HPV and Cancer

1
Anno Accademico 2016-2017
UNIVERSITÀ DEGLI STUDI DI TRIESTE
DIPARTIMENTO DI SCIENZE DELLA VITA
CORSO DI LAUREA IN SCIENZE E TECNOLOGIE BIOLOGICHE
HPV and Cancer.
The role of the Human Papillomavirus high-risk E6 oncoprotein
in malignant progression
Laureanda:
Elena Tassotti
Relatore:
Dott. Lawrence Banks
Correlatore:
Dott. Paola Massimi

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TABLE OF CONTENTS
1. INTRODUCTION
1.1. The Human Papillomavirus and its Replication
1.1.1.The HPV DNA and its gene products
1.2. The diversity of Human Papillomaviruses and the diseases that they cause
1.3. The HPV Infection
2. HPV AND CANCER
2.1. Infection and Transformation by high-risk HPV E6 and E7 oncoproteins
2.2. The high-risk HPV E6 as a potent viral oncoprotein for cell transformation
2.3. E6AP-independent targets of E6 and the PDZ binding motif: host proteins associated with
HPV E6
2.4. E6-induced perturbation of cellular pathways that sense cell polarity and trafficking
3. EXPERIMENT
3.1. Aim of the work
3.2. Materials and Methods
3.3. Result
3.4. Discussion
4. CONCLUDING REMARKS
5. ACKNOWLEDGMENTS
6. REFERENCES

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1. INTRODUCTION
1.1 The Human Papillomavirus and its Replication
Human papillomaviruses (HPV species) are small, non-enveloped, epitheliotrophic dsDNA viruses
belonging to the family Papillomaviridae, that specifically infect human epithelia and mucous
membrane (Organization, n.d.). Their circular, double-stranded genome is approximately 8-kb in
length and encodes eight ORFs, respectively for six early proteins essential for virus replication and
two late structural proteins, L1 and L2 1
, which are exclusively expressed in differentiating
keratinocytes of the superficial stratum corneum where the assembly of the protein capsid of mature
virions exclusively occurs (Stanley, Pett, and Coleman 2007). Upstream of the set of growth-related
early genes, whose transcription occurs using host cell RNA polymerase II and cellular transcription
factors2
, is located a region containing ARS (autonomously replicating sequences), several gene
expression regulatory elements (REs) and the N-terminal amino acid sequence ubiquitous in all
early proteins (E1, E2, E4, E5, E6, E7) that fulfill pleiotropic functions.
Primarily, the E1 and E2 gene products are actively involved in the replication of the viral genome
and, since they bind the viral origin of replication, are specifically required in the initiation of
replication and, eventually, in the elongation. Once DNA replication has begun, the late genes are
transcribed and translated to give rise to late structural proteins that compose the capsid with
icosahedral surface symmetry. Both late and early viral proteins are synthesized in the cytoplasm,
but are often transported back to the nucleus where both viral replication and nucleocapsid
assembly occurs.
A probable explanation of this lies in the fact that early proteins are also involved in the regulation
of viral gene expression, as they typically activate transcription of late genes, and may also down-
regulate3
their own transcription. Furthermore, early proteins are able to alter the metabolism of the
host-cell by activating pathways that induce G1-arrested cells to enter S phase, during which
cellular DNA synthesis occurs, and cellular replication enzymes are exclusively present. Because
small DNA viruses require these cellular enzymes to replicate the viral DNA, this effective
adaptation mechanism clearly represents the way by which HPVs manage to proliferate, namely by
expressing the early proteins responsible for cell cycle control and for inducing resting cells to enter
the replicative phase.
1
major capsid protein L1, arranged as 72 pentamers on a T=7 icosahedral lattice, associate with the minor capsid pro-
tein, L2. The protein-protein interaction is crucial in the replication of HPVs.
2
which enhance the synthesis of the early pre-mRNAs that are subsequently processed (capped, polyadenylated and
eventually spliced to maximize the coding potential) in the nucleus and then are transported to the cytoplasm and ulti-
mately translated into the corresponding early proteins.
3
autoregulate

4
The viral DNA replication produces thousands of new viral genomes, nonetheless the process has
its remarkable limits: because of the limited coding capacity, which is ultimately due to their
relatively small genome size, HPVs can only replicate their genomes by using the host cell DNA
synthesis machinery (Moody and Laimins 2010). As long as it needs to use host cell DNA enzymes,
plus a limited number of viral proteins, the viral chromosome replication is exclusively restricted to
the gradually differentiating keratinocytes of those body surface tissues, such as the skin or mucosal
membrane4
, which are collectively known as stratified squamous epithelia. Keratinocyte stem cells,
which replenish the outermost layers of the epithelium, are thought to be the initial target of
productive papillomavirus infections; however, production and egression of mature virions are not
accomplished until the infected cells specialize and slough off the upper epidermis (MacBride
2011). A clear demonstration of this lies in the fact that the newly formed viral particles are released
along with dead keratinocytes rising to the epithelial surface. Therefore, the viral life cycle is
strictly dependent on keratinocyte differentiation (Pyeon et al. 2009).
However, while low-risk HPVs begin replication in cells that are still proliferating, the replicative
cycle of high-risk HPV infection is confined to more differentiated cells that have already exited the
S phase and are non-permissive for DNA synthesis (Doorbar et al. 1997). In order to avoid this
problem, the high-risk HPV E7 protein targets a number of cell cycle regulatory proteins, including
the 'pocket protein' family of pRb, p107 and p130; as a direct consequence of these interactions,
genes required for G1/S transition and DNA synthesis undergo selective up-regulation.
Nevertheless, the host cell would normally respond to this unscheduled proliferation by inducing
apoptosis and/or growth arrest. In order to prevent this from happening, the high-risk E6 protein
targets a wide variety of cellular proteins involved in regulating these surveillance pathways, as
well as those involved in terminal differentiation and antiviral protection (Mantovani and Banks
2001). Although the viral life cycle would normally continue, resulting in production and release of
infectious virions, on some rare occasions, the viral life cycle is interrupted and the cell undergoes
in vivo immortalization and ultimately complete transformation.
1.1.1 The Human Papillomavirus DNA and its protein products
Considering the genome structure, which is the same in all known papillomavirus genera, each HPV
gene is contained within the positive-strand of the circular DNA molecule that serves as a template
(Conway and Meyers 2009). The circular viral genome, which is maintained as episomes at
approximately 100-150 copies per cell in the basal layer (Doorbar et al. 2012), encodes both late
and early viral proteins, which are synthesized in the cytoplasm and then imported into the nucleus
where nucleocapsid assembly occurs (Figure 1). The accumulation of viral proteins at high levels
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the inside of the cheek, air ways, genitals and conjunctiva

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can be observed as "inclusion bodies" scattered throughout the cytoplasm of infected cells (Villiers
et al. 2004; Iarc 2007).
Figure 1. Genomic organization of the HPV16 circular genome showing the location of
the early (E1 and E2) and late genes (L1 and L2), and of the long control region (LCR).
The HPV genome encodes eight well-characterized proteins, whose functions are
indicated, all of which have been proved to be genuine targets for small molecule-based
approaches for the treatment of HPV-associated diseases. Source: D’Abramo and
Archambault 2011
Herein, we review the crucial functions carried out by the proteins encoded by the human
papillomavirus DNA:
L1 protein, also referred to as major capsid protein, is the main structural component of
the viral capsid, which also contains the L2 protein with which L1 interacts. Besides, L1
mediates either humoral and CD8+ and CD4+ T cell immune responses against hrHPV infection
(Song et al. 2015)
The less abundant L2 protein, called minor capsid protein, also has a significant role in
assembling the capsomers forming the nucleocapsid (Manuscript 2014);
The major protein E1 allows episomal replication during the initial amplification phase of
the viral life cycle, and has helicase activity5
;
E2 protein is involved in the regulation of the E6 promoter, activates E1 and develops
transactivating abilities6
. In particular, E2 inhibits the transcription of E6 and E7, as long as the
5
its helicase domain is able to contact the DNA
6
transactivation is the increased rate of gene expression triggered either by natural processes or by artificial means,
through the expression of an intermediate transactivator, E2 protein in this specific case: the E2 transactivation domain
is also implicated in self-interaction and looping of DNA containing E2 binding sites

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viral genome stays separate from that of the host: in fact, integration of HPV DNA within the
human chromosomes entails DSBs of E2/E1 gene sequences, which are then no longer expressed;
the resulting downregulation of the E2 regulatory protein7
, and consequently of E1, limits DNA
synthesis and contributes to cell-cycle dysregulation through the loss of those cell-intrinsic
checkpoints that suppress carcinogenesis. Since E2 and E1 regulatory proteins are absent, the
host cell becomes particularly prone to pro-oncogenic mutations and other genomic
abnormalities such as aneuploidy, chromatid gaps and breaks that have interestingly been
detected in pre-malignant HPV-associated cervical lesions (Steinbeck 1997; Mittal and Banks
2017). These observations support the premise that the onset of genetic instability is an early
event in the development of malignancy, occurring before integration of the episomal viral
genome into host chromosomes. However, once integration occurs, E2 expression is lost, which
suppresses the inhibition of E6 and E7 which target the p53 and pRb oncosuppressor genes,
respectively. As a result, the deregulation of E6 and E7 gene expression with the consequent loss
of the oncosuppressors' homeostatic function might favor the acquisition of metastatic capacities
in infected cells (Romanczuk and Howley 1992). Alongside viral transcription, E2 also regulates
extrachromosomal genome maintenance as well as partitioning owing to multiple, sequence-
specific DNA binding site motifs found in the LCR8
or URR9
at E2’s C-terminal domain, which
has dimerization properties (Hernandez-Ramon et al. 2008). This non-coding region, also known
as the URR/LCR (upstream regulatory region/long control region), contains many regulatory
elements that control initiation of viral replication such as consensus sequences, transcription
factor-binding sites and the replication origin, which together determine the broad tissue tropism
of different HPV types (MacBride 2011).
E4 protein, only expressed at later stages of HPV infection, is essential for virion
maturation and proliferation, yet its transforming properties have not been formally proven;
withal, E4 causes cytoskeleton deformation (koilocytosis) by bonding its constituent proteins,
thereby allowing the release of preformed virions from the infected cells (Longworth and
Laimins 2004; Doorbar et al. 1997; Doorbar 2013).
E5 protein confers apoptosis resistance to the host cells thereby contributing to their
progressive transformation (Kabsch and Alonso 2002). In fact, the evasion of this programmed
cell death pathway, which the cell activates as a survival mechanism against DNA damage-
induced cell death and other stress stimuli, almost invariably leads to tumour progression and
resistance (Fulda 2010). In this respect, E5 supports the transforming activity of E6 and E7,
7
that controls cell cycle
8
long control region
9
the upstream regulatory region includes the replication origin which contains binding sites for the E2 and E1 proteins

