The Natural History of Human Papillomavirus Infection and Cervical Intraepithelial Neoplasia Among...

The natural history of human papillomavirus infections of

the mucosal epithelia

LOUISE T. CHOW,1 THOMAS R. BROKER1 and BETTIE M. STEINBERG2

1Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham,AL, USA; and 2The Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System,

Manhasset, New York, USA

Chow LT, Broker TR, Steinberg BM. The natural history of human papillomavirus infections of themucosal epithelia. APMIS 2010; 118: 422–449.

Human papillomaviruses (HPVs), members of a very large family of small DNA viruses, cause bothbenign papillomas and malignant tumors. While most research on these viruses over the past 30 yearshas focused on their oncogenic properties in the genital tract, they also play an important role in dis-eases of the upper aerodigestive tract. Rapidly accelerating advances in knowledge have increased ourunderstanding of the biology of these viruses and this knowledge, in turn, is being applied to newapproaches to prevent, diagnose, and treat HPV-induced diseases. In this introductory article, we pro-vide an overview of the structure and life cycle of the mucosal HPVs and their interactions with theirtarget tissues and cells. Finally, we provide our thoughts about treatments for HPV-induced diseases,present and future.

Louise T. Chow, Department of Biochemistry and Molecular Genetics, University of Alabama atBirmingham, 1918 University Boulevard, McCallum Building Room 510, Birmingham, AL 35294-0005, USA. e-mail: [email protected]

PART I. INTRODUCTION AND

OVERVIEW

Human papillomaviruses (HPVs) are most com-monly known for their benign and neoplasticdiseases of the anogenital tract. These viruses arealso the causative agents of laryngeal papillomasand other hyperproliferative epithelial lesions ofthe airway mucosa, collectively known as recur-rent respiratory papillomatosis (RRP; see thearticle by Larson and Derkay for a detaileddescription of this disease). HPVs can also infectother sites in the head and neck (H&N) regionsuch as the conjunctiva of the eyes, ear canals,nasal sinuses, and oral cavity, especially the

oropharynx and tonsils. Although not as preva-lent as genital tract infections, HPV infectionsand lesions of the airway tract are among themost difficult to investigate, to understand, toprevent, and to bring under therapeutic control.For biomedical research into papillomaviruses

and their associated diseases to lead to effectivepublic health measures, both viruses and hostresponses to infection must be understood inconsiderable molecular detail. This introductoryoverview considers many attributes of HPVsincluding some of the similarities and differencesof HPV genotypes, disease manifestations andthe natural history of infections as determinedfrom analyses of patient specimens. We alsodescribe the genetic and functional organiza-tion of the viral genome, the regulation of viralInvited review

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transcription and RNA processing, the mecha-nisms of viral DNA replication and long-termmaintenance, the properties of the viral proteins,the nature of virus–host interactions in supportof viral DNA amplification, and virion morpho-genesis. Then we summarize the pathogenic con-sequences across the spectrum of benign lesions,neoplastic progression, and ultimately cancers,and the clinical management of infections.We necessarily consider in detail the biologyof the host tissue for all papillomaviruses – theepithelium.One of the most challenging aspects of HPV

research has been that few, if any, infectiousviral particles can be isolated from patient speci-mens. Unlike many other DNA viruses, HPVscould not be propagated in any submerged cellculture system. As a consequence of major diffi-culties in establishing experimental systems torecapitulate the complete infection cycle, earlyinvestigations relied heavily on cloning andsequencing of various HPV types from naturalinfections. Viral RNA transcripts recoveredfrom a small number of benign patient lesionswere characterized to determine their basicorganization and major splicing patterns. Fromrepresentative HPV genotypes, DNA sequencescorresponding to the predominant mRNAexons were then used as probes to examinepatient specimens from different anatomic sitesand across the entire spectrum of lesions. Whatbecame clear from these analyses was that thepapillomavirus reproductive cycle absolutelydepends on complete squamous differentiationof the host epithelium and that squamous andglandular carcinomas do not support the pro-ductive program (1–3). Elevated levels of viralDNA and mRNA are restricted to the mid- andupper cell strata, while the capsid antigen isdetected in only a small fraction of superfi-cial keratinocytes. Viral activity is distinctlyincreased in lesions from patients with immuno-suppressive disorders. In high-grade dysplasiasand cancers, the viral genome is often integratedand only a subset of the viral genes is consis-tently expressed. Concurrently, the encodedviral proteins and their functions were identifiedusing a variety of in situ and in vitro assays. Onlywhen this portrait of viral activities and virus–host interactions in natural infections hademerged could development begin to establishappropriate experimental model systems that

recapitulate real infections or selected elementsof those infections.

