2010 The Authors
Journal Compilation 2010 APMIS
DOI 10.1111/j.1600-0463.2010.02618.x
APMIS 118: 471–493
Interaction of viral oncoproteins with cellular target
molecules: infection with high-risk vs low-risk human
papillomaviruses
DAVID PIM and LAWRENCE BANKS
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
Pim D, Banks L. Interaction of viral oncoproteins with cellular target molecules: infection with highrisk vs low-risk human papillomaviruses. APMIS 2010; 118: 471–493.
Persistent infection by a subgroup of so-called high-risk human papillomaviruses (HPVs) that have a
tropism for mucosal epithelia has been defined as the cause of more than 98% of cervical carcinomas
as well as a high proportion of other cancers of the anogenital region. Infection of squamous epithelial
tissues in the head and neck region by these same high-risk HPVs is also associated with a subset of
cancers. Despite the general conservation of genetic structure amongst all HPV types, infection by the
low-risk types, whether in genital or head and neck sites, carries a negligible risk of malignant progression, and infections have a markedly different pathology. In this review, we will examine and discuss
the interactions that the principal viral oncoproteins of the high-risk mucosotrophic HPVs and their
counterparts from the low-risk group make with cellular target proteins, with a view to explaining the
differences in their respective pathology.
Key words: Human papillomaviruses; E6; E7; pRB; p53; PDZ proteins; chromosomal instability.
David Pim, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99,
34012 Trieste, Italy. e-mail: pim@icgeb.org
More than 25 years of research into the life
cycle and transforming properties of human
papillomavirus (HPV) types that infect mucosal
epithelia has established a direct causal relationship between viral infection by a subset of these
viruses, termed ‘high-risk’ and carcinoma of the
uterine cervix (1, 2). Although infection by ‘lowrisk’ HPV types is associated with a negligible
risk of malignant progression in laryngeal and
tracheal mucosa, HPV-6 and -11 are responsible
for recurrent respiratory papillomatosis (RRP),
a rare but life-threatening condition that can
convert to cancer (3, for review). HPV-6 and -11
infection in genital mucosa causes genital warts
(condyloma acuminata) whose uncontrolled
growth in rare instances can lead to a condition
Invited review
known as Buschke-Lowenstein giant condyloma; both of these conditions, although not
life-threatening, nevertheless, impose a significant financial burden on health systems worldwide (4). The viral types associated with cervical
cancer also play a causal role in a subset of
other cancers of the anogenital region, and can
infect mucosal tissues of the head and neck
region where they are associated with a subset
of squamous cell carcinomas (SCCs). Previous
studies correlate the risk of cervical cancer with
persistent infection by these high-risk papillomavirus types, and at the molecular level, the
interaction between the major viral oncoproteins and cellular proteins provides a functional
model for the initiation and progression of
malignant disease. However, the precise parameters that govern the relationship between
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infection of squamous mucosa in various anatomical sites by various HPV types and the
development of cancer or non-malignant diseases are still not fully understood. Although
data on the host response to normal HPV infection are sketchy, it is likely that similar host
immune parameters are relevant to both highand low-risk HPV types (5, for review, and references therein) and there is a body of evidence
showing that defects in the innate immune
response are what permits a respiratory tract
infection by a low-risk HPV type to progress to
RRP (6–8); it is likely that similar defects may
lead to the progression of genital warts to condylomas and possibly also to persistent infection
by high-risk HPV types and risk of cancer. In
the past, head and neck cancers have been frequently placed in a single group, whereas the
precise tissues that comprise this region are
diverse and this is reflected in the frequency of
occurrence of different HPV types in different
cancers. For instance, HPV-18 is rarely found in
SCC of the oro-pharyngeal region, where HPV16 is most common, but more frequently found
in SCC of the oral or laryngeal regions. In addition, other high-risk HPV types are, overall,
only very rarely found in Head and Neck Squamous Cell Carcinoma (HNSCC) (9), and infections by low-risk HPV types such as 6 and 11
are still rarer. These aspects of HPV infection
will be covered in more detail in other sections
of this review issue.
One of the most significant observations linking infection by HPVs with the development of
cervical carcinoma has been the demonstration
of the retention of viral DNA in tumour cells,
many years after the initial immortalizing events
(10, 11). In the overwhelming majority of cases,
the cells derived from cervical tumours harbour
variable numbers of integrated copies of the
viral genome with some portions deleted.
Invariably, however, there is retention of the E6
and E7 open reading frames and continued
expression of the E6 and E7 proteins (12). That
maintenance of the malignant phenotype of
these cells absolutely requires continued expression of these two oncoproteins has been demonstrated by experiments where their expression
was inhibited in HPV-positive tumour cells
causing the cells to undergo apoptosis (13–15),
demonstrating unequivocally that regardless of
other changes that have taken place at the
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genetic level in these cells during malignant progression, the interaction between the viral E6
and E7 oncoproteins and cellular targets is
absolutely required to maintain this malignant
phenotype. Although HPV-16 E6 and E7 have
been shown to immortalize primary human tonsillar epithelial cells and so mirror the immortalization results for primary human keratinocytes
from genital regions (16), data formally demonstrating that oral SCCs have this same absolute
requirement for continued E6 and E7 expression
are lacking, due in part to a general lack of suitable HPV-positive tumour cell lines from the
head and neck regions. However, it is extremely
likely that the same parameters regulating HPVdriven malignancy hold true for both anatomical areas.
Low- and high-risk mucosotrophic HPVs
infect and replicate in the same general tissues,
presumably encountering the same cellular environments and therefore need to overcome the
same cellular defences to viral infection. Therefore, it is surprising that considerable differences
are observed in their respective pathologies and
in their respective cellular targets. In this review,
we shall highlight the most well-defined interactions of the viral oncoproteins with their
respective cellular targets, which seem to best
explain the differences in pathology between the
high- and low-risk HPV types.
HIGH- AND LOW-RISK HPV TYPES: THE
TRANSFORMATION STORY
One of the earliest methods used to differentiate
between high- and low-risk HPV types was the
establishment of transformation assays. Broadly,
these assays fell into three categories: transformation of established rodent cells, transformation
of primary rodent cells and immortalization of
primary human cells. While the cooperative
action of the E6 and E7 proteins to immortalize
primary human keratinocytes is regarded as the
functional ‘gold standard’ of these assays, each
system has its own merits and has contributed
to our current understanding of E6 and E7 function. As an example, it was the transformation
of established NIH3T3 cells with HPV-16 DNA
that first demonstrated that the DNA of a highrisk HPV type, found in cervical tumours, had
transforming capacity (17, 18). Extension of
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HIGH- AND LOW-RISK HPV TARGETS
these assays to primary rodent cells showed that
the HPV-16 early region, encoding E6 and E7,
together with activated ras, could transform primary baby rat kidney (BRK) cells (19); the principal oncogene responsible in these assays was
subsequently shown to be E7 (20, 21) and
importantly, that low-risk HPV-6E7 and 11E7
had no detectable transforming activity (21).