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especially if expressed in tissue culture assays (GENT 2012). Furthermore, E5 suppresses T cell-
mediated responses through the downregulation of MHC histocompatibility leucocyte antigen
(HLA class I) expression, thus allowing the virus to elude the immune system and to maintain an
undetectable viral load over the long term (Song et al. 2015). Also, because of its intrinsic
hydrophobicity, E5 localizes in plasma membrane by binding the 16K protein, thus modifying
the proliferative signaling through transmembrane receptor proteins10
responsive to EGF and
PDGF2; HPV16 E5 is also assumed to inhibit the fusion of early endosomes with acidified
vescicles thereby altering EGF endocytic trafficking and preventing endosome maturation
(Suprynowicz et al. 2010). In addition, the fusogenic activity of 16 E5 that promotes the
generation of tetraploid cells, aneuploidy and chromosomal instability (CIN) as a direct result of
cell-cell fusion, seems to facilitate integration of HPV genomes and further enhances co-
expression of the E6/E7 oncogenes, conferring strong growth advantages on cancerous cells (Hu
et al. 2009). On the basis of this evidence, the hrHPV E5-induced cell fusion and cell cycle
deregulation appears to be a critical event in the early stages of the development of cervical
cancer, providing p53 or apoptosis is perturbed (Gao and Zheng 2010).
Finally, E6 and E7 are the major oncoproteins encoded by the virus, whose actions
combine synergically to cause aberrant multipolar mitoses and abnormal centrosome
reduplication 11
that interferes with cytokinesis, eventually leading to chromosomal
missegregation in either pre-cancerous or HPV-immortalized cells (Mittal and Banks 2017).
Since increased levels of either high-risk E6 or E7 are directly proportional to an enhancement in
extent and severity of neoplasia (Doorbar et al. 201; Melsheimer et al. 2004), it is nowadays well
established that high-risk E6 and E7 genes of the HPV type 16 together provide a subset of the
minimally required carcinogenic changes to kickstart immortalization of primary human
epithelial cells (Munger et al. 1989; Hawley-nelson et al. 1989). Along with continued high level
expression of E6 and E7, additional genomic hits are absolutely required before the cells become
fully transformed, as shown by the requirement for extensive passaging in tissue culture or
addition of other activated oncogenes (Schwarz et al. 1984; Smotkin and Wettstein 1986; Banks
et al. 1987).
Against this backdrop, these two proteins represent the ideal targets for therapeutic intervention
in HPV-induced malignancies (Mittal and Banks 2017), even though many other cervical cancer-
specific biomarkers are yet to be characterized according to Yim and Park (Yim and Park 2007).
Therefore, research attempts towards understanding the molecular mechanisms underlying the
oncogenes' respective functions are of paramount importance not only for developing antiviral
10
such as EGFR which is activated
11
reduplication induced by E7 alters the normal number of centrosomes (Duensing and Munger 2004)

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treatments, such as gene therapy, but also for identifying new biological and/or potential therapeutic
targets of the oncogenic proteins. In fact, the wide variety of cellular proteins that are associated
with E6 and E7 may provide fundamental information concerning the development and progression
of HPV-associated malignancies. With the advent of more advanced proteomics technology, a
growing number of HPV oncogene interaction partners, including regulators of the cell cycle, gene
expression, DNA replication, and cell signaling, were discovered and widely used in the screening,
early diagnosis, prognostication and prediction of response to therapy (Pim et al. 2012).
The current review attempts to illustrate the multiple roles of the major viral oncoproteins during
viral life cycle and carcinogenesis, with particular emphasis on E6’s marked transforming potential
and its dramatic impact on cell polarity control networks and cell trafficking. In this respect, we
pursue assays and extensive experiments over several cellular targets of HPV E6, that shall be
closely analyzed further in this study.
1.2 The diversity of Human Papillomaviruses and the diseases that they cause
Papillomaviridae is an ancient taxonomic group of non-enveloped dsDNA viruses, collectively
referred to as papillomaviruses. Papillomaviruses were first identified in the early 20th century,
when it was demonstrated that an infectious agent could cause papillomas and carcinomas
transmitted between individuals.
To date, several hundred species of papillomaviruses, traditionally called "types", have been
identified based on the sequence of the L1 ORF, which is the most conserved region within the
HPV genome (Humans, n.d.), with over 170 human papillomavirus new specific subtypes being
fully characterized (E. De Villiers et al. 2004) and subsequently classified into 5 genera (α, β, γ, μ,
ν).
Over the past 15 years, a large number of clinical studies have compared many isolated DNA
sequences of different HPV types which, not surprisingly, show striking divergence from one
another (Humans, n.d.). Indeed, human papillomaviruses comprise a diverse family which is
ubiquitous in the human population (Thomas, Pim, and Banks 1999) and are epitheliotropic, with
specific preferences for particular locations (i.e. mucosal versus cutaneous), as well as life-cycle
schedules. In addition to being epitheliotropic, the different papillomaviruses types are also thought
to be strictly host-specific, and therefore rarely transmitted between species.
Their current diversity is due to host/virus co-evolution and recombination events which
ultimately determine different genotypes associated with various pathological conditions and
disease prevalence, which are still under investigation (De Villiers et al. 2004). In addition, HPV
species are transmitted through different strategies within the epithelium and probably differ in the
ways they interact with the host’s immune system (Doorbar et al. 2016), however most of them

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have acquired the ability to either avoid detection or suppress the innate anti-viral response, with
specific regard to Langerhans cells (LC12
) whose inhibition is dependent upon exposure to high-risk
genotype HPV16 (Fausch et al. 2002) and occurs through the deregulation of the PI3K – Akt
pathway, as demonstrated by a Fausch et al., 2005. HPV16 suppression of the phenotypic activation
and immuno-stimulatory function of LC amounts to a wider virally-mediated strategy which
involves many other mechanisms eventually resulting in immune evasion13
(Da Silva et al. 2014).
Stanley et al. estimated that 15% of women who contract high-risk HPV infection are incapable of
mounting an effective immune response (Stanley, Pett, and Coleman 2007); such failure of the
innate immunity to clear persistent HPV infections accelerates the process of malignant conversion.
As mentioned before, HPVs have been classified on the basis of their varying propensities for
advancing cancer (i.e. high-risk versus low-risk) (Lizano, Berumen, and García-Carrancá 2017),
and according to the nature of the lesions they generate, whether cancerous or benign condylomas
(Bernard 2017; E.-M. de Villiers 2013).
Of all the known genera, the Alpha PVs are the most extensively discussed. These include
cutaneous and mucosal types, with the mucosal types further divided into high-risk and low-risk
groups. The cutaneous Alpha types such as HPV 2, 27 and 57 are considered ‘low-risk’, as they
only cause common benign warts, according to AIRC14
; only a restricted set of the typically
innocuous mucosal Alpha types, which inter alia include HPV6 and HPV11 causing benign genital
lesions referred to as condyloma acuminata, have any occasional carcinogenic potential, leading to
some rare instances of papillomatosis, especially in immunocompromised-individuals (Doorbar et
al. 2016). On the other hand, the number of mucosal Alpha strains that have been defined by
WHO15
as high-risk cancer-causing types, in accordance with their ability to originate high-grade
disease, now exceeds twelve (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), with additional
types (68, 73) being recognised as ‘possibly’ carcinogenic. All of these genotypes are, together,
directly responsible for nearly 500 000 new cases of cervical cancer per year globally (zur Hausen
2002; Maxwell Parkin and Bray 2006), as WHO and ICO16
have estimated, with approximately half
of these being lethal (Maxwell Parkin and Bray 2006). Certain cutaneous beta-PV genotypes such
as HPV5 and HPV8 have interestingly been implicated in the development of non-melanoma skin
cancers (NMSC) and/or epidermodysplasia verruciformis (EV) in immuno-suppressed patients with
primary immunodeficiency (Bernard 2017), whereas gamma genera viruses are well-tolerated by
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resident antigen presenting cells in the epithelium
13
such as suppression of T cell effector function, and frequent loss of human leukocyte antigen (HLA) expression
14
Associazione Italiana per la Ricerca sul Cancro
15
World Health Organization
16
Information Centre on Human Papilloma Virus and Cervical Cancer

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host immunity and manage to complete their life-cycle without causing any apparent lesion,
conceivably because of their anciently established adaptation to the host (Doorbar et al. 2012).
The high-risk Alpha types have predominantly been associated with high-grade diseases such as
squamous cell carcinoma (SCC, mostly induced by HPV16) originating at the ectocervix and at the
transformation zone and adenocarcinoma (AC) of the endocervix (mostly induced by HPV18) (Iarc
2007). Among those, HPV-16 is by far the most prevalent mucosal high risk genotype, being
regularly found in low-grade disease and in more than 50% of the world’s cases of cervical
squamous cell carcinoma (Clifford et al. 2003), followed by HPV-18, HPV-31, HPV-45 and others
(zur Hausen 2002). Papillomas induced by HR HPVs 16 and 18 are the most likely to become
cancerous (Pim et al. 2012; Humans, n.d.; Health and Safety Authority 2015), as the high-risk
strains have, for unknown reasons, evolved the ability to persist longer so as to drive cell
proliferation in the basal and parabasal cell layers, thus interfering with maintenance of viral
genomes as episomes in the basal layer and with tight control of viral gene expression (Doorbar
2006).
The issue addressed was determining why only a small fraction of HPVs are powerful
carcinogens, whereas the vast majority of them, including most high-risk types, do not typically
induce neoplasia at the endocervix.
Although cutaneous/mucosal groupings are not tight, the Alpha lineages markedly differ in the
levels of deregulation of viral gene expression, in the concomitant efficiency with which they bind
their targets for degradation, and, finally, in the risk of persistence and association with high-grade
disease (Doorbar et al. 2012). Because functional differences imply diverse HPV-associated clinical
consequences seen in vivo, it is crucial to highlight the divergence between low-risk and high-risk
HPV types in term of pathogenesis: while low-risk HPVs are very rarely related to papillomatosis
or cancer in immunosuppressed people, they mostly cause inconspicuous infections eventually
resolved by the host’s immune system (Doorbar et al. 2012), the high-risk genotypes cause almost
all cases of cervical cancer17
. In particular, high-risk 16 and 18 subtypes can induce moderately or
severely abnormal lesions (dysplasias) on the surface of the cervix, evolving into an abortive18
, non-
lytic infection referred to as high-grade squamous intraepithelial lesion (HSIL) at the
squamous/columnar junction of the cervical transformation zone, a site where productive infection
may be inefficiently supported (Doorbar 2006).
17
however, the high-risk infections rarely progress towards malignancy since the majority of them are cleared quickly
18
even though viral components have been synthesized, no infective virus is produced