HPV taxonomy and pathobiology

Papillomaviruses are a very ancient family ofpathogens and are known to infect the epithelialtissues of amphibians, reptiles, birds, and mam-mals. These viruses appear to have co-evolvedwith their hosts. Almost all viruses are strictlyspecific to their natural host and do not infecteven closely related species.Human papillomaviruses are highly prevalent

and are medically important pathogens. Infec-tions may remain subclinical or they may beactive and induce benign, hyperproliferativelesions of the epithelia, variously called warts,papillomas, or condylomata, according to theanatomic sites of infection. Well over 120 dif-ferent genotypes of HPVs have been isolatedfrom lesions, sequenced and phylogeneticallycharacterized (4). Each genotype is character-ized as being more than 10% different from allothers in their DNA sequences. Closely relatedtypes (approximately 80–90% identical) areclassified as members of the same species, andthey tend to share such important biologicalproperties as tissue tropism, disease manifesta-tion, and pathogenicity.The a-papillomavirus group of HPV types

(comprised of 15 species distinguished to date)infects the anogenital tract, upper aerodigestivetract, and other H&N mucosa. These viruseshave received the preponderance of researchand clinical attention because they can be sexu-ally transmitted and cause significant diseases.These mucosotropic HPVs are further classifiedinto non-oncogenic or low-risk (LR) types, suchas HPV-6 and HPV-11 (species 10), and poten-tially oncogenic or high-risk (HR) viruses,including HPV-16 (species 9), HPV-18 (species7), HPV-51 (species 5), and HPV-53 (species 6).Within each of these species, there are other,closely related types. A small subset of thelesions induced by the oncogenic types can pro-gress to high-grade dysplasias and cancers,notably cervical, vaginal, penile, anal, tonsillar,and oropharyngeal cancers (5). In contrast, thenon-oncogenic or LR HPV-6 and HPV-11 arerarely found in genital cancers and are onlyassociated with pulmonary cancers in a smallfraction of patients with RRP (6).

NATURAL HISTORY OF HPV INFECTIONS AND PATHOLOGY

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PART II. KERATINOCYTES AND THE

SQUAMOUS EPITHELIA

The natural host tissue for the complete infec-tion cycle of all HPVs is the squamous epithe-lium, either the dry external cutaneous skin ormoist mucosal epithelial lining of all body open-ings. Most viral types are predominantly trophicfor one or the other of these tissue types, butcertain genotypes can infect and reproduce (orat least persist) in both. The epithelium is a largeand complex organ and is comprised primarilyof tightly interlocked sheets of keratinocytessupported by an underlying dermis consistingof fibroblasts and extracellular matrices thattogether provide mechanical stability, flexibility,and the physical, chemical, and biologicalbarriers to the external body surfaces and manyof the internal surfaces. The dermis and epi-dermis working in concert present an immuno-logic defence to infection via various infiltratingimmune cells and circulating antibodies.The proximal epithelia lining each of the body

openings are stratified squamous structures(described in detail below), while the more inter-nal epithelia are typically comprised of colum-nar epithelia, some of which are ciliated, as inpart of the respiratory tract. A band of rapidlycycling and dividing keratinocytes called themetaplastic or transformation zone establishes asquamo-columnar junction at each body open-ing. These anatomic regions are located in ornear the larynx, nasal sinuses, urethra, uterinecervix, and anal ⁄ rectal junction. Each suchmetaplastic zone is particularly susceptible topapillomavirus infection and supports variousdegrees of reproduction. If the infecting virus isa HR genotype, the metaplastic tissues can, incertain cases and at a low frequency, undergoneoplastic progression to dysplasias of increas-ing severity and to carcinomas.