When the transforming activity of the E6 proteins was assessed using these assays, it
appeared to be markedly less efficient than E7 in
established cell assays (22, 23) and to have very
little activity in BRK cells. But in primary
mouse cells, E6 was shown to be almost as efficient as E7 when cooperating with activated ras
(24, 25). This species difference probably reflects
the efficiency with which the E6 proteins from
high-risk HPV types can interact with their relevant cellular partners.
The first equivalent assays using human cells
showed that primary human keratinocytes,
from various genital sites, could be immortalized when transfected with the viral DNA from
cancer-associated HPV types without the necessity of co-expressing an activated oncogene such
as ras (26–29). These analyses were extended to
show that immortalization could be achieved
using constructs expressing only the E6 and E7
regions of high-risk HPV types (30, 31), and
subsequently, the HPV-16 E6 and E7 proteins
have been shown to immortalize human tonsillar epithelial cells (16). It is important to stress
that the cells immortalized in these assays are
not tumorigenic and to achieve this phenotype,
they either require passaging for extended periods, where they are assumed to acquire oncogene-activating mutations, or co-transfection
with activated oncogenes (32–34) thus recapitulating aspects of the multistep processes that are
believed to govern the malignant progression of
cancer in vivo. Essential additions to these
experiments have been those that showed that
the E6 and E7 from low-risk HPV types had no
immortalization capacity (35, 36). The molecular data on the mechanisms of immortalization
that have been generated by this approach will
be discussed below.
In broad terms, what these assays achieved
was first to confirm the oncogenic potential of
the viral DNA of HPV types that were actually
found in cervical tumours. Second, it identified
the viral early region, comprising the E6 and E7
2010 The Authors Journal Compilation 2010 APMIS
open reading frames as the region responsible.
Third, they showed that, at least in a given cellular context, both E6 and E7 could individually
transform primary cells and thus paved the way
for the first experiments directed at determining
the molecular interactions that might explain
the oncogenic potential of the high-risk HPVs.
These assays, critically, also showed that the E6
and E7 proteins of the low-risk types had no
appreciable transforming or immortalizing
activity.
There are two extensions of these cellular
assay systems that have greatly extended our
knowledge on how HPVs function in cells. The
first is the use of transgenic mouse models to
target the expression of viral proteins to specific
organs to assess their effects and the second is
the development of organotypic raft systems
using differentiating human keratinocytes, to
examine these same aspects of HPV biology and
ultimately to recapitulate the viral life cycle
in vivo. These systems will be briefly described
below.
ASSESSING E6 AND E7 FUNCTIONS IN
TRANSGENIC MICE
The first meaningful assays demonstrating the
same cooperative actions of the high-risk HPV
E6 and E7 proteins that were observed in primary rodent cell transformation assays led to
the growth of tumours in the murine ocular lens
of transgenic mice where the HPV-16 early
region, comprising the E6 and E7 open reading
frames, was placed under the control of the aA
crystallin promoter (37) and in neuroepithelium
(38). However, when E6 and E7 were expressed
from a keratin 14 promoter, which is activated
in cells in the basal layer, tumours arose in the
skin and most importantly in the cervix (39–41).
These assays showed a striking cooperation
between prolonged oestrogen exposure and the
presence of the HPV-16 E6 and E7 oncoproteins
in the induction of cervical tumours in these
mice (41). Indeed a recent study has shown that
oestrogen receptor antagonists can inhibit the
development of cervical cancer in these transgenic models (42). When the individual contributions of E6 and E7 to skin cancers were
assessed in these transgenic systems, E7
functioned more strongly in the stage of tumour
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initiation, whereas E6 was shown to be stronger
during the progressive stage of skin tumorigenesis (43). The situation is different in cervical tissues and when the individual contributions of
E6 and E7 are assessed in this system, E7
appears to be the dominant oncogene, since
when it is expressed alone, after treatment of the
mice with oestrogen, it produced reproductive
tract carcinomas, whereas the expression of E6
alone did not, unless the mice were treated with
oestrogen for an extended period. However, the
dual expression of E6 and E7 under the same
conditions resulted in larger tumours, indicating
that E6 promotes progression (44). In transgenic models of head and neck cancer, as with
cervical cancer, E7 appears to be the dominant
oncogene (45).
Many of the above studies have allowed
assessment of the relative contributions made to
cancer by some of the cellular targets of E6 and
E7 (Tables 1 and 2). Such studies, undertaken
with E6 and E7 mutants, have assumed that the
same interactions that occur in the mouse system are also valid in human cells. Although the
details of such molecular interactions will be
discussed in depth below, it is worth noting that
the interaction of E7 with pocket proteins has
been shown to be necessary, but not sufficient,
for its tumorigenicity (45, 46) as is the interaction with the cdk inhibitor p21 (47). Similarly,
the interaction with and degradation of p53,
though required, is not sufficient to produce
either E6-dependent tumours in mouse skin (48)
or to explain its oncogenic potential in cervical
tissues (49) and the presence of a PDZ-binding
motif has been shown to be absolutely required
for the ability of high-risk E6 to drive epithelial
hyperplasia (48, 49), although this ability is, by
itself, insufficient for E6 tumorigenicity.
ORGANOTYPIC RAFT SYSTEMS
In contrast to other DNA tumour virus types,
such as the adenoviruses and polyoma viruses,
the early days of papillomavirus research were
hampered by a lack of cellular systems capable
of infection by HPVs, which inevitably led to
the use of the transformation and molecular
strategies that have been discussed in this
review. Productive though these strategies were
in terms of defining the oncogenic properties of
HPVs, they gave little information regarding the
normal papillomavirus life cycle. The first
Table 1. Cellular targets for the E7 proteins from high- and low-risk HPV types. Listed are those cellular targets
that the authors believe are most relevant to separate the oncogenic as opposed to non-oncogenic properties of
the E7 proteins from the two HPV types
HPV E7 interactions: functional consequences
Cellular
target
pRB
p107
p130
p21
p27
Cyclin A
Cyclin E
TBP
P300 ⁄ CBP
)
o
High-risk
Low-risk
Proteasome-mediated degradation
De-repression of cell cycle genes
Cell survival
Modulation of differentiation
Proteasome-mediated degradation of p107
pRB and p130: NO
Overrides cell cycle inhibition
p21: weakly overrides cell cycle inhibition?
p27?
No
Overrides cell cycle inhibition
Transcriptional modulation
?
Modulation of acetylation and histone
Modulation of acetylation and histone
modification
modification-WEAK
MPP2
Transcriptional modulation
No
IGFBP-3
Modulation of insulin signalling
?