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1.3 The HPV Infection
Human papillomavirus (HPV) is the most prevalent viral infection of the reproductive tract
worldwide (Organization 2016), occurring at some point in up to 75% of sexually active women in
developing nations (Kahn et al. 2012) and it is the most common sexually transmitted infection in
the United States (Institute 2015).
Being “the causative agent of 5% of all human cancer” (Mittal and Banks 2017), human
papillomaviruses (HPVs) are responsible for a large number of human malignancies, the most
significant of which is cervical cancer which is a major cause of cancer-related mortality in women
living in developing countries (Pim et al. 2012). In fact, it has been estimated that the majority of
cervical cancers, more than 99% according to Yim and Park (Yim and Park 2005; Clifford et al.
2003), are directly related to previous infection in their cervical tissue with one or more of the
oncogenic types of high-risk HPV. In addition, HPV is the aetiological agent of a large number of
other anogenital cancers, including 90% of anal cancers, and increasing percentage of head and
oropharyngeal, upper respiratory, and even non-melanoma skin cancers (Humans, n.d.; Zelkowitz
2009; Zur Hausen 2009).
Although human papillomaviruses are a prerequisite for generating invasive cervical cancer
(Studies 1999), not all of these dysplastic lesions HPVs cause will necessarily evolve into
malignancy, nor will all of them cause noticeable symptoms: indeed, no more than 8% of the
precancerous changes will develop into early cancer, limited to the whole epithelial layer of the
uterine cervix (carcinoma in situ; CIS), and only a narrow subset of infected cells will develop an
oncogenic phenotype, and all those that do have been found to be infected with high-risk HPV types
(Yim and Park 2005).
Initial infection by HPVs occurs through skin-to-skin contact, since it requires micro-wounds by
which the infectious virions are allowed to access the basal lamina of the epithelial tissue. Under
normal circumstances, the host immune system prevails and therefore most HPV infections resolve
without any treatment within a few months of acquisition, and with no clinically significant long-
term effects (Bosch, Broker, and Forman 2013; Richardson, Kelsall, and Tellier 2003).
Nevertheless, the restriction of viral DNA amplification and production of viral antigens within
the superficial layers of epithelium, which occurs in approximately 10% of cases, inhibits host
immune surveillance, resulting in infections that persist for longer periods during which cell
phenotype and behavior are gradually yet markedly altered, whilst initially not in a visible way. In
fact, HPVs develop seemingly asymptomatic infections that, if not eventually cleared by the
immune system, become evident after prolonged passaging of cells in vitro culture and generally
over a span of 10-20 years in vivo (Burd 2003), during which the mechanisms of the host defence

12
apparently remain indifferent to the practically invisible pathogen (Stanley, Pett, and Coleman
2007).
By contrast to 90% of the transient infections that normally are faded in about 2 years (Winer et
al. 2011), it appears that cervical infection by high-risk oncogenic types, particularly HPV16, tends
to persist longer19
, and may take many months to years to be cleared (Ho et al. 1998; Winer et al.
2011); therefore persistent infections are more likely to develop chronic intraepithelial prominent
lesions (Richardson, Kelsall, and Tellier 2003) by virtue of DNA viral integration, which
dramatically increases cancer risk (Schiffman and Wentzensen 2013). Even non-oncogenic strains,
whose clearance may also take a long time (Giuliano et al. 2002), predispose to malignancy by
undergoing abortive infections where the productive cycle of the virus is not completed (Graham
2017), even though normal patterns of early virus gene expression are perturbed (Middleton et al.
2003).
However, the high-risk infections rarely progress towards a malignant phenotype, the majority of
them being cleared quickly by host immunity; nonetheless, failure of HPV-induced
immunosuppression may allow HPV reappearance from latency, and there may also be cases of
recurrent infection.
2. HPV and CANCER
2.1 Infection and Transformation by high-risk HPV E6 and E7 oncoproteins
Long-term, persistent high-risk human papillomavirus infection is strongly related to
carcinogenesis and, specifically, it has been indicated as a necessary, yet insufficient cause of
cervical neoplasia, according to the International Agency for Research on Cancer (Humans, n.d.).
There is now overwhelming evidence of the direct contribution of HPV E6 and E7 oncoproteins
in the occurrence of cervical carcinoma, as well as in the maintenance of the cancer phenotype
many years after the initial transforming events (Duensing and Munger 2004). Indeed, the continual
high-level expression of both E6 and E7 in cervical cancer-derived cell lines, which is consequent
to viral DNA integration events (Schwarz et al. 1984), is one of the distinctive hallmarks of HPV-
induced malignancy. By contrast, the inhibition of E6 and/or E7 expression or function leads to
cancer cell growth arrest and apoptosis, eventually restoring epithelial homeostasis.
Conversely, in low cell culture passage numbers high-risk HPV immortalized human
keratinocytes are nontumorigenic and pre-cancerous alterations (dysplastic lesions) are not frequent
nor particularly prompt to evolve towards malignancy (Pecoraro, Morgan, and Defendi 1989).
Therefore, cancer is not a typical outcome of HPV infection. Since the rate of spontaneous
19
on average from 12 up to 19 months for HPV16 (Kuhne and Banks 1998)

13
mutagenesis in normal human cells is exceedingly low, transformation requires viral oncoproteins
to be translated in order to implement the accumulation of genetic defects, with substantial time
lapse until the host cell's potent tumour-suppressive mechanisms are actually overcome (Duensing
and Munger 2004).
E6 and E7 are considered as viral tumorigenic genes that maintain replication competence and
whose products are well-known for their intrinsic transforming characteristics and highly
pleiotropic functions, which occur through the binding to multiple host-cell targets, including those
negative regulators of the cell cycle, which maintain genome integrity.
Although the roles of E6 and E7 in the viral life-cycles of both high- and low-risk HPVs are
quite well-understood, the cellular targets of the low-risk E6 and E7 proteins remain relatively
undetermined.
E6 and E7 early gene products (encoded by high-risk HPVs) typically affect diverse intracellular
events such as transmembrane signaling, immortalization of primary cell lines, transformation of
established cell lines, and dramatically subvert chromosomal stability (Yim and Park 2007).
The ability of E6 and E7 oncogenes to establish malignant conversion in infected stratified
epithelial cells basically relies upon their association with tumour suppressor proteins p53 and
p105Rb respectively, which causes deregulation of the normal cell cycle controls so that the HPV-
positive terminally differentiating suprabasal cells withdraw from the cell cycle arrest and instead
induces excessive DNA synthesis and expression of markers for cell proliferation (Jeon, Allen-
Hoffmann, and Lambert 1995; Flores et al. 1999), thus supporting amplification of the previously
integrated viral genome and subsequent generation of progeny (Humans, n.d.).
Although neither E6 nor E7, when expressed individually, have any significant capability to
immortalize primary human keratinocytes, their complementary roles in mis-directing pro-survival
(E7) and pro-apoptotic (E6) signaling pathways and stimulating cell proliferation (E7) have proved
to be sufficient and necessary for inducing rapid centrosome amplification and various
chromosomal aberrations which in many instances drive cancer onset and metastasis (Munger et al.
1989; Duensing et al. 2000; Riley et al. 2003). Most specifically, the continued combined
expression of E7 and E6 results in inactivation of pRb’s and p53 tumor suppressor’s functions
respectively, following deregulation and loss of regular cell cycle checkpoints which allows
retention of genetically aberrant cells (Bodily and Laimins 2011). In this regard, high-risk E6 and
E7 viral oncoproteins can each independently interfere with the DNA damage response (DDR) and
contribute to disrupt genome integrity in normal human epithelial cells, thus promoting overall
carcinogenesis and tumor evolution (McKinney, Hussmann, and McBride 2015; Münger et al.
2004).

14
E7's activity in promoting unscheduled DNA synthesis triggers an apoptotic response20
, which is
counteracted by E6's activity in inducing the degradation of proapoptotic signaling molecules, such
as p53 and Bak. Nevertheless the specific role of E7 in the viral life cycle has not yet been fully
elucidated, it is evident that E7 alone has a high affinity towards the so-called 'pocket domain'21
of
tumor-suppressing retinoblastoma gene protein (Rb) whose biological function is to repress the
expression of replication enzyme genes, as well as apoptosis- and cell cycle- associated genes,
through the binding to E2F-family transcription factors22
(Lipinski and Jacks 1999). Three of these
transcription activators, namely E2F1, E2F2 and E2F3a, are responsible for G1/S transition;
however, the binding of pRb to E2F1 inhibits the transcriptional activation of E2F1 by preventing it
from interacting with many S phase specific genes. As long as the dephosphorylated form of pRb
complexes E2F-DP thus deregulating E2F-mediated activity23
, transcription during S phase is
repressed and consequently the cell entry into S phase is no longer permitted; under normal
circumstances, pRb-E2F-DP complex mediates G1 arrest (Munger et al. 1989).
In the scenario of pRb-mediated cell cycle interruption, the differentiated, post-mitotic cell will
not acquire transforming potential nor any invasiveness or motility, unless the virus provides the
functional instruction for the cell to destabilise Rb tumor suppressor protein. Therefore, there must
exist a mechanism HPVs establish in order to render their host cell permissive for DNA synthesis
during the productive stage of the viral life cycle.
Since at least two mutational events have constantly been observed as a distinctive hallmark of a
large subset of malignant cells (Yokota 2000), it was plausibly hypothesized that multiple tumor
suppressor genes have to be inactivated by genetic alterations frequently occurring in human
neoplastic diseases such as retinoblastoma, which is indeed caused by recessive loss of normal anti-
oncogenes (Knudson 1985). In this respect, direct mutation of pRb or its irreversible inactivation by
viral proteins are crucial events for malignant conversion. As such, HPV-16 E7 has been
successfully revealed to directly mediate pRb degradation through interaction with the S4 subunit of
the 26S proteasome, so that no ubiquitin tagging is needed (Gonzalez et al. 2001).
Elucidation of HPV’s non-lytic life cycle and its intimate association with the diverse
differentiation stages of the host cell keratinocyte will be necessary to further comprehend the direct
contribution of E7 in the maintenance of a stem cell-like, aggressive phenotype of cervical cancer
cells.
20
E7 oncogene has been demonstrated to be necessary for triggering apoptosis (Cheng et al. 2001)
21
which is frequently inhibited in its function by E7 and is sufficient for degradation.
22
this ability to arrest cellular proliferation correlates with the tumor suppression function of Rb
23
which occurs in S, G2 and M phases