Squamous epithelia and dynamics

The squamous epithelium is a multilayeredstructure in which each stratum has a particularprofile of gene expression, protein forms, andcellular architecture that continuously changesas keratinocyte differentiation proceeds (7). Assuch, the strata can be very well identified histo-logically and by in situ probing with antibodiesfor various biomarker proteins, with nucleic

acid probes to individual mRNA species, andvia metabolic labeling of replicating host cellDNA. The basal keratinocytes are in contactwith and responsible for laying down the base-ment membrane network of the extracellularmatrix that separates the dermis from the epi-dermis. Contact with the basement membranegives basal keratinocytes special propertiesincluding cell cycling, lateral motility duringwound healing, and asymmetical cell divisionand vertical polarity once wound closure hasoccurred and contact inhibition sets in. Theintegrity of the basement membrane is a criticalbarrier to invasion of keratinocytes into thedermis and beyond. However, inappropriateup-regulation of various matrix metalloprotein-ases in the transformed keratinocytes comprisingcarcinomas in situ can lead to the proteolyticbreakdown of the basement membrane and totumor cell invasion.The continuous process of turnover and

replacement of skin cells is governed by the lim-ited number of cell divisions accorded to any cell,the so-called ‘Hayflick limit’ of about 60–120divisions before onset of senescence, a conse-quence of erosion of the telomeres of chromo-somes upon every round of replication until theyare too short to avoid chromosome instability.How then do epithelial keratinocytes manage tokeep up with the need for constant turnover andrefreshment? The basal keratinocytes in mostsquamous epithelia are quiescent and do notcycle into the S phase and divide very often.Rather, the stratum that completes a full cellcycle every day or two is the next layer up, theparabasal keratinocytes or transit amplifying(TA) cells. The TA cells are committed to differ-entiation but maintain the ability to divide. Thisis evidenced by the presence of the proliferatingcell nuclear antigen (PCNA) and pRB, the tumorsuppressor which controls G1 to S transition incycling cells, and the ability to incorporatelabeled nucleosides into newly replicated cellularchromosomes. Conversely, basal cells lackPCNA, while being often high in p130 protein,which is related to pRB and known to be abun-dant in quiescent cells, keeping them from enter-ing the cell cycle (8–10). There are additionaldeeper reservoirs, the stem cells that are amongthe basal cells or localized to the bulge of the hairfollicles. Thus, a multi-tiered system of reservecells, sporadically active basal cells, and highly

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active parabasal cells can ensure a healthy epi-thelium for a full lifetime. This division of activ-ity, function, and lineage longevity mightaccount for the temporal variability observed inpersistence of HPV infections, regression ofactive lesions, and possible reappearance of pap-illomas, a concept that remains to be proven orestablished experimentally. Already it is fullysupported by clinical observations of new cervi-cal lesions in middle-aged and elderly patientswho are immunosuppressed and by studies thathave reported the presence of HPV DNA in thebiopsies of RRP patients who have been inremission for a number of years (11, 12).Only after several months of dividing do the

parabasal cells reach their limit and undergo ter-minal differentiation, whereupon the underlyingbasal cell will divide once to replace it. The basalor parabasal keratinocytes divide asymmetri-cally; one daughter cell remains in place as a res-ervoir, while the other is pushed upward towardthe cell surface. The upward moving daughtersof TA cells permanently withdraw from the cellcycle and will not replicate their DNA againunder normal conditions. These cells, the cellsabove them, and those to follow from belowtogether establish a full thickness epithelium ofdifferentiating cells, with each stratum contrib-uting specialized functions as a consequence ofthe particular suites of genes that are turnedon or off (Fig. 1). As examples, involucrin isexpressed in all suprabasal cells, whereas the

keratin pairs expressed in the basal stratum (e.g.K5 and K14) are replaced with higher molecularweight keratins in the parabasal and spinousstrata (e.g. K1 and K10 in cutaneous skin or K4and K13 in mucosal epithelium) that contributeincreased mechanical stability to the skin. Nearthe surface, the cutaneous epithelium assumes anew, histologically identifiable feature as aresult of having keratohyalin granules com-prised of proteins such as (pro)filaggin and lori-crin that help cross-link the intracellular keratinnetwork under the direction of epithelial trans-glutaminases. These upper layers are alsoresponsible for the considerable amount of lipidsynthesis that helps establish the bidirectionalwaterproofing to the superficial strata. Externalcutaneous skin exposed to the drying effects ofair undergoes a final process of maturation andprogrammed death and the cells convert from a‘chemical reducing state’ characteristic of mostanabolically active cells to an ‘oxidizing state’associated with cell death. The nucleic acids aredegraded, the nucleus disappears, and the resid-ual fibrous proteins and outer membrane shellform a stratum corneum of highly cross-linkedcell envelopes that pile up and slough off inresponse to mechanical abrasion. In contrast,the fully differentiated keratinocytes comprisingthe uppermost layers of the squamous mucosamaintain a moist surface and do not develop astratum corneum, but the superficial cells, none-theless, slough off as they die.

