Molecular biology of human papillomavirus infection and ... · Molecular biology of human papillomavirus infection and cervical cancer John DOORBAR Division of Virology, National

Clinical Science (2006) 110, 525–541 (Printed in Great Britain) doi:10.1042/CS20050369 525

R E V I E W

Molecular biology of human papillomavirusinfection and cervical cancer

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

A B S T R A C T

HPVs (human papillomaviruses) infect epithelial cells and cause a variety of lesions ranging fromcommon warts/verrucas to cervical neoplasia and cancer. Over 100 different HPV types havebeen identified so far, with a subset of these being classified as high risk. High-risk HPV DNA isfound in almost all cervical cancers (>99.7%), with HPV16 being the most prevalent type in bothlow-grade disease and cervical neoplasia. Productive infection by high-risk HPV types is manifestas cervical flat warts or condyloma that shed infectious virions from their surface. Viral genomesare maintained as episomes in the basal layer, with viral gene expression being tightly controlledas the infected cells move towards the epithelial surface. The pattern of viral gene expression inlow-grade cervical lesions resembles that seen in productive warts caused by other HPV types.High-grade neoplasia represents an abortive infection in which viral gene expression becomesderegulated, and the normal life cycle of the virus cannot be completed. Most cervical cancersarise within the cervical transformation zone at the squamous/columnar junction, and it hasbeen suggested that this is a site where productive infection may be inefficiently supported. Thehigh-risk E6 and E7 proteins drive cell proliferation through their association with PDZ domainproteins and Rb (retinoblastoma), and contribute to neoplastic progression, whereas E6-mediatedp53 degradation prevents the normal repair of chance mutations in the cellular genome. Cancersusually arise in individuals who fail to resolve their infection and who retain oncogene expressionfor years or decades. In most individuals, immune regression eventually leads to clearance of thevirus, or to its maintenance in a latent or asymptomatic state in the basal cells.

HPVs (HUMAN PAPILLOMAVIRUSES)AND DISEASE

HPVs cause a diverse range of epithelial lesions. Over 100different HPV types have been identified based on DNAsequence analysis [1], with each being associated withinfection at specific epithelial sites [2]. At an evolutionarylevel, HPVs fall into a number of distinct groups or

genera (Figure 1), and the lesions they cause have differentcharacteristics.

The two main HPV genera are the Alpha and Betapapillomaviruses, with approx. 90 % of currently char-acterized HPVs belonging to one or other of thesegroups. Beta papillomaviruses are typically associatedwith inapparent cutaneous infections in humans but,in immunocompromised individuals and in patients

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

C© 2006 The Biochemical Society

526 J. Doorbar

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

suffering from the inherited disease EV (epidermodys-plasia verruciformis), these viruses can spread uncheckedand become associated with the development of non-

melanoma skin cancer [3,4]. EV patients carry mutationsin their TMC6 (previously known as EVER1) or TMC8(previously known as EVER2) genes, which renders themsusceptible to these viruses [5,6].

The largest group of HPVs comprise the Alpha papi-llomaviruses, and it is this group that contains the genital/mucosal HPV types (Figure 1). The Alpha papilloma-viruses also include cutaneous viruses such as HPV2,which cause common warts, and which are only veryrarely associated with cancers [7]. More than 30 differentHPV types are known to infect cervical epithelium, witha subset of these being associated with lesions that canprogress to cancer. These cancer-associated HPVs areclassified as high-risk HPV types. HPV16 is the mostprevalent high-risk HPV in the general population, andis responsible for approx. 50 % of all cervical cancers. Theremaining mucosal types are classified as intermediate orlow risk depending on the frequency with which they arefound in cancers. Low-risk HPV types, such as HPV11,are associated with cervical cancer only very rarely,but are still medically important because they causegenital warts. Genital warts are a major sexually trans-mitted disease in many countries and can affect 1–2 % ofyoung adults [8].

The remaining HPVs come from three genera(Gamma, Mu and Nu) and generally cause cutaneouspapillomas and verrucas that do not progress to cancer(but see [9,10]). The relationship between the differentgenera of HPVs, and in particular the high-risk HPVtypes associated with cervical cancer, is shown in Figure 1.

NORMAL PRODUCTIVE INFECTION

Many HPV types produce only productive lesions fol-lowing infection and are not associated with humancancers. In such lesions, the expression of viral gene prod-ucts is carefully regulated, with viral proteins beingproduced at defined times and at regulated levels as theinfected cell migrates towards the epithelial surface.The events that lead to virus synthesis in the upperepithelial layers appear common to both the low- andhigh-risk HPV types, with protein expression patternsin low-grade CIN (cervical intraepithelial neoplasia) 1resembling the patterns found in benign warts causedby other papillomaviruses. Productive infection can bedivided into distinct phases, with the different viralproteins playing specific roles.

Establishment of HPV infectionAll papillomaviruses share a number of characteristicsand contain double-stranded circular DNA within anicosahedral capsid (Figure 2). Although the viral genomecan vary slightly in size between different HPV types,it typically contains around 8000 bp [7904 bp for HPV16(GenBank® accession number NC 001526)], and encodes


Papillomavirus molecular biology 527

eight or nine ORFs (open reading frames) (Figure 2). Thevirus shell is made up of two coat proteins. The L1 proteinis the primary structural element, with infectious virionscontaining 360 copies of the protein organized into 72capsomeres [11]. L2 is a minor virion component, andit is thought that a single L2 molecule may be presentin the centre of the pentavalent capsomeres at the virionvertices [11,12]. Both proteins play an important role inmediating efficient virus infectivity.