15
Initial infection requires that the virus gains access to the proliferating basal cells of the
epithelium, presumably through microabrasions (IARC) (Figure 2). Once internalized24
, the viral
genome undergoes a transient phase of rapid mitoses (the so-called "establishment replication"),
which enables its establishment within the host cell. The viral genome does not integrate in a
normal life cycle, but rather it replicates as an episome (MacBride 2011): the subsequent
"maintenance replication" in concert with the cellular replication maintains the viral episome at a
steady state in daughter cells, keeping it at low copy number (generally of 50-100 genomes per cell).
Following viral DNA synthesis and cytokinesis in undifferentiated, mitotically active
keratinocytes25
, the offspring cells begin migrating towards the uppermost cell layers where
epithelial differentiation leads to cell cycle exit (Cheng et al. 1995), and the genes encoding
components of the host DNA replication machinery are no longer transcribed. Therefore, the host
DNA replication machinery is not able to sufficiently sustain the high proliferation rate the virus
demands for its episomal26
DNA amplification. Accordingly, cervical cancers consists of cells that
have lost their ability to differentiate (Thomas et al. 2008). Since HPV replication requires epithelial
differentiation, which is indeed essential for a productive viral life cycle, cervical cancers cells do
not produce new virions. This is consistent with HPV’s attempts to ensure its own survival by
avoiding malignant transformation, which is a "dead-end" event for the virus. Instead, HPV just
wants to complete its life-cycle. Therefore, high-risk HPV types have evolved to maintain their
infected cells differentiation-competent, in a sort of artificial S-phase, in order to establish a
persistent infection which only on rare occasions may result in cancer. In this regard, E6 and E7
have fundamental roles in the virus life cycle27
, and are rarely oncogenic. In fact, transformation
only occurs as a mistake.
Notwithstanding, as a consequence of persistence of HR HPV lesions, the accumulation of
genetic alterations over extended periods of time has permitted to bypass the existing constraints on
cell cycle progression, mainly through E6’s and E7’s immortalizing activity, which however has
been shown not to be tumorigenic in nude mice, nor in primary human keratinocytes assays
(Pecoraro, Morgan, and Defendi 1989), thus proving that HPV infection is necessary but not
sufficient for cancer promotion (Peters, Jan-Michael. Harris, J. Robin. Finley 1998).
Whether genome instability provides any benefit to the virus is still an open question,
nonetheless, as outlined in the previous sections, it has been proved to arise as an accidental,
undesired outcome of suppressing the p53 and pRb pathways (Bodily and Laimins 2011).
24
by nuclear translation
25
which are under immune surveillance
26
viral DNA has to exist episomally in order to be replicated
27
mainly in preventing apoptosis and tumour evolution

16
Figure 2. Model of infected stratified epithelium. The multiple layered sheets of
keratinocytes with various shapes, in different stages of differentiation, are, from
bottom: stratum basale made of mitotic cells that stably maintain the viral episomes,
stratum spinosum, stratum granulosum (with brown keratohyalin granules), and stratum
corneum. When the basal infected cells divide, the HPV genome replicates in synchrony
as plasmids (dark blue circles). As these cells detach and progress upwards as part of the
differentiation program, increased levels of HPV E6 and E7 promote unscheduled cell
cycle progression and subsequent viral genome amplification (dark blue particles).
Source: McKinney, Hussmann, and McBride 2015
In order to keep the host cell replicative genes active as long as possible, thereby establishing an
environment conducive to its own amplification, HPV reprograms and subverts various signaling
pathways that regulate host cells replication, principally driving S-phase re-entry in the upper
epithelial layers: in fact viral replication only takes place as the infected cell moves from an S to a
G2-like phase before committing to full differentiation (MacBride 2011).
The normal terminal differentiation of multiple-layered keratinocytes is retarded primarily by the
activities of the E6 and E7 oncoproteins, whose key role is to overcome the restrictions on cell cycle
progression and ensure a high viral genome copy number28
during the productive phase of the viral
28
up to thousands per cell (Flores and Lambert, 1997)

17
life cycle (Flores et al. 2000); the high-risk HPV types are also assumed to account for cell
proliferation in the basal and parabasal layers (Doorbar et al. 2016).
The maintenance of viral replication competence upon differentiation to provide cellular
replication factors is achieved through diverse yet coordinated activities such as manipulation of the
DDR, which allows the recruitment and usurpation of replication factors, abrogation of growth
arrest signals carried out through the inactivation of the pRb pathway by E7 as well as ablation of
of p53 by E6, and through the deregulation of other cell cycle checkpoint systems, cyclins and
cyclin‐dependent kinase inhibitors, all events which cumulatively induce hyperplasia (Funk et al.
1997; Jones, Alani, and Munger 1997; Noya et al. 2001).
HPV 16 E7 has been shown to be necessary and sufficient to induce suprabasal DNA synthesis
by uncoupling cellular differentiation, which is perturbed, from proliferation (Jones, Alani, and
Munger 1997; Flores et al. 2000). This occurs owing to the fact that pRb, in particular its most
conserved region called “pocket domain”, is one of the targets of E7. The high-risk E7 oncoprotein
is thereby able to disrupt the interaction between pRb and E2F, accordingly E2F factors are released
in their transcriptionally active forms (Chellappan et al. 1992), thus promoting the expression of
genes encoding the DNA replication machinery and allowing the cell to progress out of G1 and into
S phase, as has been observed in fully differentiated, yet still proliferative, human keratinocytes.
As a result of HR E7's efficient abrogation of pRb function, stabilization of p53 is heightened;
nonetheless, apoptosis is hindered as the HR E6 proteins have evolved to induce degradation of p53
(Howie, Katzenellenbogen, and Galloway 2009).⁠
Moreover, anti-proliferative responses mediated by DDR genes are evaded by HPV oncoproteins.
In fact, a large number of viruses disable some components of the DDR which normally prevent the
cell cycle from further advance, and take advantage of others to synthesize viral DNA in productive
infection (Luftig 2014).
The DDR pathway accurately preserves genomic integrity, but also activates specific
downstream checkpoints during each phase of the cell cycle, stalling it until DNA repair is
completed. For instance, a DDR deals with increasing stabilization of p53 with consequent
transcriptional upregulation of its target, namely the cyclin-dependent kinase (cdk) inhibitor p21,
which arrests cells in G1 phase by preventing the cyclin E/cdk2 and cyclin A/cdk2 kinases from
promoting S-phase entry (Levine 1997; Lakin and Jackson 1999; Besson, Dowdy, and Roberts 2008)
(Figure 3).

18
Figure 3. Diagram of the DNA Damage and Repair Response Pathway. DNA breaks
and collapsed replication forks are detected by sensors that mark the site of damage and
activate three kinase signaling cascades (ATM, ATR, and DNA-PK). The signal is
transduced and amplified by several mediator and effector proteins, eliciting a huge
remodel chromatin, cell cycle arrest and/or apoptosis. Source: Noguchi 2010
Likewise, the ATM/ATR-related protein kinases which sense DNA damage relay the anti-
proliferative signal to ATR and ATM, which phosphorylate downstream kinases such as Chk1 and
Chk2 affecting p53 and Cdc25 activities, respectively. More specifically, phosphorylation by Chk1
effector inhibits Cdc25 family members, which are responsible for progression at G1-S stage of the
cell cycle, and activates Wee1 (Carr 2002; Nyberg et al. 2002). On the other hand, activation of
ATM in response to double-strand breaks (DBSs) and, subsequently, of Chk2 downstream kinase,
triggers pro-apoptotic signaling by p53 with the concomitant growth arrest that would be
detrimental for viral replication in dividing cells. In either case, the cells cease to proliferate and
enter either apoptotic or senescent states.
Since E7-induced cervical neoplasia formation was detected even after the E7-pRb interaction
was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele, pRb
inactivation appears to be insufficient for HPV E7 to contest and hijack the carefully crafted DDR,
suggesting that other E2F regulators besides pRb are indispensable targets of E7.

19
So as to cope with differentiation-induced or DDR-induced cell cycle-arrest, E7 mediates
upregulation and accumulation of MRN, as well as stimulation of ATM through the constitutive
activation of STAT-5, which properly engages the DDR machinery which has been demonstrated to
be required for HPV genome amplification (upon differentiation) as well as late gene expression in
differentiating keratinocytes (Hong and Laimins 2013; McKinney, Hussmann, and McBride 2015).
In addition, E7 binds E2F6 (Mclaughlin-drubin, Huh, and Mu 2008) as well as the DNA damage
sensor ATM (Moody and Laimins 2009) in order to hinder anoikis induction (Bodily and Laimins
2011). Both low risk and high risk E7 confer resistance to anoikis and anchorage independent
replication by interfering with the microtubule associated protein p600, thus disturbing the integrity
of keratin organization (Huh et al. 2005). Furthermore, E7 promotes mitotic entry despite the
presence of DNA damage signaling by accelerating the degradation of the Chk1 binding protein,
claspin, which is required for the ATR-dependent activation of Chk129
(Spardy et al. 2009).
HPV-16 E7-expressing suprabasal cells show delay in cellular differentiation and elevated cdk2
kinase activity despite high levels of p21 (Cip1) and p21-cdk2 complexes that would normally
suppress cervical cancer promotion (Shin et al. 2009). This is due to the ability of high risk E7 to
interact with the cyclin kinase inhibitors p21 (Cip1) and p27, deregulating their cell cycle inhibitory
functions, and with cyclins A and E to enhance their activities: p21-mediated inhibition of cdk2
activity, as well as of cyclin A and E-associated kinase activities are then abrogated (Jones, Alani,
and Munger 1997; He et al. 2003; Nguyen and Münger 2008).
Overall these strategies allow E7 to create a unique cellular milieu that maintains viral
proliferation despite activated DNA damage checkpoints in infected differentiated keratinocytes and,
at the same time, that render cells resistant to apoptosis. However, this inadvertently leaves cells
highly susceptible to mutation and genetic instability, as much as deregulation of E2F1 and
subsequent nucleotide deficiency by E7 promotes replication stress (Bester et al. 2011).
2.2 The high-risk HPV E6 as a potent viral oncoprotein for cell transformation
As was pointed out in the previous paragraphs, the E6 oncogene is required for progression to
the late productive stages of the viral life cycle, in addition to being implicated in cancer onset.
Even though E7 exerts extensive mutational impact on benign tumor initiation, multistage
carcinogenesis needs the high-risk E6 oncoprotein in order to drive the end stages of malignancy
(Song et al. 2000): the expression of the cutaneous beta-HPV E6, for instance, equally manipulates
the host DDR network at many levels, thus rendering cellular genomes vulnerable to destabilizing
events (Wallace, Robinson, and Galloway 2014).
29
briefly Cdk2 is the engine that draw cells towards division and it is regulated by the CKIs p21 and p27.