Fig. 1. The HPV productive program in a mucosal squamous epithelium. A hematoxylin and eosin-stained tissuesection of a laryngeal papilloma. The cellular differentiation profile and viral productive program are indicatedon the left and right sides, respectively. Photograph provided by Dr. Hsu-Kun Wang from the Chow ⁄Brokerlaboratory.



HPV can infect the squamous epithelium whenbasal or parabasal cells are exposed by wound-ing. Viral early genes are active during woundhealing. In turn, the expression of viral early pro-teins E6, E7, and E5may prolong the duration ofcell proliferation, thereby expanding the infectedcell population. Accordingly, warts, papillomas,and condylomata are often sequelae to a scratch,cut, abrasion, or microbial infection.

Metaplastic zone – the squamo-columnar junction

The dynamic cycling and cell division of thekeratinocytes comprising the metaplastic zonecontribute to the more external squamous epi-thelium and to the more internal columnar epi-thelium. As these keratinocytes do not have aprotective overlay of non-cycling cells, they canbe directly infected by papillomaviruses andother pathogens, a vulnerability that helpsaccount for the particular susceptibility of thesquamo-columnar junction to viral infection. Ifthe metaplastic zone of the cervix becomesinfected with HPV, its cell division, lateralmigration, and glandular differentiation canprovide a conduit for HPV to be conveyed intoendocervical glandular mucosa. The columnarepithelium of the endocervical mucosa is just asingle cell thick with polarized orientation, andit often forms an invaginated glandular net-work. This secretory tissue produces mucus thatbathes the columnar epithelium exocervix andmore external squamous epithelia. However, thecolumnar epithelium is not a supportive host forthe HPV reproductive program as it does not

establish the differentiation gradient character-istic of squamous epithelia. Rather, columnarendocervical tissue infected with HR HPVs candevelop into carcinoma in situ (CIS) and can-cers. In the airway, LR HPVs may infect cells ata laryngeal squamo-columnar junction andmigrate into the ciliated columnar epithelium ofthe trachea, but there is also good evidence thatthey can directly infect the columnar epithelium.Without the appropriate squamous host factorsto support virus expression, these tracheal infec-tions remain latent, with no evidence of disease(12).

PART III. PAPILLOMAVIRUS GENOME

ORGANIZATION, RNA TRANSCRIPTION,

AND PRODUCTIVE DNA

AMPLIFICATION

Synopsis

Papillomavirus genome organization and func-tions have been perfected over millions ofyears of evolution and selection and are exqui-sitely sophisticated and interconnected. Eachelement of the biology of HPV raises fascinat-ing questions and provides an intriguing exam-ple of diversion of cellular control in normalepithelia. HPV is the object of investigationand equally the informant concerning many ofthe fundamental attributes of vertebrate cellbiology.The genome organization is conserved among

human and animal papillomaviruses, but with

Fig. 2. Genomic organization of HPV-11. The genome contains a non-coding upstream regulatory region(URR), also called the long control region (LCR), an Early (E) region and a late (L) region. The open readingframes (ORF) are denoted as boxes. The three major promoters (P1, P2, P3), a minor promoter (P4), and twopoly-adenylation sites are indicated. Alternative splicing of the primary RNA transcripts, coupled with the utili-zation of alternative promoters and poly-A sites, allows the translation of viral proteins as the first or the secondORF in the messenger RNAs. This genomic organization is highly conserved except for the absence of P2 inhigh-risk HPV genotypes, where the E7 protein is translated from mRNAs with one of two alternative intragenicsplices in the E6-coding region.