Infection by papillomaviruses requires that virus part-icles gain access to the epithelial basal layer and enter thedividing basal cells. At present there is some controversyas to the precise nature of the receptor for virus entry[13], but it is thought that heparan sulphate proteoglycansmay play a role in initial binding and/or virus uptake[13–15]. As with other viruses [16,17], it seems that HPVinfection requires the presence of secondary receptors forefficient infection, and it has been suggested that this rolemay be played by the α6 integrin [18–20]. Papillomavirusparticles are taken into the cell relatively slowly followingbinding [21] and, for HPV16, this occurs by clathrin-coated endocytosis [22]. This mode of entry may not beconserved among all HPV types, however, and it has beensuggested that HPV31 may gain entry via caveolae [23].Papillomavirus particles disassemble in late endosomesand/or lysosomes, with the transfer of viral DNA tothe nucleus being facilitated by the minor capsid proteinL2 [22,24]. In experimental systems, viral transcripts canbe detected as early as 12 h post-infection, with mRNAlevels increasing over the course of several days [24].

Infection leads to the establishment of the viral genomeas a stable episome (without integration into the host cellgenome) in cells of the basal layer, and this is thought torequire expression of the viral replication proteins, E1 andE2. The E2 protein plays several roles during productiveinfection and, in basal cells, is required for the initiationof viral DNA replication and genome segregation. E2 isa DNA-binding protein that recognizes a palindromicmotif [AACCg(N4)cGGTT] in the non-coding region ofthe viral genome ([25], and Figure 2). HPV16 has foursuch motifs, one of which lies adjacent to the viral originof replication. E2 binding is necessary for the recruit-ment of the E1 helicase to the viral origin, which bindsin turn to cellular proteins necessary for DNA repli-cation, including RPA (replication protein A) and DNApolymerase α primase [26–29]. The E2 protein sub-sequently dissociates from the viral origin, allowing theassembly of E1 into a double hexameric ring that func-tionally resembles the hexameric ring structures thatassemble at cellular replication origins and which arebuilt from the cellular MCM (multicopy maintenance)proteins.

In the basal cells, it appears that the viral genomereplicates with the cellular DNA during S-phase, withthe replicated genomes being partitioned equally duringcell division. The role of E2 in anchoring viral episomes

to mitotic chromosomes is critical for correct segregation[30] and, in some papillomavirus types, involves thecellular Brd4 protein, which directly associates throughits C-terminus with the viral E2 protein [30,31]. Forthe high-risk HPV types that are associated with humancancers, association appears to be via the spindle, withadditional cellular proteins being involved. In additionto its role in replication and genome segregation, E2 canalso act as a transcription factor and can regulate the viralearly promoter (p97 in HPV16; p99 in HPV31) and con-trol expression of the viral oncogenes (E6 and E7). Atlow levels, E2 acts as a transcriptional activator, whereasat high levels E2 represses oncogene expression by dis-placing SP1 transcriptional activator from a site adjacentto the early promoter (Figure 2). It is unclear whetherE2-mediated transcriptional regulation is important inbasal epithelial cells, where the nucleosomal structureand/or methylation status of the viral DNA may notbe compatible with efficient activation of the early pro-moter [32,33]. The analysis of genome maintenance inBPV (bovine papillomavirus)-transformed keratinocytesand cell lines derived from cervical lesions [34,35] hassuggested that viral episomes may be maintained at 10–200 copies in basal cells, although, as yet, this has not beenadequately demonstrated in lesions. Knockout mutants inthe viral genome have suggested that both E1 and E2 arerequired for viral genome maintenance in the basal layer[36,37].

Stimulation of cell proliferationA number of model systems have been used to examinethe papillomavirus productive cycle during in vivo infec-tion. Following experimental inoculation of mucosalepithelial tissue by ROPV (rabbit oral papillomavirus)or COPV (canine oral papillomavirus), an increase incell proliferation in the basal and suprabasal cellularcompartments is readily apparent, with mature wartsbecoming visible by 4 weeks post-infection [38,39]. Thetime between initial infection and the appearance of pro-ductive papillomas can vary depending on the titre ofthe infecting virus and possibly also on the nature of theinfecting papillomavirus type, and it has been suggestedthat latency may result when inoculating titres are low[40,41]. In cervical lesions caused by HPVs, the increasedproliferation of suprabasal epithelial cells is attributedto the expression of the viral oncogenes, E6 and E7.During natural infection, the activity of these genesallows the small number of infected cells to expand, in-creasing the number of cells that subsequently go on toproduce infectious virions. The ability of E6 and E7to drive cells into S-phase is also necessary, along withE1 and E2, for the replication of viral episomes abovethe basal layer. Suprabasal cells normally exit the cellcycle and begin the process of terminal differentiation inorder to produce the protective barrier that is normallyprovided by the skin [42]. In HPV-infected keratinocytes,


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

(A) The HPV16 genome (7904 bp) is shown as a black circle with the early (p97) and late (p670) promoters marked by arrows. The six early ORFs [E1, E2, E4 andE5 (in green) and E6 and E7 (in red)] are expressed from either p97 or p670 at different stages during epithelial cell differentiation. The late ORFs [L1 and L2(in yellow)] are also expressed from p670, following a change in splicing patterns, and a shift in polyadenylation site usage [from early polyadenylation site (PAE)to late polyadenylation site (PAL)]. All the viral genes are encoded on one strand of the double-stranded circular DNA genome. The long control region (LCR from7156–7184) is enlarged to allow visualization of the E2-binding sites and the TATA element of the p97 promoter. The location of the E1- and SP1-binding sites isalso shown. (B) The key events that occur following infection are shown diagrammatically on the left. The epidermis is shown in colour with the underlying dermisbeing shown in grey. The different cell layers present in the epithelium are indicated on the left. Cells in the epidermis expressing cell cycle markers are shown withred nuclei. The appearance of such cells above the basal layer is a consequence of virus infection, and in particular, the expression of the viral oncogenes, E6 andE7. The expression of viral proteins necessary for genome replication occurs in cells expressing E6 and E7 following activation of p670 in the upper epithelial layers(cells shown in green with red nuclei). The L1 and L2 genes (yellow) are expressed in a subset of the cells that contain amplified viral DNA in the upper epithelial