20
Essentially, E6 accelerates expansion and malignant conversion of tumours promoted by E7
(Song et al. 2000) which most relevantly affects the cervix, head and neck (Mittal and Banks 2017).
As a direct response to E7-induced stabilization of p53 in the suprabasal layers of the rafts
(Flores et al. 2000), E6 mediates ATP-dependent p53 degradation through ubiquitin-mediated
proteolysis, thereby preventing induction of apoptosis in response to unscheduled S‐phase entry
stimulated by both oncoproteins30
(Howie, Katzenellenbogen, and Galloway 2009; Scheffner et al.
1990; Stubenrauch and Laimins 1999).
The initial expression of viral oncogenes undermines the normal p53 proteasomal turnover by
the ubiquitin ligase Mdm2 (Honda, Tanaka, and Yasuda 1997; Kubbutat, Jones, and Vousden 1997),
so that p53 is both stabilized and activated (Ashcroft and Vousden 1999). As a consequence, in
HPV-positive cancer cells p53 degradation depends entirely on E6 (Hengstermann et al. 2001). This
indicates that E6 is able to reactivate degradation of p53 under conditions when this would be
normally inhibited, e.g. after DNA damage (Thomas, Pim, and Banks 1999). In fact, mucosal high
risk E6 oncoprotein31
binds to the tumour suppressor gene product p53 determining its proteasome-
dependent ablation, and therefore abolishing its surveillance activities, which inter alia include
DNA damage repair (DDR) mechanisms, protection from mutation and genetic aberration, control
of apoptotic demise of transforming cells (Levine 1997; Lepik et al. 1998). The functional loss of
the tumour suppressor protein p53 is certainly a major driver of cancer development, as it directly
inactivates the checkpoint systems’ response to DNA damage32
, eventually causing hyperplasia
(Lakin and Jackson 1999; Kessis et al. 1993). In addition, E6-mediated dysregulation of p53 is
absolutely required for the amplification of viral DNA in a stratified epithelium (Kho et al. 2013).
Wild-type p53 is recruited, by/via the E3 ubiquitin ligase E6-associated protein, to a trimeric
complex comprising E6, p53 and E6-AP, which is driven towards ubiquitination and rapid turnover
in the exclusive presence of HR E6: HR E6 targets E3 Ub ligase E6AP before associating with p53
so that ubiquitin peptides are specifically transferred from E6AP to p53, which is finally redirected
towards the 26S proteasome (Banks, Pim, and Thomas 2003).⁠
It has been documented that in HPV-positive cells, the nuclear localization of p53 in response to
DNA damage is blocked even if proteasome degradation is inhibited (Mantovani and Banks 1999).
Even though E6 functionally inactivates p53 principally through the ubiquitin-proteasome pathway
(Scheffner et al. 1990), alternative strategies are adopted to counteract p53 growth suppressive
activity, hence p53 levels are invariably low in E6-expressing cells (Matlashewski et al. 1986).
30
E7 has been shown to augment the levels of the cellular proteins mdm2, and p21. However, the absence of p53 in the
basal layer of BC16/E7(+) rafts indicates that E6 is performing its expected role of targeting p53 for degradation there
(Flores et al. 2000)
31
low risk and cutaneous HPV E6 types are unable to target p53 for degradation through the proteasome pathway, even
though LR E6 can interact with E6AP
32
including growth arrest and apoptosis

21
Firstly, cytoplasmic sequestration may be due to masking p53's nuclear localization signal by E6
binding to the p53 C-terminus, or due to enhanced nuclear export of p53 (Mantovani and Banks
2001). Whilst both high- and low-risk mucosal HPV E6 proteins are able to bind the p53 C-
terminus, it is not such interaction that induces degradation of p53 in vivo, which rather appears to
be the result of high risk E6 protein's stronger33
association with the core region of p53 (Li and
Coffino 1996). Secondly, the E6AP-independent mechanism by which high-risk E6 abrogates
transactivation of p53 target genes does not only depend on p53 destabilization34
, since E6 mutants
defective for degradation can abrogate transcriptional activation by p53 in vivo (Pim et al. 1994).
This is, at least in part, explained by the ability of E6 to prevent p53 from binding its DNA
recognition site (Lechner and Laimins 1994; Thomas et al. 1995) and/or of the high-risk HPV-16
E6 to repress p53-responsive promoters by binding to the transcriptional coactivator p300 CBP
(Zimmermann et al. 1999). Nonetheless, promoting p53 degradation is absolutely necessary to
prevent p53-induced apoptosis, proving that this activity of p53 remains unrelated to its role as a
transactivator (Thomas et al. 1996).
Despite all these cooperative strategies, not all p53 is degraded in cervical cancer lesions, as a
series of experimental findings have reported (Cooper et al. 1993; Lie et al. 1999; Mantovani and
Banks 1999). These detectable levels of p53 in HPV-infected cells might be indispensable to allow
a fine-tuning of the activity of E6 with respect to p53 during viral replication (Mantovani and Banks
2001). In fact, in order to elicit a productive infection, viral DNA amplification needs to be
controlled and it is plausible that the activity of E6* could ensure the presence of a limited amount
of p53 at the replication sites, where it could both prevent overreplication of the viral genome and,
possibly, enhance the replicative fidelity of DNA polymerase by means of its proofreading 3'→ 5'
exonuclease activity (Albrechtsen et al. 1999).
The exact nature of the E6/p53 interaction may vary according both to HPV type, and to
intratypic variations in E6 sequence (Thomas, Pim, and Banks 1999). Alternatively spliced variants
of E6 have been recognized as a means by which the virus could potentially manipulate the E6/p53
interaction. High-risk HPV transcription patterns include a series of alternative splices which
generate a multitude of mRNAs (Doorbar et al. 1990), among which those encoding four
truncations of the full-length E6 protein (called E6*I-IV) commonly referred to as the E6* proteins.
E6* proteins are generally unstable when translated in vitro (Shally et al. 1996), indicating that
rapid turnover may explain the low levels of E6* protein. Since no HPV-16 E6* immortalization
capacity has emerged from previous investigations on primary human keratinocytes, the splicing
event was believed to be merely intended to increase the efficiency of E7 translation (Sedman et al.
33
enhanced by the presence of E6AP
34
it is indeed partially independent of targeting p53 for degradation

22
1991). This general assumption has been confuted by the contrary evidence that E7 is translated
equally effectively from spliced and unspliced transcripts (Stacey et al. 1995), and in vitro studies
have attested that translation of HPV-16 E6*IV (Shally et al. 1996) and HPV-18 E6*I (Pim,
Massimi, and Banks 1997) can inhibit the E6-directed, ubiquitin-mediated degradation of p53, thus
suggesting the prospective role of E6* proteins in reducing the risk of hyperplasia by modulating
the E6-p53 interaction. Most specifically, p53-null cell assays showed that coexpression of HPV-18
E6*I with p53 and full-length E6 resulted in an inhibition of E6-directed degradation of p53, which
was not observed in cells lacking either p53 or E6 (Pim, Massimi, and Banks 1997). These
observations provide compelling evidence that HPV-18 E6*I can bind in vitro to full-length E6 and
also to E6-AP, but not to p53 itself, and that this interaction hampers the formation of the E6-AP/E6
E3 ligase that is specific for p53. As it is no longer poly-ubiquitinated, p53 is still able to perform
its tumour suppressor function and, accordingly, to neutralize E6-promoted transformation.
Since pathways both upstream and downstream of p53 are intact in cervical cancers (Butz et al.
1995), the E6-p53 interaction could be of utmost importance for therapeutic intervention (Thomas,
Pim, and Banks 1999). Up to now, genotoxic35
treatments have oftentimes resulted in down-
regulation of E6 mRNA and elevation of p53 levels, and subsequently of p53 mediated
transcriptional activation and apoptotic response (Butz et al. 1996). Therefore, it would be tempting
to speculate that blocking E6 mediated degradation may be adequate to reactivate p53 within the
infected cells, nonetheless, abstracting from a few rare cases where it could have therapeutic
potential, this is not necessarily the case: the inhibition of E6-induced ablation does not always lead
to increased p53 levels nor to p53 reactivation, since E6 uses diverse other deleterious strategies to
affect p53 function. Indeed, despite increased levels of p53 protein36
, the subsequent correct nuclear
localization of p53 appears to be perturbed and additional stimuli are needed to activate p53. In
several cervical cancer cell lines p53 can be stabilized only after additional genotoxic insult,
indicating a lack of intrinsic signals for activating p53 despite the presence of viral oncogenes
(Mantovani and Banks 1999). This experimental evidence validates the notion that E6 has activities
other than E6–E6AP interaction which are equally required for its contribution to malignancy.
2.3 E6AP-independent targets of E6 and the PDZ binding motif: host proteins associated
with HPV E6
Interestingly, mutants of E6 that fail to abrogate p53 function are still able to immortalize,
suggesting that other alternative activities of E6 also contribute to its transforming capability (Pim
et al. 1994; Nakagawa et al. 1995). Notwithstanding the prominent function of HPV E6 is the
35
with DNA damaging agents
36
as a consequence of the inhibition E6 induced degradation

23
proteolytic inactivation of certain pro-apoptotic factors, such as p53, Bak37
or Bax, through the
ubiquitin-proteasome pathway, it has been extensively demonstrated that E6 is a multifunctional
protein affecting a plethora of additional host proteins which are involved in cellular controls for
proliferation and differentiation, and possibly also represent valid targets for therapeutic
intervention (Thomas, Pim, and Banks 1999).
Besides complementing E7’s activity by preventing p53 accumulation in the nucleus, and
altering epithelial differentiation to promote continual viral proliferation, high-risk HPV E6 proteins
perform a wide range of oncogenic activities that are independent of p53 38
(Figure 4) and
undermine additional pathways to overcome anti-proliferative effects, even when p53 is not
destabilized (Yim and Park 2005). In fact, E6 proteins act primarily as scaffolds for many other
interacting cellular substrates involved in diverse processes, as they lack any known enzymatic
activities (Bodily and Laimins 2011). Ongoing research is proposed to provide further mechanistic
insight into the transduction pathways and the way by which they are perturbed or are consistently
rendered dysfunctional to sustain HPV propagation.
A deeper understanding of the process needs further analyzation of E6 molecular structure,
which is depicted in Figure 5. The HPV E6 proteins are 18-kD polypeptides that consist of 158
amino acid residues and contain four Cys-X-X-Cys highly conserved motifs which permit the
formation of two zinc-finger domains (Cole and Danos 1987; Barbosa, Lowy, and Schiller 1989).
37
in particular, the over-expression of Bcl-2 suppresses the death function of Bak (E. H. Cheng et al. 2001)
38
which do not include p53 deregulation