Mi2
Recruitment of HDAC1 and 2 modulation of
No
histone acetylation and transcription
NuMA
Dissociation of NuMA ⁄ Dynein complex
Dissociation of NuMA ⁄ Dynein complex
Mitotic defects
Mitotic defects
p600
Transformation, anchorage-independent growth
?
HPV, human papillomavirus; NuMA, nuclear mitotic apparatus protein 1.
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Table 2. Cellular targets for the E6 proteins from high- and low-risk HPV types. Listed are those cellular targets
that the authors believe are most relevant to separate the oncogenic as opposed to non-oncogenic properties of
the E6 proteins from the two HPV types
HPV E6 interactions: functional consequences
Cellular target
p53
High-risk
Inhibition of transcription
Proteasome-mediated degradation
Overriding cdk inhibitors and apoptosis
Bak
Proteasome-mediated degradation
Inhibition of apoptosis
myc
Transcriptional activation of hTERT
Co-immortalization of primary cells
E6AP
Mediates E6 stability
Proteasome-mediated degradation
UbE3 ligase for substrate targeting
E6BP ⁄ ERC55
Binding yes; modulation of calcium-mediated
differentiation?
P300 ⁄ CBP
Binding to three domains of p300 ⁄ CBP
Inhibits intrinsic transactivation function;
modulation of acetylation
PDZ proteins
Proteasome-mediated degradation, loss of cell
polarity, changes in actin kinetics, modulation
of signal transaction
hTERT
Activation of telomerase
Tyk2
Interference with host immune system by blocking
interferon-a activation of Jak ⁄ STAT signalling
hAda3
Proteasome-mediated degradation
Abrogation of p53 ⁄ p14Arf pathway
HPV, human papillomavirus.
organotypic raft experiments undertaken examined the stratification of primary keratinocytes
immortalized by high-risk HPV DNA allowing
the analysis of the phenotypic changes that
occur during differentiation. These experiments
showed that the changes in cellular differentiation that occurred with extended passage mirrored the cellular phenotypes typical of the
progression of pre-malignant to malignant
lesions observed in patients and thus linked the
effects observed during real cancer progression
with the effects seen when immortalized keratinocytes are grown in normal culture (50). These
experiments, although useful, failed to recapitulate the viral life cycle as no viral particles were
synthesized. In the first study demonstrating
virion production, cells were treated with
phorbol ester to induce differentiation of an
HPV-31b-containing keratinocyte line grown in
raft culture (51), a strategy that allowed some
of the first analyses of the link between cellular
differentiation and changes in viral transcription
to be made (52). These initial studies with HPV31b were ultimately repeated with HPV-18 to
2010 The Authors Journal Compilation 2010 APMIS
Low-risk
Inhibition of transcription
Proteasome-mediated degradation
Overriding cdk inhibitors and apoptosis
Proteasome-mediated degradation
Inhibition of apoptosis-WEAK
?
Proteasome-mediated degradation?
UbE3 ligase for substrate targeting
Confirmed for HPV-11 E6 only
?
Binding to one domain of p300 ⁄ CBP
Consequences?
No
No
Weak interaction, modulation of host
immune response?
No
produce infectious virions (53) and subsequently
with HPV-16 (54). Interestingly, collagen raft
systems were also shown to function for HPV11-positive laryngeal cells (55). However, the
first study that allowed a comparison between
the biology of high- and low-risk HPV types in
raft cultures also showed that, although HPV11 cannot immortalize keratinocytes, their lifespan was considerably extended, and in addition
the differentiation pattern of the raft cultures
was also altered, albeit to a lesser degree than
seen with HPV-31 (56). While no major changes
were seen between HPV-11-containing and normal keratinocytes in the levels of cell cycle-relevant proteins such as cyclins or cdk inhibitors, a
major difference was the lack of repression of
interferon-regulated genes that is seen with
high-risk HPV types, although these comparisons were made for cells in monolayer rather
than under conditions of differentiation. While
global changes in cellular gene expression have
been assayed by microarray analysis for highrisk HPV type E6 and E7 proteins (57), this type
of comprehensive analysis has not been
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undertaken for low-risk type E6 and E7. Subsequent studies have obtained viable organotypic
raft systems capable of recapitulating the HPV11 life cycle, but only in cells that have been
immortalized by ectopic expression of TERT,
the catalytic subunit of telomerase (58). It is evident that there are a number of gaps in the use
of such systems regarding the analysis of lowrisk HPV life cycles, but it is anticipated that
recent studies describing efficient production
and passaging of HPV-18 in raft culture systems
(59, 60) should pave the way for a more comprehensive comparative analysis between the life
cycles of high- and low-risk HPV types.
INTERACTIONS WITH CELLULAR
TUMOUR SUPPRESSORS
Some of the first molecular explanations for the
malignant potential of the high-risk HPV types,
16 and 18, came with the observations that their
E6 and E7 oncoproteins could interact with the
key cellular tumour suppressors p53 and pRB.
p53 is a multifunctional transcriptional modulator and inducer of apoptosis. In response to
DNA damage, nucleotide depletion or hypoxia,
it becomes activated by acetylation and phosphorylation, functioning as a nuclear transcription factor to induce genes involved in cell
cycle inhibition or apoptosis, or it can induce
apoptosis more directly by interacting with
proteins in the cytoplasm at mitochondrial
sites. Its multiple functions have been studied
extensively for more than a quarter of a century (61, for review) and one attractive model
to explain the oncogenicity of high-risk HPV
types might be its functional abrogation by
papillomavirus E6 proteins, thereby allowing
mutations that activate cellular oncogenes to
pass unchecked.
Similarly, pRB is also a key regulator, whose
interaction with the members of the E2F family
of transcription factors regulates both cell cycle
progression and apoptosis. As with p53, pRB
has been extensively studied over a period of
many years (62, 63 for reviews) and it is clear
that one model to explain the transforming
capacity of the E7 proteins of high-risk mucosal
HPVs in established and primary cells might be
their abrogation of pRB function. Both p53 and
pRB mutations are common in many types of
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cancer, but occur very rarely in early-stage cervical cancers, inviting the speculation that their
functional abrogation by the E6 and E7 oncoproteins from high-risk mucosal HPVs is, to a
degree, functionally equivalent to mutation in
other cancer types. Because of the functional
complexity and importance of these two key cellular proteins, their interaction with the viral
oncogenes will be described below.
E7 AND ‘POCKET PROTEINS’
Analysis of the amino acid sequence of the E7
proteins from high-risk mucosal HPVs shows
regions of conservation with both adenovirus
E1a and SV40 large T antigen. It was this observation that led to experiments showing that
HPV-16 E7 could interact with pRB (64) and
that the E7 proteins from low-risk types could
also interact with pRB, but less efficiently (65,
66). Subsequently, it was shown that high-risk,
but not low-risk, E7 could induce the proteasome-mediated degradation of pRB (67).