however, the restraint on cell-cycle progression is lost andnormal terminal differentiation does not occur [43]. Thebasic mechanism by which papillomaviruses stimulatecell-cycle progression is well known and is similar to theway that other tumour viruses deregulate cell growth.E7 associates with pRb [Rb (retinoblastoma) protein]and other members of the pocket protein family anddisrupts the association between pRb and the E2F familyof transcription factors, irrespective of the presence ofexternal growth factors (Figure 3). E2F subsequentlytransactivates cellular proteins required for viral DNAreplication such as cyclins A and E. E7 also associates withother proteins involved in cell proliferation, includinghistone deacetylases [44], components of the AP1 tran-scription complex [45] and the cyclin-dependent kinaseinhibitors p21 and p27 [46]. During natural infection,however, the ability of E7 to drive cell proliferation isinhibited in some cells, depending on the levels of the p21and p27 cyclin-dependent kinase inhibitors (Figure 3).High levels of p21 and p27 in differentiating keratinocytescan lead to the formation of inactive complexes with E7and cyclinE within the cell. It appears that the ability ofE7 to drive cells through mitosis in differentiating epi-thelium may be limited to those cells which express p21and p27 at low level or which express sufficient E7 toovercome the block to cell-cycle progression [47]. This isimportant given that deregulated expression of the viraloncogenes is a predisposing factor in the developmentof HPV-associated cancers (Figure 3). The function ofthe viral E6 protein complements that of E7 and, in thehigh-risk HPV types, the two proteins are expressedtogether from a single polycistronic mRNA species [48].A primary role of E6 is its association with p53 which, inthe case of the high-risk HPV types, mediates p53 ubi-quitination and degradation. This is thought to preventgrowth arrest or apoptosis in response to E7-mediatedcell-cycle entry in the upper epithelial layers, which mightotherwise occur through activation of the ARF (ADP-ribosylation factor) pathway (see Figure 3). The generalrole of E6 as an anti-apoptotic protein is emphasizedfurther by the finding that it also associates with Bak [49]and Bax [50]. This role of E6 is of key significance in thedevelopment of cervical cancers (discussed in more detailbelow), as it compromises the effectiveness of the cellularDNA damage response and allows the accumulation ofsecondary mutations to go unchecked. The E6 proteinof the high-risk HPV types also plays a role in mediating

cell proliferation independently of E7 through its C-terminal PDZ ligand domain [the name PDZ is derivedfrom the first three proteins in which these domainswere found: PSD-95 (a 95 kDa protein involved insignalling), Dlg (the Drosophila discs large protein), andZO1 (the zonula occludens 1 protein which is involved inmaintaining epithelial cell polarity)] [51]. E6 PDZ bindingcan mediate suprabasal cell proliferation [52,53] and maycontribute to the development of metastatic tumours bydisrupting normal cell adhesion. In productive lesions,cells are driven into cycle only in the lower epitheliallayers and extend towards the epithelial surface to varyingextents depending on lesion grade and the nature of theinfecting HPV type [54]. In benign warts caused byHPV1, proliferating cells are restricted to the epithelialbasal layer, with genome amplification beginning as soonas the infected cell enters the suprabasal cell layers. HPVs,such as HPV6 and HPV16 (Alpha papillomaviruses),typically have a region that may be many cell layers thick,where cells are retained in cycle prior to the onset ofvegetative viral genome amplification [54]. In the caseof the high-risk HPV types that are associated withcervical cancer, the relative thickness of these layersincreases with the grade of neoplasia, while the extentof epithelial differentiation decreases.

Genome amplificationAlthough cell proliferation is required for lesion form-ation and the maintenance of viral episomes, all papi-llomaviruses must eventually amplify and package theirgenomes if infectious virions are to be produced. Whattriggers the onset of late events is not yet fully understood,but appears to depend in part on changes in the cellularenvironment as the infected cell moves towards theepithelial surface. Critical for this is the up-regulationof the differentiation-dependent promoter, which formany HPV types is contained within the E7 ORF (P670in HPV16; p742 in HPV31) [55,56]. The activation of thedifferentiation-dependent promoter depends on changesin cellular signalling, rather than on genome ampli-fication [55,56], and leads to an increase in the level ofviral proteins necessary for replication (i.e. E1, E2, E4and E5). Cells supporting productive infection can bevisualized using antibodies to E4 (Figure 4), whereasthose supporting genome amplification also contain E7[57].