24
Figure 4. The high risk HPV E6 protein interacting partners and associated activities.
Since either E6 and E7 have a very large number of cellular substrates whose identity
differs between HPV types of the same high-risk clade, as well as between the high- and
low-risk arrays themselves, there is apparently no single characteristic that could define
high-risk types as cancer-causing. Accordingly, research has outlined little concordance
between cancer risk, and the capacity of the E6 oncoproteins from the high-risk types to
degrade p53 or PDZ domain-containing substrates, and thereby to induce keratinocyte
immortalization. Source: Mittal and Banks 2017
These zinc-finger motifs, whose integrity is essential for E6 function (Kanda et al. 1991), are
separated by a hydrophobic domain and are followed by a short carboxy-terminal domain, which, in
the case of the high-risk mucosotropic HPV types, contains a PDZ-binding motif (Kiyono et al.
1997; Lee, Weiss, and Javier 1997) carrying, in turn, an overlapping site for protein kinase A (PKA)
phosphorylation that can negatively regulate the association of E6 with its PDZ domain-containing
substrates39
(Kühne et al. 2000). This complex multimeric conformation allows E6 to dimerize quite
39
once phosphorylated by PKA, E6 proteins are no longer able to interact with their PDZ-domain-containing substrates

25
effectively in vitro40
, and therefore to associate with multiple interacting partners at any given time
(Nominé et al. 2006), whose deregulation equally contributes to HPV pathogenesis.
Figure 5. In vitro mutational analyses of the high risk HPV-18 E6 protein have defined
different binding sites along with the PKA consensus phosphorylation motif, two of
which appear to be required for p53 binding and degradation (Pim et al. 1994).
Subsequently, Li and Coffino (Li and Coffino 1996) detected two distinct regions of
p53 that are able to associate to E6: one is situated within the core region of p53
(between amino acids 66-326), and actually correlates with the induction of p53
degradation, and another, situated at the C terminus (amino acids 376-384), is bound by
both benign and oncogenic types of E6 without having any effect upon p53 stability.
Furthermore, additional cellular host proteins have been reported to interact with E6,
and thereafter categorized by their site of interaction: the N-terminal binding proteins
(amino acids 30-70) E6AP, Mcm7 and p53; the C-terminal binding proteins (amino
acids 100-140) E6AP, E6TP1, p300/CBP, Bak, AMF-1/Gps2, Paxillin, PDZ proteins
and p53. Source: Thomas, Pim, and Banks 1999
2.4 E6-induced perturbation of cellular pathways that sense cell polarity and trafficking
In summary, HPV E6 has been shown to be intimately implicated in the perturbation of:
the cellular controls of DNA replication, through hMcm741
, E6TP1, p300/CBP, Gsp2, the
interferon regulatory factor IRF-3 and ADA3;
chromosomal structure, through the upregulation of telomerase42
activity at a transcriptional
level is required for life span extension and immortalization of primary human keratinocytes
40
in vivo E6 dimerization still lack formal demonstration (Pim et al. 2012)
41
DNA replication licensing factor Mcm7 is degraded by the ubiquitin pathway via E6-AP (Kuhne and Banks 1998)⁠
42
which ensures telomere integrity upon rapid mitoses

26
(Klingelhutz, Foster, and McDougall 1996; Stöppler et al. 1997; Kiyono et al. 1998;
Galloway et al. 2005)⁠⁠
cytoskeletal structure, through the focal adhesion protein paxillin;
cell-cell adhesion, polarity and proliferation control, by means of proteins which contain a
PDZ motif, such as hDlg, hScrib, PKN, MAGI-1, MAGI-2, MAGI-3 or MUPP1;
signal transduction, via E6TP1, paxillin, c-Myc and E6BP;
differentiation, through the EF-hand calcium-binding protein E6-BP/ERC55/RCN2
(reticulocalbin 2) and c-Myc;
immune evasion, through TNF receptor 1;
p53-independent43
apoptosis (Pan and Griep 1995)⁠, partially as a result of E6-induced
ubiquitin-mediated degradation of the c-Myc and Bak pro-apoptotic proteins (Gross-
Mesilaty et al. 1998; Thomas and Banks 1998⁠).
Therefore, since the vast majority of the effects observed in cells over-expressing E6 are detached
from the proteolysis of p53, it is evident that E6 hijacks additional, p53- and E6AP-independent
signaling routes to abrogate (p53’s) growth suppressive activities. This occurs by means of the
highly conserved C-terminal PBM domain which indeed is not involved in p53 binding and
degradation (Crook, Tidy, and Vousden 1991; Pim et al. 1994; Li and Coffino 1996) but instead
mediates the interaction with a series of PDZ domain-containing proteins (Zhang et al. 2007). Many
of these cellular proteins are membrane-associated and/or involved in the regulation of the same
signaling pathways, which are implicated in control of cell polarity (Bilder, Li, and Perrimon 2000;
Thomas et al. 2008) cell proliferation44
(M. L. Nguyen et al. 2003)⁠, and cell attachment (Woods et
al. 1996), as well as in clustering ion channels, receptors, and adhesion molecules to specific
structures at the membrane-cytoskeletal interface of polarized cells (Kim 1997)⁠. All of these
interacting partners are characterized by the common presence of the PDZ domains (they are listed
in Table 1).
PDZ (PSD95/Dlg1/ZO1) domains are 80-90 amino acid-long regions that are sites for protein-
protein interaction, and which are recognised by highly conserved PDZ-binding motifs (PBMs).
The high-risk HPV E6 proteins have a type 1 PBM at their extreme C-terminus whose canonical
sequence (-x-S/T-x-V/L) is conserved in all high-risk types. Actually many crystallographic and
NMR studies showed that at least 7 more non-canonical residues lying outsite the precise PBM
consensus sequence contributes towards determining substrate specificity45
(Charbonnier et al. 2011;
Zhang et al. 2007)⁠. The high degree of variation of the PBM region among the different high risk
43
occurring without inducing ubiquitin-mediated p53 proteasome degradation
44
the PBM has been demonstrated to induce abnormal lens cell growth (Nguyen et al. 2003)
45
E6’s high substrate specificity in the recognition of both Dlg and MAGI-1 is due to the diversity of PBMs and their
kinase recognition sequences

27
E6 genotypes, suggests that E6 proteins differ significantly in the way they recognize diverse PDZ
domain-containing substrates whose identity tends to vary; this is consistent with the large number
of different PDZ domain-containing proteins targeted exclusively by the mucosally derived high-
risk E6 proteins, with the twelve up-to-date identified ligands being directed to proteasome
degradation (Massimi et al. 2004; Thomas et al. 2016).⁠
Table 1. Known PDZ domain-containing ligands of E6; several of them harbour
multiple PDZ domains or other interaction motifs so that they can function as molecular
scaffolds for the assembly of multifunctional protein complexes. Twelve of these PDZ
partners are targeted for proteasome-mediated degradation by mucosal high-risk E6
oncoproteins: Dlg (Gardiol et al. 1999), Scrib (Nakagawa and Huibregtse 2000;
(Thomas et al. 2005)⁠, MAGI-1 (Glaunsinger et al. 2000; Thomas et al. 2001)⁠, MAGI-2
& MAGI-3 (Thomas et al. 2002)⁠, PDS95 (Handa et al. 2007)⁠, NHERF1 (Accardi et al.
2011)⁠, MUPP1 (Lee et al. 2000)⁠, PATJ (Latorre et al. 2005; Storrs and Silverstein 2007)⁠
PTPH1/PTPN3 (Jing et al. 2007), PTPN13 (Spanos et al. 2008)⁠, PDZRN3 (Thomas and
Banks 2015)⁠. Source: Ganti et al. 2015
The multifunctionality of high risk E6's PBM is also due to its potential phospho acceptor site, a
highly dynamic region whose phosphorylation by different kinases results in turn in a dramatic

28
inhibition of E6's ability to recognize and interact with its PDZ substrate (Kühne et al. 2000; Boon
et al. 2015)⁠. HPV 18E6 is exclusively phosphorylated by PKA, whose levels are high upon
differentiation, whilst HPV 16E6 can be phosphorylated by PKA or AKT which is abundant in
proliferating cells (Boon and Banks 2013; Boon et al. 2015)⁠. Because the subtle mechanisms
modulating PBM-PDZ interactions are species-specific, it is plausible that the E6 PBM function
would be differentially regulated through the progression of the viral life cycle, both in the
recognition of PDZ-containing substrates and in its interaction with phospho-dependent cellular
proteins (Boon et al. 2015)⁠. Recent studies demonstrated that phosphorylation of the PBM confers
an additional function upon the E6 protein: a strong direct association with a family of versatile
molecular regulators referred to as 14-3-3 proteins (Boon and Banks 2013)⁠. These acidic proteins
function as signal transducing adapters which bind to a large repertoire of proteins involved in
control of metabolism, cell cycle, trafficking, apoptosis, cytoskeletal maintenance, tumor
suppression, and transcription (Benzinger et al. 2005)⁠. Therefore, it is conceivable that the
interaction of E6 with 14-3-3 proteins in a phospho-specific manner raises modulation of their
function so as to maintain an environment favorable for viral genome amplification. It has been
hypothesized that phosphorylated E6 could be sequestered by the 14-3-3 proteins and, consequently,
be unable to target the PDZ proteins that are crucial for maintaining structural integrity of the
infected cell. In this way, this intricate phospho-regulation which compartmentalises E6's function
during the various stages of the viral life cycle appears to be required for promoting cellular
proliferation (Ganti et al. 2015).
Although E6’s multifunctional structure entails overlapping interaction sites (Nominé et al. 2006),
the ordered conformation of the PBM allows specific mutations to be introduced within the PBM
without affecting any of E6’s other activities: studies on E6 mutants lacking a PBM and expressing
E6 demonstrated that the PBM is required for the expansion of replication-competent cells and for
the maintenance of long term viral episomal DNA (Delury et al. 2013)⁠; Park, 2002), and, possibly,
for the contribution to the loss of cell polarity46
and cell contact regulators, thus driving the cell
towards hyperproliferation and, eventually, invasion (McCaffrey et al. 2012)⁠. In fact, altered
polarity can profoundly impact the trafficking of proteins to the apical and/or basolateral regions,
resulting in aberrant signalling which is due to the mislocalization of receptors as well as to the
inappropriate distribution of cell adhesion molecules or matrix degradative enzymes at the cell
surface. All these events may in turn promote cytoskeletal defects, migration and transition towards
a transformed phenotype (in EMT) (Goldenring 2013)⁠⁠.
46
the establishment and maintenance of cell polarity, which is essential for the cellular pathways to organize and inter-
pret external signals, hinges on the correct spatio-temporal distribution and levels of expression of polarity control com-
plexes (the Scribble-Dlg, the Par-aPKC, and the Crumbs) (Ganti et al. 2015)⁠, perturbed by both viral oncoproteins