Although the precise pathway involved in E7directed degradation of pRB remains to be
mapped, recent studies have begun to unravel
its complexity (68, and references therein). It
has also been shown that pRB is ubiquitinated
by an HPV-16 E7-cullin 2 ubiquitin ligase complex (69), and so E7 may function as a ligase
adapter to target pRB by a mechanism that is
analogous to the way in which high-risk E6
functions towards p53, as described below. The
other pocket protein family members p107 and
p130 were also shown to be targeted by HPV-16
E7 (70–72). Interestingly, the degradation of
p130, but not of either pRB or p107, has also
been recently shown for the E7 proteins from
low-risk HPV types (73) demonstrating at least
some conservation of function between the E7
proteins from high- and low-risk HPV types. E7
interacts principally with the active, unphosphorylated form of pRB (74) and in broad
terms, this leads to the de-repression of E2Frepressed genes, whose expression is required
for cell cycle progression from the G1 to the S
phase. By analogy with the conserved regions 1
and 2 (CR1: amino acids 37-49; and CR2:
amino acids 117-137) of adenovirus E1A, the
amino terminus of E7 can be broadly divided
into conserved domains 1 and 2 (CD1: amino
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HIGH- AND LOW-RISK HPV TARGETS
acids 1-15; CD2: amino acids 17-37) and it is the
LXCXE motif in CD2 of E7 that interacts with
the pocket domain of pRB (75, 76). The key feature of the E7–pocket protein interaction is that
it leads to deregulation of the normal cell cycle
and loss of checkpoint controls (77, 78); cells
that have divided upwards from the basal layer
would normally, permanently exit the cell cycle
and start to differentiate. HPVs must re-initiate
or maintain a functional cell cycle in these cells
to have a supply of proteins required for
S-phase progression, and to amplify viral DNA.
HPV E7 therefore is able to uncouple keratinocyte differentiation from cell cycle progression
and it is a central tenet of the current model for
HPV-induced transformation that this unscheduled DNA synthesis is what activates the cellular apoptotic pathways by a mechanism that has
been termed the ‘trophic sentinel response’ (79)
and this response is inactivated by the high-risk
HPV E6 proteins. Low-risk E7s do not appear
to activate this response despite their interaction
with pocket proteins.
pRB-INDEPENDENT FUNCTIONS OF THE
E7 PROTEINS
The E7–pRB interaction and its consequences
have been thoroughly reviewed (80, 81, for
reviews), but there are other interactions
between E7 and cellular proteins that contribute
to its driving of, or inhibition of withdrawal
from, the cell cycle, as mutations in other
regions of E7, particularly the carboxy-terminal
zinc-binding finger, can also abolish transforming ability (82, 83). For example, E7 binds to
Mi2, a component of histone deacetylase
(HDAC) NURD complexes (84), and this interaction is thought to mediate the interaction
between high-risk E7 and HDACs 1 and 2 – an
interaction that may allow E7 to modulate histone modification and transcription of cellular
genes relevant to cell cycle deregulation (85) or
to immune evasion (86). Another example is
that during keratinocyte differentiation, loss of
contact with the basal membrane is accompanied by increased levels of the cyclin ⁄ cdk inhibitors p21 and p27 (87, 88), and increased levels
of both p21 and p27 are found associated with
specific cyclin ⁄ cdk complexes (88, 89). High-risk
E7 can overcome the cell cycle inhibition
2010 The Authors Journal Compilation 2010 APMIS
mediated by both of these proteins. In the first
instance, high-risk E7 can interact with p21 and
block its ability to inhibit cyclin E ⁄ cdk2 activity
(90, 91); low-risk E7 also appears to interact
with p21, but appears to have a reduced ability
to relieve p21-mediated cell cycle arrest (77). A
similar mechanism appears to operate for E7
relief of p27 inhibition (92). High-risk E7s also
appear to help drive cell survival pathways by
increasing Akt phosphorylation, both by pRBdependent (93) and -independent pathways (94).
The binding of high-risk E7 protein through its
amino terminus with pRB-associated factor
p600 has been shown to be independent of any
interaction between E7 and pocket proteins,
and appears to contribute to the transforming
capacity of E7 and the ability of tumour cells to
grow anchorage-independently (95).
In addition to these functions, E7 proteins
have properties that modulate cellular transcription. For example, both low- and high-risk
E7 proteins have been shown to interact with
the cellular histone-modifying machinery, such
as pCAF, in this case disrupting its acetyl
transferase functions, resulting in reduced
transformation and transactivation (96), and
both low- and high-risk E7 interact with p300,
also disrupting its transactivating capacity (97).
E7 has also been shown to associate with
MPP2, a member of the forkhead transcription
factor family (98), and AP-1 (99) as well as the
basal transcription machinery (100, 101). The
finding that high-risk E7 can interact with insulin-like growth factor-binding protein 3 (IGFBP-3) (102) and accelerate its proteolytic
turnover (103) links high-risk HPV to modulation of the insulin signalling pathway, and it
has also been shown that IGFBP-2 and 3 levels
are increased in HPV-16 E7 expressing cells
under low growth factor conditions in an NFjB-dependent manner (104), the situation seen
when basal cells divide upwards from the basal
layer. This observation may also be linked to
the ability of infected cells to evade the host
immune system.
It is the link between high-risk HPV E7 and
the induction of aneuploidy that has become the
focus of attention more recently (105, for
review). It has been known for several years
that expression of the E7 oncoprotein from
high-risk HPV types caused chromosomal
changes (106), a property that seems not to be
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shared with the low-risk HPV types (107, 108).
Amongst the chromosomal damage observed in
cells expressing HPV oncogenes, perhaps the
most prominent are multipolar mitoses, due to
aberrant numbers of centrosomes resulting
from centrosome duplication errors (109, 110).
The molecular mechanism that leads to such
defects has been shown to be in part due to a
pRB degradation-dependent increase in CDK2
activity (111) and partly due to an association
with gamma-tubulin (112). Although high-risk
E7 has been shown to cause delocalization of
dynein from mitotic spindles (113), this seems
not to be directly associated with the induction
of mitotic defects. However, there is an interaction with nuclear mitotic apparatus protein 1
(NuMA), an interaction that is shared with
the E7 protein from low-risk HPV types and
which results in a prometaphase delay (114).
Aneuploidy that results from such mitotic
defects is considered to play a significant role in
DNA damage of the type that might push HPVimmortalized cells towards malignancy and
would be complemented by those aspects of E6
function that have also been shown to lead to
mitotic abnormalities (108, 115). There are,
however, potential problems with such a model.