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


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Figure 3 Stimulation of cell-cycle progression by high-risk HPV typesHPV infection leads to deregulation of the cell cycle. Regulation of protein expression in uninfected epithelium is shown in (A). In the presence of high-risk HPV(B), the regulation of proteins necessary for cell proliferation is altered, allowing HPV to stimulate S-phase entry in the upper epithelial layers. (A) Uninfected epi-thelium. The expression of proteins necessary for cell-cycle progression is controlled by pRB, which in non-cycling cells associates with members of the E2F transcriptionfactor family (centre). In the presence of growth factors, cyclinD/Cdk4/6 is activated, which leads to Rb phosphorylation and the release of the transcription factorE2F, which drives the expression of proteins involved in S-phase progression. p16 regulates the levels of active cyclinD/Cdk in the cell, providing a feedback mechanismthat regulates the levels of MCM, PCNA (proliferating-cell nuclear antigen) and cyclinE. p14Arf , whose expression is directly linked to that of p16, regulates the activityof the MDM (murine double minute) ubiquitin ligase, which maintains p53 at a level below that required for cell cycle arrest and/or apoptosis. (B) HPV-infectedepithelium. In cervical epithelium infected by high-risk HPV types, progression through the cell cycle is not dependent on external growth factors, but is stimulatedby the E7 protein, which binds and degrades pRB and facilitates E2F-mediated expression of cellular proteins necessary for S-phase entry. Although p16 levels rise,normal feedback is by-passed, as HPV-mediated cell proliferation is not dependent on cyclinD/Cdk4/6. The rise in the level of p14Arf , which occurs in the absenceof p16-mediated feedback, leads to the inhibition of MDM function and an increase in the level of p53. This is countered by E6, which associates with the E6APubiquitin ligase in order to stimulate the degradation of the p53 protein and prevent growth arrest and/or apoptosis. In low-grade cervical disease, where E7 levelsare carefully regulated, it is thought that E7-mediated cell proliferation can sometimes be inhibited as a result of association with p21 and cyclin E/Cdk. The highlevels of E7 found in cervical cancer cells are thought to overcome this block by binding and inactivating the Cdk inhibitor p21.

The role of E1 and E2 in viral genome amplificationis well established. E1 is highly conserved among papi-llomaviruses and has a weak affinity for a consensusmotif (AACNAT) repeated 6 times in the viral origin.During natural infection, E1 is expressed at very lowlevels and requires the presence of E2 in order to be effi-ciently targeted to its binding sites. E2 associates withE1 primarily through its N-terminus and binds to DNAas a dimer through its C-terminus [58,59] (Figure 2).The formation of an E1–E2 complex at the origin ofreplication induces localized distortion in the viral DNA,which facilitates the recruitment of additional E1 mol-ecules and the eventual displacement of E2 [60]. E1 canalso associate with cellular Hsps (heat-shock proteins),

in particular Hsp40 and Hsp70, and this contributes tothe formation of E1 dihexamers [61]. E1 and E2 also actto regulate the viral early promoter (p97 in HPV16 andp99 in HPV31), with high levels of E2 acting to down-regulate the expression of E6 and E7 in experimentalsystems. The ability of E2 to either repress or activateearly viral gene expression according to its abundanceis thought to result from differences in the affinity ofE2 for its various binding sites [62]. In HPV16, it isthought that binding site 4 is the primary site that is occu-pied when E2 is present at low levels and that binding tothis site and to binding site 3 (Figure 2) leads to pro-moter activation [63]. As E2 increases in abundance,occupancy of the remaining sites leads to the displacement



Figure 4 Characterization of events during productivepapillomavirus infection by immunostainingThe pattern of viral protein expression is conserved in productive lesions caused bypapillomaviruses of diverse origins. Typical immunofluorescence staining patternsare shown. (A) Productive papilloma showing the typical expression profile of PCNA[a surrogate marker of E6/E7 gene expression (in red)] and E4 (in green), whichis thought to reflect the sites of expression of E1 and E2. Nuclei are counter-stained with DAPI (4,6-diamidino-2-phenylindole; in blue). A small area of un-infected tissue is apparent on either side of the papilloma. (B) Comparison ofthe sites of viral genome amplification [in red; detected by FISH (fluorescencein situ hybridization)] and E4 expression (in green) reveal that the two events cor-relate very closely. Nuclei are counterstained with DAPI (blue). (C) The viral capsidprotein (L1; in red) is expressed in a subset of the cells that express E4 (in green)in the upper epithelial layers. Nuclei are counterstained with DAPI (in blue).

of basal transcription factors, such as Sp1 and TBP(TATA-box-binding protein), that are necessary for pro-moter activation [64]. It appears that the increase in E2

expression that is important in stimulating viral genomeamplification will lead eventually to the down-regulationof E6/E7 expression and to the eventual loss of thereplicative environment necessary for viral DNA syn-thesis. This curious link between replication and tran-scription provides a mechanism by which the virus canlimit the timing and duration of genome amplification. Incervical lesions caused by HPV16, cells supporting viralgenome amplification are often scarce and vary greatlyin their location within the lesion [57]. By contrast, inverrucas caused by HPV1, cells supporting genomeamplification are relatively prevalent and are consistentlyfound immediately above the basal layer.

Although E1 and E2 are key players in viral genomeamplification, the viral E4 and E5 proteins also contribute[65–69]. E5 mutant genomes of HPV16 and 31 exhibitlower levels of genome amplification than wild type,and it is thought that the ability of E5 to modulate cellsignalling is responsible for this. HPV E5 is a trans-membrane protein that resides predominantly in the ER(endoplasmic reticulum), but which can associate withthe vacuolar proton ATPase and delay the process ofendosomal acidification [70–72]. It is thought that thisaffects the recycling of growth factor receptors on the cellsurface, leading to an increase in EGF (epidermal growthfactor)-mediated receptor signalling and the maintenanceof a replication competent environment in the upperepithelial layers [73]. By contrast, the role of E4 ingenome amplification is not yet fully established. E4accumulates in the cell at the time of viral genome ampli-fication, and its loss has been shown to disrupt late eventsin a number of experimental systems [67–69]. Part ofthe effect may be related to the ability of certain HPVtypes to associate with cyclinB/Cdk2 and to relocatethe complex to the cytoplasm [74,75]. This prevents thenuclear accumulation of cyclinB/Cdk2, which is neces-sary for progression through mitosis. The ability of E4to cause cell-cycle arrest in G2 and to antagonize E7-mediated cell proliferation is a common feature of theE4 proteins encoded by several HPV types, includingHPV16, HPV11 [75], HPV18 [76] and HPV1 [77]. Inter-estingly, recent work has suggested that the E4 proteinsof HPV16 and HPV18 can also associate with E2, whichsuggests an additional mechanism by which E4 may act[78].