29
The E6 C-terminus harbours a Class 1 PBM, or PDZ Binding Motif, through which it interacts with
proteins containing the PDZ protein-protein interaction domains. PDZ domains are named after
three proteins in which they were originally identified: the Post Synaptic Density (PSD95), the Disc
Large (Dlg) and the Zona Occludens 1 (ZO-1) proteins (hence the name PDZ), the most notable of
which is the human homologue of the Drosophila tumour suppressor Dlg (hDlg). E6 interacts with
components of the Scribble tri-partite polarity complex (consisting of Scribble, Dlg1 and HuGL1),
which is located on the basolateral membrane of the cell at the Adherens junction (AJ). The Scrib
polarity module is required for the regulation of cell-cell and cell-substratum attachment,
basolateral polarity, asymmetric cell division and cell invasion in epithelial tissues (Woods et al.
1996; Kiyono et al. 1997; Lee, Weiss, and Javier 1997). Dlg1 is a multi-PDZ domain-containing
member of a family of proteins referred to as membrane-associated guanylate kinases (MAGUKs)
(Roberts, Delury, and Marsh 2012)⁠ that function as scaffolds, orchestrating the assembly of large
signal transduction networks at specific sites, including plasma membrane (Tight Junction [TJ]
proteins) and cytoskeleton (Bilder 2001)⁠.
Since functional disruption of Drosophila melanogaster Dlg resulted in defects in apical junctional
complex establishment and loss of apico-basal cell polarity, and therefore was concomitant with
uncontrolled overproliferation and neoplastic transformation, Discs Large (Dlg), in concert with
Scribble, has been described as a tumour suppressor protein necessary for the maintenance
homeostasis and polarized architecture of epithelial cells (Woods and Bryant 1989; Bilder, Li, and
Perrimon 2000). Analysis of null mutations of either of these gene homologs in a mouse strain have
highlighted that Dlg and Scrib47
are required for preserving the normal pattern of growth and
differentiation, as well as for cell cycle control, in the Mouse Ocular Lens Epithelium, suggesting
their potential anti-neoplastic in vivo functions in vertebrates (M. M. Nguyen et al. 2003).
Interestingly, since E6 proteins carrying a PBM mutation that impairs the ability to degrade Dlg are
no longer able to transform rodent cells, the ability of the high-risk HPV E6 oncoproteins to
recognize and deregulate cellular PDZ host proteins might be relevant for the transformation
potential of E6, both in vitro (Kiyono et al. 1997; Watson et al. 2003)⁠ and in vivo (M. M. Nguyen et
al. 2003). Indeed, HPV-16 E6’s capacity to induce skin hyperplasias in vivo is strictly dependent on
the integrity and functionality of the carboxyl-terminal PBM and is directly correlated to the loss of
Dlg-promoted growth suppressive restrictions (M. L. Nguyen et al. 2003)⁠.
The most compelling evidence of tumour suppressor activity in humans came from the
identification of Dlg1 as a target of three viral oncoproteins, amongst them HPV E6 (Pim et al.
47
overexpression of hScrib inhibits the transformation of rodent epithelial cells by both viral oncoproteins, indicating
hScribs’s potential role as a tumor suppressor (Nakagawa and Huibregtse 2000⁠; Thomas et al. 2005)⁠

30
2012). Mucosal high-risk E6, preferentially the HPV-18 type48
, targets various functions of Dlg149
at several different points in the viral life cycle, directing its degradation through the 26S
proteasome (Banks, Pim, and Thomas 2003; Massimi et al. 2004) even in systems that lack E6AP,
probably by enhancing a physiological process (Mantovani, Massimi, and Banks 2001)⁠; hDlg,
hScrib and MUPP1 are also labeled for ubiquitin-mediated proteolysis (Gardiol et al. 1999;
Nakagawa and Huibregtse 2000; Massimi et al. 2004)⁠. This action might account for the frequently
observed reduction in Dlg expression during advanced stages of invasive cervical cancer
(Mantovani, Massimi, and Banks 2001; Watson et al. 2002⁠; Cavatorta et al. 2004)⁠, thus indicating
that its possible role as a tumour suppressor is not merely a general phenomenon due to
overexpression. Other PDZ substrates exerting a strong inhibition of oncogene-induced cell
transformation are MAGI-150
, and MUPP1 (Massimi et al. 2004)⁠, in line with their being efficiently
degraded by E6. This assumption comes from the evidence that patterns of expression of hDlg,
hScrib and MUPP1, which are severely perturbed during cervical cancer development (Cavatorta et
al. 2004)⁠, are consistent with their being substrates for E6-induced degradation (Massimi et al.
2004)⁠. Dlg1 exists as diverse isoforms produced by alternative splicing and exhibits diverse patterns
of expression which are differently susceptible to degradation by E6 (Massimi and Banks 2011)⁠.
Treatment of HPV-positive cells with proteasome inhibitors reveals a remarkable increase in level
of hDlg accumulating at the nuclear regions (Massimi et al. 2004)⁠. These experiments prove that
HPV-18 E6 preferentially targets phosphorylated nuclear and/or cytoplasmic fractions of hDlg,
while membrane-associated bound forms, which probably found within multimeric protein
complexes, remain unaffected by E6. It is plausible that the soluble forms of Dlg that are eliminated
by E6 are those involved in signalling. Further analysis is required to clarify these hypothesis (see 3.
EXPERIMENT).
In the absence of E6, the majority of Dlg is situated at the sites of cell-cell contact, where the viral
oncoprotein has minimal effect. However, addition of HPV-18 E6 which can bind to nuclear hDlg
efficiently overcomes the growth suppressive activity of hDlg, consistent with its being a substrate
for E6-induced degradation. Furthermore, this activity of E6 may be carefully regulated during the
virus life cycle, thus determining dramatic changes in Dlg1’s subcellular localization (Kühne et al.
2000). In the light of the above findings, it is clear that Dlg has a more complicated function than
simply being a tumour suppressor (Massimi et al. 2004)⁠: in the cancer-causing HPV types, the
mislocalized forms of Dlg1 (as well as those of Scribble) found in premalignant cervical lesions can
paradoxically acquire oncogenic attributes, and consequently contribute to the early stages of cancer
48
contains a better consensus Dlg-binding motif (Kiyono et al. 1997)⁠
49
also the other MAGUKs are degraded by recruiting a cellular E3 ubiquitin ligase
50
the most strongly bound of all junctional proteins which is involved in tight junction assembly

31
development in some specific contexts (e.g. the presence of viral oncogenes51
), and depending upon
the precise subcellular (mis)localization. In fact, as discussed in the previous sections, distinct
subcellular pools are subjected to different degree of (de)regulation by E6. Deregulation of Dlg’s
function in HPV-positive cancer cells is conforming to the general trend of an abnormal
cytoplasmic redistribution of Dlg observed in early dysplasia, as opposed to the cell-cell contact
localization seen in normal tissue. The levels of the so-called ‘Jekyll and Hyde’ of the epithelial
polarity proteins (Roberts, Delury, and Marsh 2012)⁠ are therefore subjected to a steady reduction as
the tumor progresses, until the complete loss of Dlg occurs in more advanced stages of cervical
cancer (Cavatorta et al. 2004)⁠.
The pattern of Dlg1 expression during the cell cycle is, to some extent, regulated by
phosphorylation 52
. This post-translational modification modulates the potential molecular
interactions occurring within Dlg1, possibly influencing its accessibility to either E6 or the
ubiquitin proteasome machinery. For example, the hyperphosphorylation by Cdk 1 and Cdk 2
induces the degradation of those nuclear forms of Dlg1 that appear to be involved in controlling cell
proliferation (Narayan, Subbaiah, and Banks 2009)⁠. Additionally, recent assays have shown that
hyperphosphorilarion of Dlg, in response to the cell's exposure to osmotic shock, alters its
subcellular localisation, causing its accumulation within the cell membrane at sites of cell contact,
and rendering Dlg more susceptible to degradation induced by the HPV-18 E6 oncoprotein
(Massimi et al. 2006).
Certainly, altered patterns of phosphorylation of Dlg may reflect changes in cellular signal
transduction pathways and be a prognostic marker for the predisposition to invasive cancer. This in
turn could enhance or restrict malignant progression. The profile of Dlg's interactors also includes
the tumour suppressors APC and PTEN, as well as beta-catenin, a proto-oncogene. In each case,
these interactions are mediated by PDZ binding motifs at the C-terminus of these proteins.
Furthermore, recent data have shown that in addition to these well-known interaction partners, Dlg1
may also recruit components of the vesicle trafficking machinery either to the plasma membrane or
to transport vesicles, bringing them in close proximity to specific cargoes. In this context, Dlg1-
mediated coupling between vesicle components and cargoes could facilitate their specific delivery
to microdomains of the plasma membrane or to endosomes (Walch 2013)⁠.
Obviously, over the last few decades great emphasis has been placed on identifying the cellular
PDZ domain-containing targets of E6, bringing about the discovery of novel potential interacting
51
the presence of the Ad9 E4-ORF1 oncogene induces the translocation of Dlg1 isoform to the plasma membrane,
where it constitutively activates PI3K, and mediates Akt signaling which is associated with tumorigenesis (Roberts, De-
lury, and Marsh 2012; Feigin et al. 2014)⁠
52
also MAG-1 and Scribble are all subjected to post-translational modifications that regulate E6-PDZ domain interac-
tions