First, the effects demonstrated occur rapidly,
whereas in our current understanding of HPVdriven malignancy, the effects that occur during
persistent infection are on a scale of years. Second, the viral E2 protein has been shown to
inhibit the E7-dependent centrosome duplication errors by direct interaction with E7, rather
than by any transcriptional-repressive means
(116) and third, the generation of at least some
mitotic defects is shared by the E7 proteins from
low-risk HPV types because of their interaction
with NuMA (114); if low-risk types induce such
defects why are they not associated with malignancy? However, this remains an attractive
model for HPV-induced DNA damage if one
considers that levels of E6 and E7 are likely to
be very low during normal, even persistent
infection, but that at some stage the viral E2
protein loses the ability to repress the viral
early promoter and E6 and E7 levels rise to a
high enough level to perturb centrosome duplication. Aneuploidy that results from these
errors might activate DNA damage repair
mechanisms that increase the risk of viral integration, a model that has been proposed in the
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past (117), and integration of the viral genome
is an event that occurs frequently during cervical
cancer progression. Indeed, recent evidence has
shown that HPV-31 E7 interacts with ATM
kinase and leads to the activation of downstream pathways such as CHK2, BRCA1 and
NBS1 (118), and HPV-16 E6 has been shown to
dysregulate CHK1 activity (119). Although activation of these pathways is thought to be
required for viral genome amplification upon
host cell differentiation, it is possible that their
perturbation in the context of aneuploidy-driven DNA damage might have more serious consequences.
E6 AND p53
HPV-16 and 18 E6 were shown to interact with
the cellular tumour suppressor p53 (120) and
then subsequently to direct its degradation
(121). While the intracellular levels of p53 are
normally regulated by the cellular ubiquitin
ligase hdm2 (122, 123), high-risk HPV E6 proteins interact with p53 and direct its degradation
by recruiting a different cellular ligase, E6AP
(124, 125). Subsequent studies showed that the
E6 proteins from low-risk HPV types could also
interact with p53, but failed to increase its rate
of turnover (126, 127), showing that low-risk E6
employs a different mechanism for the abrogation of p53 function, most likely by reducing its
reported ability to both repress TATA-containing promoters (128) and to activate p53-responsive promoters (25, 129). These early studies
suggested that the E6 from low-risk HPV types
failed to direct the degradation of p53 because
they were unable to interact with and recruit
E6AP, but more recent data have changed this
model for low-risk E6 function as will be discussed later on in this review. Studies had
already suggested that the region of E6 involved
in binding p53 might be separate from that
involved in inducing its degradation (126, 129),
but conclusive data showing precisely which
regions of high-risk E6 bind to p53 and which
bind to E6AP have been lacking, with many
publications showing conflicting results. However, when looking at the regions of p53 that
interact with E6, an explanation for the functional difference between the E6 proteins from
high- and low-risk types came from studies
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HIGH- AND LOW-RISK HPV TARGETS
showing that the E6 from high-risk types could
bind to p53 at two regions, a carboxy-terminal
region (amino acids 376-384) and a core region
(amino acids 66-326), whereas the E6 from lowrisk types bound to p53 only at the carboxy terminal region (130). More recent data suggest
that the ability of the E6 proteins from low-risk
HPVs to interact with p53 and modulate some
of its functions occurs by sequestering it in the
cytoplasm (131). Given that some of the functions of p53 also occur in the cytoplasm, and
that the E6 proteins from high-risk types export
p53 from the nucleus to the cytoplasm when
directing its degradation (132), it would be interesting to re-assess the effect of E6 from low-risk
HPV types on these functions.
Another aspect of HPV E6 function independent of its direct interaction with p53, but highly
relevant to the p53 pathway, is p53 acetylation.
High-risk, but not low-risk HPV E6 interacts
with hAda3, a component of histone acetyltransferase complexes that is involved in driving
the p53 ⁄ p14Arf pathway by acetylation of p53.
High-risk E6 directs its degradation in an
E6AP-dependent manner thereby inhibiting
p53-mediated senescence (133, 134). However,
both high- and low-risk HPV E6 proteins seem
capable of binding to CBP ⁄ p300 and this interaction has been shown to prevent the acetylation of p53, independently of any ability to
direct E6AP-mediated degradation of p53,
thereby inhibiting its ability to transcriptionally
activate promoters (135). This observation suggests a higher degree of functional conservation
between the E6 proteins of high- and low-risk
HPV types, with respect to p53 function, than
was previously considered.
increase its rate of proteolytic turnover through
the E6AP ⁄ proteasome pathway (138, 139) and
it is significant that for cutaneous HPV types,
this interaction with Bak is the only apoptosisrelevant pathway so far clearly defined (140).
High-risk E6 binds strongly, but, low-risk E6
only weakly to p300 ⁄ CBP (141, 142). Only highrisk E6 proteins bind to E6BP ⁄ ERC-55 (143),
MCM7 (144) and c-Myc (145). Both bovine papillomavirus type 1 (BPV-1) and high-risk, but
not low-risk, HPV E6 proteins bind to Paxillin
(146), a component of the focal adhesions that
attach keratinocyte basal cells to the basal
membrane and which is required for driving
integrin-mediated proliferation and survival signalling through focal adhesion kinase (FAK).
However, while BPV-1 E6 degradation of Paxillin
is required for its transforming capacity (147) the
significance of the interaction with high-risk HPV
E6 is likely different because in both HPV-positive
carcinoma cells and in HPV-immortalized keratinocytes, paxillin levels and FAK-driven tyrosine
phosphorylation are increased rather than
decreased (148). Of relevance to the way in which
HPVs can evade host immune systems was the
observation that high-risk E6 could inhibit the
interferon-a activation of the Jak-STAT pathway
by interacting with tyrosine kinase Tyk2 – an
interaction that was observed to occur less
strongly for low-risk E6 types (149). Finally, there
are those cellular interactions that are relevant to
the reactivation of telomerase in infected cells
and a growing list of cellular proteins containing
PDZ domains, understanding the consequences
of which may be more relevant in modelling
immortalization ⁄ transformation ⁄ cancer progression functions. Both of these will be discussed in
detail below.
P53-INDEPENDENT FUNCTIONS OF E6
E6 AND TELOMERASE ACTIVATION
There are several lines of evidence, based on
analysis of the function of E6 mutants in cellbased assays, that point to the involvement of E6
in other aspects of cell biology, independently of
its interaction with p53, that contribute towards
its oncogenic potential (25, 136, 137). Indeed, a
large number of cellular binding partners have
been described for E6 that might contribute to
its oncogenic character, some of which are
shared by the E6 proteins from low-risk HPV
types. High- and low-risk E6s bind to Bak and
2010 The Authors Journal Compilation 2010 APMIS
Isolated primary cells undergo a finite series of
population doublings before embracing death
(150) by the activation of senescence pathways.