Virus assembly and releaseThe final stage in the papillomavirus productive cyclerequires that the replicated genomes are packaged intoinfectious particles. Capsid proteins (L1 and L2) accumu-late after the onset of genome amplification, with L2expression preceding the expression of L1 [79,80]. Theevents that link genome amplification to the synthesisof the capsid proteins are not yet fully understood, butare dependent on changes in mRNA splicing and onthe generation of transcripts that terminate at the late


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(rather than the early) polyadenylation site (Figure 2).During epithelial cell differentiation, the timing of capsidsynthesis is regulated both at the level of RNA processingand at the level of protein synthesis [81–83]. Negativeregulatory elements that control RNA stability arepresent in the coding regions and in the late untranslatedregion of HPV16 [84,85], whereas a splicing silencer inthe HPV16 L1 gene leads to the preferential synthesisof early transcripts in proliferating cells [86]. In additionto this, the pattern of codon usage within the HPV16L1 and L2 genes is distinct from the pattern usuallyfound in mammalian cells, which contributes further tothe inhibition of capsid expression in the lower epitheliallayers [82,87,88]. Similar regulatory mechanisms are alsothought to exist in HPV1 [89], HPV31 [90] and BPV [91]and are likely to extend to other papillomavirus types.

The assembly of infectious virions in the upper epi-thelial layers is thought to require E2 in addition tothe capsid proteins L1 and L2 [92,93], and it has beensuggested that E2 may improve the efficiency of genomeencapsidation during natural infection [93,94]. L2 local-izes to the nucleus by virtue of nuclear localizationsignals located at its N- and C-termini and, once there, itassociates with PML (promyelocytic leukaemia) bodies.Although some papillomavirus L2 proteins can associatedirectly with DNA [95], the specific recruitment ofviral genomes to PML bodies is thought to require E2,which can associate with viral DNA through its specificrecognition sites. L1 assembles into capsomeres in thecytoplasm prior to nuclear relocation and is recruited intoPML bodies only after L2 has bound and has displacedthe PML component sp100 [79]. The assembly of virus-like particles in experimental systems requires the pres-ence of the cellular protein Hsp70 [96], which is alsoinvolved in recycling the viral E1 protein during repli-cation [97], but does not appear to be strictly dependenton PML localization [98]. Although papillomavirus part-icles can assemble in the absence of L2, its presence con-tributes to efficient packaging [99] and enhances virusinfectivity [100]. Loss of L2 in the context of the HPV31genome results in a 10-fold reduction in packaging effi-ciency and a 100-fold reduction in virus infectivity whencompared with wild-type HPV31 [101]. L2 associateswith L1 through a hydrophobic region near the C-ter-minus of the protein that is thought to insert into thecentral hole in the pentavalent L1 capsomeres [102].The interaction between capsomeres requires the C-terminus of the L1 protein [11] with virus maturation andstabilization occurring as the infected cells approach theepithelial surface as a result of disulphide cross-linking.Although papillomaviruses are resistant to desiccation[103], it is thought that their survival may be enhancedby being shed from the epithelial surface as a cornifiedsquame [104]. The retention of papillomavirus antigensuntil the infected cell reaches the epithelial surface isthought to limit the ability of the immune system to

detect infection. Ultimately, virus release requires effi-cient escape from the cornified envelope at the cell surface,which may be facilitated by the E4 protein. E4 can disruptthe keratin network [105,106] and can affect the integrityof the cornified envelope [104,107].

ABORTIVE INFECTION AS A PRECURSORTO CANCER

Virus-induced cancers often arise at sites where pro-ductive infection cannot be properly supported. CRPV(cottontail rabbit papillomavirus) induces productivepapillomas in its natural host (the cottontail rabbit),but gives rise to lesions that cannot support virus syn-thesis when inoculated into domestic rabbits. Such abor-tive infections progress to cancer more frequently thando infections in cottontails. A similar situation is seenfollowing the inoculation of SV40 (simian virus 40) andadenovirus type 5 (which are also considered to beDNA tumour viruses) into inappropriate hosts, suchas hamsters and rats, which support early, but not late,viral gene expression. The papillomavirus types thatusually cause benign cutaneous warts in humans and areconsidered low risk (e.g. HPV2 and 4) can be associatedoccasionally with cancers at mucosal sites. The restrictedtissue tropism of the different HPV types, coupled withtheir ability to infect non-optimal sites, may provide apartial explanation as to why otherwise benign HPVtypes are occasionally associated with human cancers.

The high-risk papillomavirus types that are associatedwith human cancers more frequently come predomin-antly from the Alpha 9 and Alpha 7 groups (see Figure 1),with HPV16 and HPV18 being the most prevalenttypes [108]. These viruses are found in women with nocytological abnormalities, as well as in women with LSILand HSIL (low- and high-grade squamous intraepithelialneoplasia lesions respectively) and/or cancer [109–111].By PCR, HPV16 DNA is apparent in approx. 26 %of LSIL [112], but can be seen in as many as 63 % ofSCCs (squamous cell carcinomas) of the cervix [113].HPV18 is the second most common HPV type associatedwith this disease (causing 10–14 % of all cases ofcervical SCCs), but is the primary HPV type associatedwith cervical adenocarcinoma, causing 37–41 % (HPV16causes 26–37 % of all such cases [113]). The prevalence ofhigh-risk HPV infection amongst young women (meanage, 25 years) is typically approx. 20–40 % dependingon geographical location [111,114], with the incidencedeclining with age as infections are either resolved orbrought under control by the host immune system. HPVinfections are very common in this age group, withcumulative incidence of infection over 5 years being ashigh as 60 % in some populations [110]. Most women(80 %) infected with a specific HPV type will, however,show no evidence of that type after 18 months, and it