32
partners playing a role in pathways other than cell polarity, such as endosomal transport (Belotti et
al. 2013; Ganti et al. 2016). For instance, it has been documented that E6 interacts, via its PBM
motif, with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways.
Although mediated by a classical PBM-PDZ scheme, this interaction does not induce SNX27
degradation; instead, E6 maintains the constant expression of a SNX27 cargo, the glucose
transporter GLUT1, thus perturbing SNX27’s association with components of the endocytic
transport machinery (and with a concomitant marked increase in glucose uptake). E6-induced
alteration of the recycling of cargo molecules, implies modulation of nutrient availability in HPV
transformed tumour cells.
3. EXPERIMENT
3.1 Aim of the work
There is overwhelming evidence of the high risk E6 oncoprotein's ability to target and degrade via
the proteasome the endogenous hDlg protein, thereby inactivating its function as a scaffolding
protein tethering signal transduction components into complexes subsequently recruited at the
junctional sites (Woods et al. 1996)⁠. This interaction is herein assumed to disturb Dlg's key role in
coordinating vesicle formation, protein sorting, targeting and distribution in HPV-positive cells,
thus interfering with both the exocytotic and endocytotic pathways (Walch 2013)⁠.
In the light of the common inability of cancer cells to properly internalize, recycle or degrade cell-
surface proteins such as RTKs, or to reuptake cadherins and other cell adhesion components, which
are constantly removed from the cell surface, thereby disrupting tissue polarity and instigating
motile phenotypes, it would be tempting to speculate that the functional inactivation of PDZ protein
by E6 could impact the deregulation of the endocytic pathway, a multicomponent process that is
profoundly enhanced and skewed in cancer. To assess a possible role of Dlg as a potential oncogene
we propose that Dlg might bind to Rabip4, an effector of Rab4 which controls early endosomal
trafficking, possibly by activating a backward transport step from recycling to sorting endosomes.
In addition, it has been shown that the expression of Rabip4 could modify the kinetic parameters of
receptor recycling (Cormont et al. 2001)⁠. The identification of this protein-protein interaction, in
concert with Rabp4 functional abrogation, should provide an evidence of the oncogenic activity of
Dlg, a PDZ ligand of HPV E6 oncoprotein implicated in signal transduction and control of
basolateral cell polarity. Therefore, E6 may affect the secretory, as well as the endocytic pathway
thus driving the infected cells towards the late stage of cancer. In fact, the small GTPase Rab4 has
been implicated in the regulation of the recycling of internalized receptors back to the plasma
membranes (Seachrist, Anborgh, and Ferguson 2000)⁠, by interacting with a downstream effector(s)

33
that specifically recognizes their GTP-bound conformation. The identification53
of the ubiquitous
69-kDa protein Rabip4 as the effector of Rab4 may provide further information concerning the role
of Rab4 in regulation of vesicular trafficking. Rabip454
, is an hydrophilic protein which contains
two coiled-coil domains and a C-terminal, cysteine-rich, ZnF-like FYVE-finger motif that
coordinates two zinc atoms and, additionally, functions as a phosphatidylinositol 3-phosphate
binding motif, also found in several endosome-associated mammalian proteins (e.g. Hrs, EEA1, and
PIKfyve). The enrichment of PI3P in endosomes (Gillooly et al. 2000)⁠ has been demonstrated to
drive Rabip4 endosomal localization: Rabip4 localizes in early sorting endosomes and, when
overexpressed in CHO cells, leads to modifications of endosomal compartment morphology,
specifically to the enlargement of early vesicles, by increasing the degree of colocalization of
markers of sorting (Rab5) and recycling (Rab1155
) endosomes labeled with active Rab4. When
expressed in CHO cells, Rabip4 is prensent in early endosomes, whereas is absent from Rab11-
positive recycling endosomes and Rab-7 positive late endosomes: because the coexpression of
Rabip4 with active Rab4 visibly results in the expansion of early endosomes, Rabip4 could have a
cooperative role with Rab4 in regulating the overlap of sorting and recycling endosomes which is
associated with the appearance of enlarged early endosomal structures. Furthermore, the expression
of Rabip4 leads to the intracellular retention, and increase in amount, of a recycling molecule, the
glucose transporter Glut 1, which recycles through the endocytic pathway. Recent studies (Walch
2013)⁠ have reported an emerging role of Dlg1 in several exocytotic and endocytotic pathways by
controlling specific vesicle trafficking steps.
If perinuclear Dlg could participate in endosomal routes, it would be plausible that such pro-
oncogenic functions of PDZ proteins are manipulated by the HPV E6 protein to create a favorable
environment for malignant progression. Hence, we have performed studies to investigate the
consequences of the interaction between E6 and Dlg, and, most specifically, to determine whether
functional inactivation of PDZ proteins might negatively affect the endocytic pathway involving
recycling of proteins of the secretory pathway and vescicular dynamics in the complex endosomal
system.
3.2 Materials and Methods
1. Expression of GST-Dlg fusion protein. Pull-down assays with GST fusion proteins attached
to glutathione beads was used as screening technique for the identification of protein-protein
53
by screening a cDNA library in the yeast two-hybrid system
54
its variant rabip4’ is a 80-kDa peripheral membrane protein which colocalizes with internalized transferrin and EEA1
on early endosomes is an effector that coordinates rab5 and rab4, regulating spatial distribution of lysosomes
55
Rab 11, colocalized with E-cadherin in the recycling endosome, regulates diverging and transport of E-cadherin to
basolateral membrane

34
interaction. Purification of proteins fused to glutathione S-tranferase allowed inducible,
high-level protein expression from bacterial cell lysates. Bound proteins were eluted with
1.5 M NaCl or boiled off in reducing sample buffer. Succeeding interaction measurement
was performed through Western Blot analysis which indicates the affinity with which these
proteins interact. No interaction was detected for the control GST column, even though a
greater amount of protein was used.
2. Rabip4 was produced by in vitro transcription/translation in the presence of 35S-methionine
and directly used in binding assays.
3. In vitro binding. The radiolabelled bound proteins were resolved by autoradiography after
separation by SDS-PAGE.
4. Transfection and in vivo binding. Hela 293 cells expressing rabp4’ were washed with ice-
cold PBS.
5. Co-immunoprecipitation (co-IP). Lysates were subjected to immunoprecipitation and bound
proteins were separated by SDS-PAGE and analyzed by Western blot.
3.3 Result
The full-length Rabp4 protein interacts with Dlg protein (Figure 6). We detected a small subset of
cells in which Rabp4 is visibly retained adjacent to perinuclear recycling compartments, probably of
early endosomal origin, which might be compatible with cytosolic forms of Dlg.
3.4 Discussion
In vitro translated purified Rabip4 proteins associate in vitro with GST fusions of Dlg fragments.
Subsequent in vivo binding experiments showed similar association. These data provide the
evidence that a protein disrupted by HPV high-risk E6 oncoprotein, namely Dlg, can associate with
the endosomal trafficking protein Rabp4, although they do not provide information about the nature
of the interaction. Further mutational analysis of both Dlg and rabip4 proteins might define the
specific regions of Dlg and rabip4 necessary for complex formation.
In the light of these findings, we speculate that the E6-induced deregulation of perinuclear pools of
Dlg is possibly associated with deviant signal transduction along the endocytic pathway. The Dlg-
Rabip4 interaction suggests that Dlg might act as an oncogene contributing to the deregulation of
the normal endocytic trafficking in both high- and low-risk HPV E6 expressing cells, conceivably
by ablating Rabip4’s ability to redistribute receptors to the cell surface. Again, this would attest the
loss of complex pattern of hDlg regulation as a significant step in the development of malignancy.
However, whether this is due to a lack or gain of phosphorylation is an aspect requiring further
investigation.

35
Figure 6. GST binding assay shows interaction between Dlg and Rabip4. In vitro-
translated radiolabelled Rabip4 protein was incubated with GST alone or GST-Dlg
fusion protein bound to agarose beads. After extensive washing the bound protein was
analysed by SDS-PAGE and autoradiography. 50% input Rabip4 protein was included
as control and the proteins are arrowed.
4. CONCLUDING REMARKS
The multiple transforming activities of high-risk HPVs represent a consequence of a viral
replication strategy that is driven by the necessity to replicate viral genomes in suprabasal, normally
growth-arrested cells and to establish long-term maintenance in differentiating keratinocytes which
are rapidly turned over and shed; low-risk HPVs also induce epithelial productive infections
causing benign hyperplasia, but express oncoproteins lacking PBM whose weak transforming
activities do not induce genomic destabilization, nor telomerase activity.
HPV biology is organized around the ability of the virus to persist in an infected host cell often
without causing clinically overt injuries, thus implementing the likelihood for malignant
progression to occur. Although most productive virus life cycles are completed within a few months
of acquisition, almost all of the persistent infections are cleared and transformation occurs only as a
mistake.
To complement suprabasal cells defects in sustaining viral replication, high-risk HPV types have
evolved to create a replication-competent cellular milieu in infected differentiated keratinocytes (to
maintain their infected squamous epithelial cells differentiation-competent, in a stem cell-like state),
possibly favouring the acquisition of transforming properties sustaining cervical cancer phenotype,
with each of the viral proteins contributing in some way to these process: carcinogenic progression

36
requires the joint action of HPV E6 and E7 oncoproteins which enhance genomic destabilization
through the inactivation the two hallmark tumor suppressor p53 and pRB pathways, respectively,
consequently facilitating viral genome integration. Later stages of malignancy are in part driven by
E6’s ability to target the PDZ-domain containing cellular substrates via its C-terminal PBM for
proteasome degradation. The aim of current research is to determine whether any of these PDZ
targets may have therapeutic potential, searching for possible inhibitors of E6-PDZ interactions.
The E6/Dlg complex represents an intriguing prototype of viral hijacking of both the polarity and
the endocytic pathway, and therefore provides a consistent framework for the development of
inhibitory therapies against oncogenesis mediated by human papillomavirus. Indeed, abrogation of
Dlg by E6 probably perturbs its emerging role in the vescicular trafficking pathways, contributing
to metastasis and death of the host. Thus, the evidence of a functional duality (oncogenic and
tumour suppressor dual role) for nuclear pools of Dlg1 is an intriguing matter of investigation, that
could clarify the effects/the contribution of E6’s deregulation of Dlg1 on/towards human
carcinogenesis.
Overall, the complex spectrum of E6's activity is not yet fully characterized. Hopefully, future
efforts shall be directed towards elucidating how E6 PDZ ligands can affect the endocytic pathway,
as well as towards identifying new, hallmark host proteins associated with HPV E6 and E7
oncoproteins, whose functional verification may not only shed light on the molecular basis
underlying transformation, but also, and most importantly, help to develop a promising new
approach to cancer treatment in general (Roberts, Delury, and Marsh 2012; David Pim et al. 2015;
James and Roberts 2016).⁠⁠ Designing strategies to block the viral perturbation of these host cellular
proteins remains a major challenge in cancer therapy and in antiviral therapeutics as a whole.
Conflicts of Interest: The author declares that no competing interests exist.
5. ACKNOWLEDGMENTS
I am most grateful to Lawrence Banks and Paola Massimi for letting me join their work group, as
well as to all PhD Students and Senior Researchers working at the Tumour Virology Laboratory at
ICGEB Trieste. I also want to thank Miranda Thomas for comments on the manuscript. This work
was supported by my parents and all the people of my family. Lastly, I am grateful to my dear
friends and future colleagues Cristina M., Federico P., Alessandra L., Gilda S., Giulia B. for the
support and friendship they have always shown to me.

37
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