Those cells that survive this ‘crisis’ stage and
continue to grow are found to have activated
telomerase, the enzyme complex that maintains
the telomeric repeats at the ends of chromosomes (151). Typically, the mechanism involves
transcriptional activation of the gene for
hTERT, the catalytic subunit of the telomerase
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PIM & BANKS
complex. Tumour cells also achieve immortality
in a great number of cases by a similar mechanism (152). When primary human keratinocyte
immortalization assays with high-risk HPV E6
and E7 were first set up, it was realized that their
telomere lengths were restored in contrast to
non-immortalized cells (153) and that the reason
for this was due to reactivation of telomerase
activity (154). The mechanism by which telomerase becomes activated during immortalization by E6 and E7 is complex, but is becoming
clear. For instance, the use of E6 mutants shows
that the mechanism is independent of the ability
of E6 to bind and direct the degradation of
either p53 (154, 155) or PDZ proteins (49), but
involves transcriptional upregulation of the
hTERT promoter by a mechanism involving
SP1 binding and down-regulation of p300 (156)
and depends on Myc (157). Myc is found bound
directly or indirectly to high-risk E6 in complexes at the E-boxes within the hTERT promoter (158), an observation that appears to be at
odds with the original finding that suggested
that Myc was targeted for proteasome-mediated
degradation by high-risk E6 in an E6AP-dependent manner (145). In addition, a recent report
has demonstrated that E6 can itself also make a
direct interaction with the telomerase complex
(159). The question of whether binding to the
ubiquitin ligase E6AP is required seems to be
controversial, some reports showing it to be
required (160) and that E6AP-dependent degradation of the transcriptional repressor NFX1-91
is necessary for hTERT activation (161),
whereas other reports seem to show no requirement for high-risk E6 to interact with E6AP to
activate hTERT (162). While E7 is by itself
unable to activate telomerase, it is clear that it
augments the activity of E6 by transcriptional
activation of the hTERT promoter in a pRBdependent manner (163). Evidently, activation
of telomerase appears to be an important facet
of high-risk HPV-induced immortalization and
an aspect of the viral properties not shared with
low-risk types; however, how this relates to the
normal viral life cycle and why low-risk HPV
types seem not to activate hTERT remain to be
clarified. It may be a function related to episomal maintenance because telomeric proteins
have been shown to be involved in the episomal
maintenance of Epstein–Barr virus origins of
replication (164).
480
OTHER HIGH-RISK VS LOW-RISK
INTERACTIONS
From the data presented above, it should be
clear that many of the interactions with cellular
targets that are made by the E6 and E7 proteins
from the high-risk mucosotrophic HPV types
are shared by their counterparts from the lowrisk group, albeit to a generally lesser degree. A
plausible argument for the difference in their
oncogenic potential could be that it is due to
these differences in the efficiency of their targeting of the same set of cellular proteins. Data
that might support this come from the observation that in extremely rare cases, RRP can have
lung involvement, and that a surprisingly high
proportion of these lung-involvement cases can
result in lung cancer. Analysis of the HPV in
these cases shows that the virus has undergone
substantial mutation, such as amplification of
the viral URR, which might lead to much higher
levels of E6 and ⁄ or E7 being expressed and thus
showing that given the right circumstances, in
the right tissue type, even low-risk HPV types
have oncogenic potential (B. Steinberg, personal
communication). However, there is a set of
interactions that is exclusive to only the E6 proteins of the high-risk HPV types and these will
be discussed below.
HIGH-RISK E6 PROTEINS AND PDZ
DOMAINS
The extreme carboxy termini of the E6 proteins
from all of the high-risk human mucosotrophic
papillomaviruses contain a consensus sequence
that is able to bind to PDZ domains
(PSD95 ⁄ Dlg ⁄ Zo-1). The PDZ-binding sequence
is absent from the E6 proteins of the low-risk
HPV group and both this sequence and the ability to bind PDZ domains are therefore molecular hallmarks of the E6 proteins from oncogenic
HPV types. These domains, 80-100 amino acids
in length, serve as docking modules for a large
variety of different cellular proteins, including
signalling molecules (165, for review). A subset
of these proteins, the Membrane Associated
Guanylate Kinases (MAGUKs), has multiple
PDZ domains and can serve as molecular
scaffolds to conjugate incoming signals at the
plasma membrane and transmit them to
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HIGH- AND LOW-RISK HPV TARGETS
multiple downstream effector pathways. These
PDZ proteins are found at a variety of cell
junctions and synapses, where they are also
involved in defining and maintaining cell polarity, as well as themselves being involved in
nuclear-cytoplasmic shuttling and signalling,
and mediate the kinetics of actin polymerization
and depolymerization at the leading edge during
cell migration. Most importantly, loss or mislocalization of these complexes during malignant
progression of cancer correlates with the loss of
cell polarity and invasiveness that is generally
observed with metastasis. Because of the large
number of identified PDZ proteins and the roles
that they play in almost every conceivable
aspect of cell biology, it can be seen that the E6
proteins from oncogenic HPV types have the
potential of modulating a large variety of cellular pathways. However, so far, although the list
of PDZ domain partners for E6 is growing, it is
also evident that there is a set of defining parameters that govern which PDZ domains may be
bound by E6 and which not. In particular, the
efficiency with which a particular E6 may direct
degradation of a given PDZ protein may
be defined by its exact PDZ-binding motif
(166, 167), and the observation that even for
proteins with multiple PDZ domains E6 seems
to interact with only one PDZ domain per
target protein (167–171) suggests selectivity in
targeting.
The first identified PDZ domain-containing
target to bind to E6 was Dlg, the human homologue of Drosophila discs large (172), and E6
was subsequently demonstrated to direct its degradation along a proteasome-mediated pathway
(173). Other studies identified hScrib as a target
for E6-directed degradation (174) and subsequently MAGI-1, -2 and -3 (169, 175), MUPP1
(168) and many others (1, 176, for review).