Figure 5 Changes in expression patterns that accompany progression to cervical cancerDuring cancer progression, the pattern of viral gene expression changes. In CIN1 (LSIL), the order of events is generally similar to that seen in productive lesions(shown diagrammatically on the left). In CIN2 and CIN3, however, the onset of late events is retarded, and although the order of events remains the same, theproduction of infectious virions becomes restricted to smaller and smaller areas close to the epithelial surface. Integration of HPV sequences into the host cell genomecan accompany these changes and can lead to further deregulation in the expression of E7 (and the loss of the E1 and E2 replication proteins). In cervical cancer(shown on the right), the productive stages of the virus life cycle are no longer supported and viral episomes are usually lost.

Figure 6 Detection of viral proteins in cervical intraepithelial neoplasia(A) The expression of surrogate markers of E7 (in this case MCM; in red) and E4 (in green) in HPV16-infected cervix resembles the pattern of expression seen inproductive papillomas caused by other papillomavirus types (see also Figure 4). Nuclei are counterstained with DAPI (in blue) and uninfected tissue is apparent eitherside of the area of infection. (B) During cancer progression, the expression of surrogate markers of E7 (in this case MCM; in red) extends towards the epithelial surfaceand the expression of E4 is restricted to isolated pockets close to the upper epithelial layers. CIN3 is shown on the left, whereas the pattern seen in many productiveCIN1 lesions is shown on the right. Nuclei are counterstained with DAPI (in blue).

is generally thought that re-infection by the same HPVtype is uncommon [115]. The development of high-grade cervical neoplasia arises in women who cannotresolve their infection and who maintain persistentactive infection for years or decades following initialexposure. In such women, the continuous stimulationof S-phase entry and cell proliferation by E7, coupledwith the loss of p53-mediated DNA repair pathways asa result of E6 expression, allows the accumulation ofsecondary point mutations in the cellular genome thateventually lead to cancer. Previous work from a num-ber of laboratories has suggested that not all cervicalcancers develop through this route, however, and that, insome instances rapid onset of HSIL can arise after initialinfection [110,116]. Given the prevalence of genital HPVtypes in the general population and the high life-time riskof infection (estimated at approx. 80 %), the incidenceof cervical cancers is very low [typically approx. 0.03 %

in the absence of screening (0.018–0.044 %)] with mostinfections being successfully resolved [117].

Low-grade cervical lesions (i.e. LSIL or grade 1 CIN)caused by high- and low-risk HPV types are similar toproductive infections in the pattern of viral gene expres-sion, and viral coat proteins can usually be detected incells at the epithelial surface [57] (Figure 5). High-gradelesions (HSIL or grade 2 or 3 CIN) have a more extensiveproliferative phase, with the productive stages of the viruslife cycle being supported only poorly (Figures 5 and 6).It has been estimated that approx. 20 % of CIN1 willprogress to CIN2, and that approx. 30 % of these lesionswill progress to more severe neoplasia if left untreated.Approx. 40 % of CIN3 lesions can progress to cancer[118], with cervical neoplasia generally arising withinthe cervical transformation zone where the columnarcells of the endocervix meet the stratified squamousepithelial cells of the ectocervix. The extent of columnar


534 J. Doorbar

and stratified epithelium in this region changes markedlyduring a woman’s life, and it is in this region of change thatmost cervical neoplasias develop. It has been suggestedthat the transformation zone may be a sub-optimal sitefor completion of the productive life cycle of high-riskHPV types such as HPV16. Interestingly, high-risk HPVinfections can be found at many sites in the anogenitaltract, including the vagina, vulva and penis, but, despitethis distribution, the incidence of cancer at these sites isgenerally low (0.001 %). Only at the anus, in men whohave sex with men, does the incidence of HPV-associatedcancer rise to levels that are similar to those found at thecervix (0.035 %) [119], and it is interesting that at boththese susceptible sites there is a transformation zone. Inthe cervix, the reserve cells of the transformation zoneeventually form the basal cells of a stratified squamousepithelium, and the higher frequency of cancers at suchsites may reflect the fact that the virus can access thesebasal cells more readily than those that are protectedfrom infection by a permanent stratified epithelial layer.Despite this possibility, it appears that the transformationzone may in fact be an epithelial site where high-risk HPVtypes cannot properly regulate their productive cycle,and that variation both in the level and in the timing ofexpression of viral proteins may underlie the developmentof cancers at these sites.

MOLECULAR EVENTS DURING CANCERPROGRESSION

The identification of cervical lesions as flat condyloma,LSIL, HSIL or invasive cervical cancer reflects molecularchanges in the normal programme of epithelial cell differ-entiation that occur following infection. Virus productionat the epithelial surface depends on the ordered and timelyexpression of viral gene products (Figure 2) [54,57],with the timing of such events becoming progressivelydisturbed during neoplastic progression (Figures 5 and6). In HSIL, viral genome amplification occurs closerto the epithelial surface than in condylomas and LSIL,and the expression of viral coat proteins is retarded[57] (Figure 5). The molecular bases for these changesare not fully understood, but may in some instancesreflect changes in the levels of E6 and E7 expressionthat occur following integration of the viral genomeinto the host cell chromosome. Integrated HPV DNAis found in most invasive cancers and in a subset ofhigh-grade lesions [120,121], but can also be found insome CIN1 lesions, and it has been suggested thatintegration may be an early event in cancer progression[122]. Indeed, p16INK4A expression, which is considereda marker of elevated E7 expression, can be detectedin some CIN1 lesions, as well as in CIN2 and CIN3lesions that show evidence of integration [123,124].Integration of the HPV genome into the host cell

