it has been suggested that this may be
an unstable site for productive infection by high-risk
HPVs. Although our understanding of HPV-associated
cancer progression and productive infection is now well


C 2006 The Biochemical Society
developed, little is known as to the influence of the
infected cell type and the consequence of different cellular
environments on viral gene expression. The factors that
regulate viral persistence and the events that lead to
latency are other areas that are only poorly understood,
but which are very important in fully understanding the
consequences of infection in humans. Such topics are
difficult to research, but will need to be addressed if our
understanding of papillomavirus molecular biology is to
be advanced further.
ACKNOWLEDGMENTS
J. D. is a programme leader in the Division of Virology
at the MRC National Institute for Medical Research and
is supported by the U.K. Medical Research Council. The
leadership and vision provided by the present Director,
Sir John Skehel FRS, is gratefully acknowledged, as
is the continued support and advice provided by
Dr Jonathan Stoye, Head of Virology. The commitment
and enthusiasm of all members of the Papillomavirus
laboratory in carrying out their work and in contributing
to our understanding of papillomavirus biology is greatly
appreciated.
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