Many PDZ proteins as well as the MAGUK targets mentioned above localize to sub-membrane
sites where they play a role in maintaining junction integrity and cell polarity. It has been
regarded as central to the philosophy of the current model for cervical cancer progression, that
degradation of these proteins relates to metastasis. However, at least in the case of Dlg, if
cells derived from cervical tumours are treated
with proteasome inhibitors, the Dlg that is rescued is predominantly localized in the nucleus
(177), suggesting that it is a nuclear pool of Dlg
2010 The Authors Journal Compilation 2010 APMIS
that is targeted by high-risk E6. As studies have
also shown that nuclear forms of Dlg are phosphorylated by cyclin-dependent kinases and
preferentially targeted by E6 (178, 179), this suggests that nuclear forms of Dlg may be playing
an antiproliferative role and that in HPV-positive cervical tumour cells, this is being inhibited,
in part, by its E6-directed degradation. This
observation does not by any means rule out the
possibility that membrane-localized forms of
Dlg, or other PDZ domain-containing proteins,
may be targeted during the normal viral life
cycle; a speculative model might be that by loss
of these proteins and a corresponding loss of
polarity, infected basal cells might divide in a
horizontal rather than a vertical orientation,
thereby expanding the population of infected
cells that are still attached to the basal membrane. This kind of model suggests that highrisk E6s may target a specific set of PDZ proteins in a particular subcellular localization, that
are involved in maintaining cell polarity during
the normal viral life cycle, but target PDZ proteins in a different subcellular localization during malignant progression. That MAGUK
proteins mediating cell junction integrity might
be the target of choice for the normal viral life
cycle is supported by the finding that these proteins are generally targeted by high-risk mucosal
viruses, whether or not there is any evidence for
their nuclear localization. Dlg and hScrib are
members of a complex found at adherens junctions (180, for review). PATJ, another target for
high-risk E6 is found localized to tight junctions
(181) as is MUPP1 (182), and the MAGI-1, 2
and 3 proteins. Although the MAGI proteins
are implicated in a wide variety of cellular activities such as the MAGI-1 regulation of b-catenin
signalling (183) and the MAGI-2 regulation of
the PTEN pathway (184), they are also membrane localized for many of their functions. A
recent study of another high-risk mucosal papillomavirus type, rhesus papillomavirus type 1
(RhPV-1), shows the presence of a PDZ-binding
motif on the carboxy terminus of the E7, rather
than the E6 protein. In this case, the preferred
target for degradation is Par-3, a component of
the apical polarity complex (185). All these data
suggest that high-risk HPV types generally target polarity-regulating complexes as part of their
normal life cycle. While it is currently unclear
what roles these interactions play, there is good
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PIM & BANKS
evidence for its essential requirement because
removal of the PDZ-binding motif, in the context
of the whole viral genome, results in the loss of
episomal maintenance (186).
New PDZ targets of the E6 oncoproteins from
high-risk HPV types are continually being identified and of particular note are a subset of tyrosine phosphatases. Given their involvement in
the inhibition of signal transduction through
receptor and non-receptor tyrosine kinases from
membrane-proximal sites, driven by external
stimuli, a reduction in their activity by E6-directed degradation might be expected to drive
signal transduction pathways that relate to cellular proliferation. The first phosphatase to be
identified as a target was PTPH1, also known as
PTPN3 (187, 188), where its degradation by
oncogenic E6 was shown to correlate with
reduced growth factor requirements. Perhaps
more important, due to its reported relevance in
head and neck cancers, is the protein tyrosine
phosphatase PTPN13 (189). In this case, degradation of the phosphatase (also known as
PTPL1, PTP-Bas and FAP-1) appears to be relevant for anchorage-independent growth, where
it results in increased signalling along the mitogen-activated protein kinase pathway (190).
Although these studies used oral cells for analysing E6 function, it is most likely that E6-mediated disruption of tyrosine phosphate function
plays a role in the life cycle of these high-risk
HPVs in cervical keratinocytes. Some of the cell
junction-localizing substrates of high-risk E6
also have consensus sites for tyrosine kinases
and are likely to be tyrosine phosphorylated. We
speculate that E6 might therefore be exerting a
two-pronged attack on some of its substrates, by
degrading a subset of tyrosine phosphatases,
and thus enhancing upstream kinase pathways
for these MAGUK targets, altering their
phosphorylation and ⁄ or cellular localization
and, as a result, also possibly altering their
susceptibility to E6-mediated degradation. That
this aspect of the biological properties of E6 is
essential for its oncogenicity is clearly demonstrated by the finding that in the transgenic
mouse models discussed above, E6 mutants with
deletions in the PDZ-binding motif are unable
to induce hyperplasia (49) and E6 lacking a
PDZ-binding motif cannot cooperate with E7
to immortalize human tonsillar epithelial cells
(189).
482
THE E6 PROTEINS FROM LOW-RISK HPV
TYPES; POTENTIAL LIGASE
INTERACTIONS
Although the presence of a PDZ-binding motif
on the carboxy terminus of high-risk HPV E6
proteins and its absence from the low-risk HPV
E6s might be regarded as a defining means of
separating their relative risk factors, and while
the E6s from low-risk HPVs were originally
regarded as being unable to recruit ubiquitin ligases for degrading cellular targets, the cloning
of the last six amino acids from a high-risk E6
onto the carboxy terminus of a low-risk E6 fully
enables it to direct the degradation of certain
cellular targets (191). This study demonstrated
not only that E6 from low-risk HPVs are able to
conjugate to cellular proteolytic systems but
also that the way in which individual cellular
proteins are targeted may be via different degradatory systems, or via different ubiquitin ligases.
The human tumour suppressor p53 has two
common polymorphic forms encoding either
arginine or proline at amino acid position 72
(192), and previous studies have hinted tantalizingly at a modest ability for HPV-11 E6 to
degrade at least the Arg form of p53 (193).
Interestingly, a more recent study has demonstrated an interaction between HPV-11 E6 and
E6AP (194), suggesting the means by which
HPV-11 E6 may do this. The story of ubiquitin
ligase recruitment is, however, far from complete. Previous studies have suggested rather
strongly that the high-risk HPV E6 proteins
may recruit ligases other than E6AP to direct
the degradation of certain cellular targets (166,
191) and several additional studies have supported this. First, synthetic peptides that inhibited the binding and degradation of some of E6s
targets were unable to block the degradation of
others (195) and second, certain cellular targets
were found to be still degraded when incubated
together with E6 after translation in vitro in
wheat germ extract, which lacks E6AP, or when
rabbit reticulocyte lysate had been immunodepleted of E6AP (196). More recently, the
observations that high-risk E6 can direct p53
degradation without inducing its ubiquitination
(197) and that E6-directed degradation of some
substrates can occur in E6AP-null mouse fibroblasts (198) suggest that we are only just beginning to understand the complex series of
2010 The Authors Journal Compilation 2010 APMIS
HIGH- AND LOW-RISK HPV TARGETS
interactions by which the high-risk E6 proteins
may target different cellular substrates.
The search is on for new interacting ubiquitin
ligases and ubiquitin pathway components that
may be involved in substrate targeting. New
data have recently shown that the high-risk E6
protein is in fact stabilized by E6AP (199) and
that a novel ubiquitin ligase, EDD, interacts
with E6 and has been shown to regulate the stability of E6AP (V. Tomaić, personal observations) and the ubiquitin-specific peptidase
USP15 has also been shown to be involved in
the regulation of E6 stability (200). Doubtless
new ubiquitin ligase discoveries are on the horizon and will help to unravel the complex set of
interactions and pathways defining the molecular parameters that govern the targeting of the
cellular substrates of E6.
OTHER HIGH-RISK SPECIFIC
FUNCTIONS
High-risk and low-risk mucosal HPV types
express their E6 and E7 open reading frames
differently. For HPV-6 and 11, E6 and E7 have
two separate transcriptional start sites, whereas
for types 16 and 18, there is only one, so that E6
and E7 are transcribed from the same promoter.