chromosome is a critical event in the development of mostcervical cancers and, although this can occur randomlythroughout the genome, several studies have indicateda preference for integration at common fragile sitesand have suggested that changes in the expression ofgenes at or near the integration site may participatein cancer development [125]. It is clear, however, thatintegration (which often results in the loss of E2) canlead to the deregulation of E6/E7 expression, and thatthis is critical for the enhanced growth characteristicsof cervical cancer cells. The requirement of these genesfor the maintenance of the cancer phenotype is shownmost dramatically by studies that have aimed to inhibitviral oncogene activity in cervical cancer cells. Cell linessuch as HeLa and SiHa, which have been grown in thelaboratory for many decades, will undergo apoptoticcell death in the presence of molecules that inhibitE6 function [126–128]. Similar results are obtainedfollowing the reintroduction of E2 into such cell lines,which suppresses the expression of the viral oncogenesby binding to the URR (upstream regulatory region),preventing continued cell proliferation [129,130]. Theubiquitous retention of the E6/E7 region of the viralgenome following integration into the host chromosomeis usually accompanied by the loss or disruption of viralsequences encoding most of E1, as well as E2 and E4. Theviral E2 protein has a negative effect on cell proliferationby regulating the viral URR and can also cause cell-cyclearrest at the G2 phase of the cell cycle [131,132]. Similarly,the E4 protein of HPV16 is able to inhibit mitosisby preventing the nuclear localization of cyclinB/Cdk1,and it is not surprising therefore that the expression ofthese proteins is abrogated in HPV-associated cancers. Inaddition to the loss of regulatory proteins, integrationalso leads to the loss of sequences at the 3′-end of theviral early transcripts that can suppress the productionof viral mRNA species encoding E6 and E7 [133–135], contributing further to the deregulation of viraloncogene expression. Although integrated HPV DNAis found in the vast majority of cervical cancers, otherfactors are likely to influence the development of theprecancerous changes seen in squamous intraepithelialneoplasia. These include exposure to glucocorticoids andprogesterones, which can affect viral oncogene expression[136–138], and the regulation of viral gene expression byDNA methylation and chromatin organization [139].Both factors can also regulate expression from integratedsequences and, in instances where integration occurs asconcatamers, it is often only one copy of the viral genomethat is transcriptionally active [140].

Although many HPV types can infect the cervix, onlythe high-risk HPV types are consistently associated withcervical cancers because of the specific activity of theironcogenes. HPV16 gene expression facilitates integrationof foreign DNA into the host cell chromosome, whichmay be a consequence of the increased level of host



genome instability in these cells [141]. The high-risk E7proteins, but not the E7 proteins encoded by the low-risk HPV types, induce centrosomal abnormalities in cellculture and in transgenic animals [142,143], and it hasbeen suggested that high-risk E7 may act as a mitoticmutator, which acts to increase the chance of errorsduring each round of cell division [144]. Although themolecular basis for E7-mediated genome instability is notfully understood, it appears in part to be independent ofthe well-characterized association of E7 with the pocketproteins Rb, p107 and p130. The association of E7 withthese proteins does, however, contribute to the ability ofE7 to stimulate cell proliferation, with the high-risk E7proteins binding Rb more efficiently than the E7 proteinof the low-risk HPV types [145,146]. The high-risk E7proteins are also capable of mediating Rb degradationthrough a proteosome-dependent mechanism [147,148],which is important for E7-mediated cell transformation[149]. As with E7, the E6 protein also differs in itsfunction between high- and low-risk HPV types. Oneof the most important of these with regard to cancerprogression is the ability of high-risk E6 proteins to forma tripartite complex with p53 and the cellular ubiquitinligase E6AP (E6-associated protein), which leads toproteosome-mediated p53 degradation [150]. Low-riskE6 proteins bind p53 with a lower affinity than the high-risk types and have no significant ability to bind E6APand to stimulate p53 degradation [150,151]. The loss of thep53-mediated DNA damage response in cells expressinghigh-risk HPV E6 predisposes to the accumulation ofsecondary changes in the host cell chromosome thateventually lead to cancer. The high-risk E6 proteins alsodiffer from those encoded by the low-risk HPV types inhaving a C-terminal PDZ-binding domain. High-risk E6proteins bind and stimulate the degradation of severalcellular targets that contain PDZ motifs, such as hDlg(human homologue of Dlg) and hSrib (human homologueof the Drosophila scribble protein), which are thoughtto be involved in the regulation of cell growth andattachment [152]. PDZ binding is a conserved feature ofall high-risk E6 proteins, and it has been suggested thatthe loss of cell–cell contacts mediated by tight junctionsmay contribute to the loss of cell polarity seen in HPV-associated cervical cancers [153]. PDZ binding, which isdistinct from the ability of E6 to bind and degrade p53,is important for cell transformation [154] and has beenshown to be necessary for the stimulation of epithelialhyperplasia in transgenic animals [53]. Another importantfunction for high-risk E6 is its ability to activate thecatalytic subunit of telomerase [hTERT (human telo-merase reverse transcriptase)], which adds hexamerrepeats to the telomeric ends of chromosomes [155].Telomerase activity is usually absent in somatic cells,leading to the shortening of telomeres with successive celldivisions and, eventually, to cell senescence. Althoughthe precise mechanism by which E6 mediates hTERT

activation is not known, it is clear that such an ac-tivity may predispose to long-term infection and thedevelopment of cancer. Interestingly, recent studies haveshown that the viral oncogenes E6 and E7 can antagonizeBRCA-mediated inhibition of the hTERT promoter[156].

Although aberrant expression of high-risk oncogenescan predispose to the development of cervical cancer,their expression alone is not considered sufficient, andthe viral proteins cannot fully transform human keratino-cytes in culture [157]. It is generally accepted that papi-llomavirus-mediated oncogenesis requires the accumu-lation of additional genetic changes that occur over timefollowing initial infection. The average age of womenwith invasive cervical cancer is approx. 50 years, whereasthe mean age of women with HSIL is approx. 28 years,which suggests in most cases a long precancerousstate that allows the accumulation of secondary geneticchanges. Although secondary genetic changes may occurrandomly, the presence of tobacco metabolites in cervicalsecretions is considered a risk factor in the develop-ment of cervical cancer [158], with certain smoking car-cinogens being found at significantly higher levels in thecervical secretions of cigarette-smoking women [159].Multiparity and the long-term use of oral contraceptivesare also associated with increased risk [160,161].

REGRESSION, LATENCY ANDASYMPTOMATIC INFECTION

In contrast with our understanding of HPV-associatedcancers, our knowledge of the molecular events thatregulate the development of latent and/or asymptomaticinfections is only poorly developed. Experimental infec-tions using animal papillomaviruses such as ROPV orCOPV lead to the development of lesions that persistfor months rather than years [38,162]. Lymphocyteinfiltration and lesion regression take place between 8and 12 weeks post-infection and, by 16 weeks, infectedsites regain the appearance of uninfected epithelium [162].A similar pattern of events occurs in cattle [163], andregression of HPV-induced lesions in humans is alsothought to follow this path [164]. The immune systemis clearly important in controlling viral persistence, andpatients with immune defects can develop widespreadlesions that are refractory to treatment. Although theimmune responses involved in clearance of HPV in-fections are considered in depth elsewhere [165,166],it is worth noting that Alpha papillomaviruses (andin particular those associated with cervical cancer) cancause persistent infections and have evolved a numberof mechanisms to limit the chance of detection by theimmune system. Among these are the regulation of E-cadherin expression and Langerhans cell density by E6[167,168], the interference with MHC presentation by


536 J. Doorbar

E5 [167] and interference with the function of inter-feron response factor 3 by E7 [169]. Perhaps equallyimportant, however, is the fact that papillomavirusesdo not cause a lytic infection, with infectious particlesbeing produced only in the upper epithelial layers incells that are eventually lost from the epithelial surfaceat the end of their life span. The viral early proteinsthat mediate cell proliferation in the lower epitheliallayers are thought to be expressed at levels below thoserequired to reliably trigger an effective host immuneresponse. When it occurs, the stimulation of a cell-medi-ated immune response that can clear infection appearsto depend on cross-priming of dendritic cells by viralantigens expressed in keratinocytes. The frequent de-tection of HPV DNA in cervical lesions in the absenceof any obvious disease may represent latent infection inwhich viral genomes are maintained in the basal layerin the absence of detectable productive infection. Immunesurveillance is thought to be important for this, as lesionsproliferate following immune supression, but this maynot be the only way in which viral latency is maintained.Recent studies have suggested that the generation ofan asymptomatic infection in which viral DNA canbe detected in the absence of any abnormal pathologymay be a normal consequence of infection under somecircumstances, and may result from the silencing of viralgene expression by methylation [139]. Latent infection isthought to require the expression of the viral E1 and E2proteins necessary for genome maintenance in the basallayer, with the E6 and E7 genes not being required [40].

CONCLUSIONS

The consequence of papillomavirus infection dependson the infecting HPV type and site of infection, aswell as on host factors that regulate virus persistence,regression and latency. The characteristics of differentHPV types have been studied extensively and it is nowwell known that high-risk types, such as HPV16, en-code genes that can contribute to cancer progressionwhen aberrantly expressed. During productive infection,however, these genes are carefully regulated and playimportant roles in virus synthesis and in avoidingdetection by the host immune system. It seems thatpapillomaviruses, like many other DNA tumour viruses,cause cancers when their regulated pattern of geneexpression is disturbed. Viral oncoproteins such as E6and E7, which are involved in cell proliferation andthe regulation of cell death, can be extremely dangerouswhen inappropriately expressed. This can happen at thecervical transformation zone, where most cervical cancersoriginate, and it has been suggested that this may bean unstable site for productive infection by high-riskHPVs. Although our understanding of HPV-associatedcancer progression and productive infection is now well

developed, little is known as to the influence of theinfected cell type and the consequence of different cellularenvironments on viral gene expression. The factors thatregulate viral persistence and the events that lead tolatency are other areas that are only poorly understood,but which are very important in fully understanding theconsequences of infection in humans. Such topics aredifficult to research, but will need to be addressed if ourunderstanding of papillomavirus molecular biology is tobe advanced further.

ACKNOWLEDGMENTS

J. D. is a programme leader in the Division of Virologyat the MRC National Institute for Medical Research andis supported by the U.K. Medical Research Council. Theleadership and vision provided by the present Director,Sir John Skehel FRS, is gratefully acknowledged, asis the continued support and advice provided byDr Jonathan Stoye, Head of Virology. The commitmentand enthusiasm of all members of the Papillomaviruslaboratory in carrying out their work and in contributingto our understanding of papillomavirus biology is greatlyappreciated.

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Received 15 December 2005/24 January 2006; accepted 27 January 2006Published on the Internet 11 April 2006, doi:10.1042/CS20050369


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Molecular biology of human papillomavirus infection and ... · Molecular biology of human papillomavirus infection and cervical cancer John DOORBAR Division of Virology, National