An examination of the transcriptional patterns
from HPV-positive cell lines has shown a complex pattern of polycistronic mRNAs that can
be multiply spliced to encode a range of viral
early proteins (201). For types 16 and 18, the
presence of a consensus splice donor site within
the body of the E6 open reading frames of such
early transcripts means that, in addition to other
open reading frames that might be encoded,
removal of an intron within the body of the E6
open reading frames can generate one or more
small truncated E6 transcripts known as E6*.
Where these introns terminate and the number
of potential E6* transcripts that can be encoded
in this way is defined by the siting of the downstream splice acceptors, but invariably these run
back into a different, intron-derived reading
frame, so that E6* transcripts encode a tail of
amino acids derived from neither E6 nor E7,
and terminate at the first available stop codon.
This pattern for the siting of the first splice
donor and acceptor is strongly conserved
among most, but not all, high-risk mucosal
2010 The Authors Journal Compilation 2010 APMIS
HPV types; HPV types 52, 59 and 67 lack these
strongly conserved donor ⁄ acceptor patterns
(202) and it has not been shown whether or not
they can make E6* transcripts. However, these
conserved splicing modules within the E6 open
reading frame are found neither in low-risk
HPV types nor in cutaneous HPVs. It has been
an open question whether these truncated E6
forms are ever translated to express the E6* protein, although the majority of early transcripts
found in HPV-positive cell lines are spliced and
encode E6* (11, 203). Several in vitro studies
have suggested that E6* proteins could bind to
their full-length E6 counterparts and were able
to inhibit the E6-directed degradation of p53
(204, 205). Additionally, data suggest its
involvement, together with full-length E6 in
modulating components of the extrinsic death
pathway (206). The most recent data on E6*
function suggest that it can decrease the half-life
both of PATJ (207) and Dlg when expressed by
itself at high levels, and may also affect the stability of other PDZ proteins (202). Compelling
evidence suggests that it provides a helper function for full-length E6 in activating the degradation of differentially phosphorylated targets,
because Dlg phosphorylated along the p38
MAPK pathway, but not along the SAPK ⁄
JunK pathway, is markedly more susceptible to
degradation by E6 when co-expressed with E6*
than it is to degradation induced by either
protein alone (202). It is unlikely that the E6*
protein reaches high enough levels in vivo to
activate degradation by itself; although it is not
an intrinsically unstable protein, it is expressed
at very low, although detectable, levels in HPVpositive HeLa cells (D. Pim, personal observations). Clearly, existing in vitro models for HPV
transformation do not predict a major role for
the E6* proteins; however, it is quite possible
that in vivo, E6* augments the effect of
full-length E6 by modulating diverse upstream
signalling pathways.
CONCLUSIONS
Any contemporary model that seeks to differentiate between the malignant potential of highand low-risk HPVs in terms of their molecular
interactions is faced with a number of problems.
One such problem is the relative lack of
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PIM & BANKS
available data for the molecular interactions
made by the E6 and E7 proteins of the low-risk
types. Regardless of the clinical implications of
infection by the low-risk types, their lack of
association with malignant disease has inevitably made them less attractive fields in terms of
funding and research. Similarly, the low-risk
types have failed to produce viable working
models, or yield significant data in key research
systems such as cellular transformation models
and in raft culture, leaving substantial gaps in
our understanding. Nevertheless, where molecular studies have been possible, the low-risk types
have proved to be every bit as interesting to
study as the high-risk.
So how can we distinguish between these viral
types? Are there life cycle differences that correlate with differences in oncogenicity? Clearly,
the pathology of these groups is different, with
the high-risk mucosal HPVs producing planar
warts in genital tissues, as opposed to the papillomas produced by low-risk types, and yet these
differences are not invariably reflected in the
sequential pattern of expression of their various
proteins or the tissue layers where viral genome
amplification takes place during the viral life
cycle. An example of such analysis shows that
low-risk HPV-11 appears strikingly similar to
high-risk HPV-16 (208). Where low-risk HPV
E6 and E7 interactions with cellular proteins
have been studied, the consensus has been that
many interactions that are seen with the highrisk oncoproteins either do not occur, or are
very much weaker; this itself could partly
explain their lack of correlation with malignancy. However, perhaps the two most significant differences between high- and low-risk
HPV types are the ability of the high-risk E7
proteins to induce chromosomal damage by
interfering with the centrosomal duplication
pathway and the interactions between the highrisk E6 proteins and cellular targets containing
a PDZ domain. Of relevance to this latter interaction, it is probable that malignant transformation by the high-risk HPVs may arise under
conditions of abortive infection and it has been
suggested that the transition zone, where most
cervical carcinomas originate, may be of a tissue type that is less competent to support viral
replication (209). It is of note that the two most
prevalent high-risk types, HPV-16 and HPV-18,
produce tumours of different cytology, SCCs by
484
HPV-16 and adenocarcinomas by HPV-18 and
in distinct regions of the uterine cervix. Given
that the transition zone delineates these two
regions, it may well be that infection by highrisk HPV types at this transition zone has a
higher risk of malignant progression and that
the differences in the targeting specificity of
polarity-determining PDZ proteins by the E6
oncoproteins of these two HPV types may affect
the migration of infected cells away from this
transition zone. Future molecular studies will
certainly refine our understanding of the precise
mechanisms involved.
Although this review has concentrated on the
functions of the E6 and E7 proteins, it is worth
bearing in mind that the expression of other
viral non-structural proteins and any interactions they make with E6 and E7 and their
association with cellular proteins is likely to
have a bearing on function and any precise definition of oncogenic potential.
OPEN QUESTIONS AND FUTURE
CHALLENGES
Far more basic and clinical research has
focused on the high-risk HPV types than on
the low-risk types despite the substantial financial burden imposed by low-risk HPV types on
the health systems of many western countries
that have good working cervical cancer screening systems in operation (4), and it is to be
hoped that despite the initiation vaccination
programmes that also include a tetravalent
HPV vaccine that targets HPV types 6 and 11
as well as 16 and 18, investment in research
into the biology of low-risk HPV types will be
seen as more essential. One significant aspect
of our current understanding of the HPV life
cycle is that infections, whether by high- or
low-risk types, that are able to be cleared by
the host immune system in a short period pose
much less of a threat in terms of malignancy.
In such cases, defects in the host immune
response may be a contributory factor and
therefore, probably the most challenging
future strategy, at least in terms of developing
new therapeutic strategies, will be to acquire a
better understanding of the molecular interactions that are involved in the way these
viruses interface with host immune systems
2010 The Authors Journal Compilation 2010 APMIS
HIGH- AND LOW-RISK HPV TARGETS
and how the genetic background of the host
may affect the outcome of infection.
The authors acknowledge the support from the Associazione Italiana per la Ricerca sul Cancro and the
International Agency for Research on Cancer and
thank Dr Lutz Gissmann for helpful comments.
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