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some instructive differences (for a review, see13) (Fig. 2). The double-stranded circularDNA, which ranges from 7600 to nearly 8000base pairs in length, replicates as multi-copyextrachromosomal plasmids in the nucleus ofinfected keratinocytes. The genome has (i) anupstream regulatory region (URR) or long con-trol region 400–700 base pairs in length thatdoes not encode proteins, (ii) six or more E(early) region open reading frames (ORFs), and(iii) two L (late) region ORFs (see Fig. 1). TheURR contains the origin of DNA replication,early promoters, and binding sites for core tran-scription factors and various enhancer andrepressor regulatory proteins (for a review, see14). All transcription takes place in the samedirection [left to right (5¢–3¢) on the conven-tional linear map or clockwise on the circularmap] using multiple promoters. The E regionand L region are both followed by a poly-Aaddition site.Low copy maintenance replication of HPV

DNA takes place in the dividing basal and para-basal keratinocytes. Elevated viral RNA tran-scription and DNA amplification to high copynumbers occur primarily in differentiating mid-to upper spinous cells, whereas the capsid pro-tein is only detected in relatively few superficialcells (Figs 1, 3 and 4). As these differentiatedcells have withdrawn from the cell cycle and nolonger produce the enzymes and substrates thatare essential for DNA replication, the virusmust reactivate deoxyribonucleotide triphos-phate (dNTP) synthesis and the production ofthe host DNA replication machinery to repli-cate or amplify its DNA. The E region proteinsare devoted to this ultimate goal. Briefly, E1and E2 proteins are directly involved in viralDNA replication, whereas E6, E7, and mostlikely E5, the three viral oncoproteins, conditionthe differentiated cells to support viral DNAamplification by inactivating major tumor sup-pressor proteins and activating signal transduc-tion. The viral oncoproteins also modulate hostimmune surveillance to establish persistence. L1and L2 assemble the newly replicated viralDNA into progeny virions in the superficialstrata. The regulation of the promoters, mRNAprocessing, and a more complete description ofthe functions of each viral protein will be dis-cussed.

Fig. 3. HPV-11 gene expression and replication inan unusually active laryngeal papilloma. The tissueswere fixed in 10% buffered formalin and embedded inparaffin. Serial 4 lm sections were subjected to in situanalyses. Top panel: immunohistochemistry revealingthe induction of proliferating cell nuclear antigen(PCNA) (reddish stain) in the differentiated strata.Middle panel: in situ hybridization with 35S-labeledsense-strand RNA probes demonstrates that viralDNA amplification occurs in the differentiated strata.Bottom panel: in situ hybridization with 35S-labeledantisense-strand RNA probes shows that elevatedviral RNA expression occurs in the differentiatedstrata. In situ analyses were performed by Ms. Mar-tha R. Hayes from the Chow ⁄Broker laboratory.



The genome organization and viral protein functions

All HPV types have two major promoters. P1 islocated immediately upstream (5¢) of the E6gene, and P3 is located within the E7 gene.There is also a universally conserved P4 pro-moter immediately upstream of the tiny E8ORF. Rare RNA species have 5¢ ends in theURR or in other coding regions, suggestive ofadditional minor promoters. The viruses relyheavily on alternative mRNA splicing to accessthe various ORFs, and some bi- or poly-cis-tronic mRNAs can or may encode more thanone protein. One feature that distinguishes HRand LR viruses is the presence of E6 intragenicsplices from one splice donor to one or twoalternative splice acceptors. The splice leads to atranslational frame shift and premature termi-nation of E6 translation. Truncation of E6 pro-tein is thought to allow more efficienttranslation initiation of the downstream E7ORF in the same mRNA (15). However, muta-tion of the splice donor did not abolish E7translation (9). It was not established whether acryptic splice donor was used in the latter case.The LR HPVs such as HPV-11 or HPV-6 havea dedicated P2 promoter located in the E6 geneto transcribe the E7 mRNA (16). The criticalelements that control the P1 promoter arelocated at the 3¢ end of the URR, overlappingthe origin of replication (ori). The ori consists ofa cluster of (usually three) E2 protein-bindingsites (E2BS), flanking an AT-rich region

containing an array of E1 protein-binding sites(E1BS). These features are illustrated in Fig. 2.In this article, we briefly review the functions

of the viral proteins. A more detailed discussionof the E6 and E7 proteins, with comparisonsbetween the LR and HR HPVs, is provided inthe article by Pim and Banks (this issue).

E6 – Numerous reports have described the bio-logical activities of the HR HPV E6 protein(about 150 amino acids in length) in proliferat-ing primary human keratinocytes (PHKs) andin cell lines (reviewed by 17). The best-knownproperty of the HR HPV E6 is its ability todegrade the major tumor suppressor protein,p53, which monitors and guards the integrity ofthe genome by inducing genes to effect cell cyclearrest, DNA repair or, alternatively, senescenceor apoptosis (see article by Pim and Banks). TheHR HPV E6 proteins also destabilize a numberof PDZ domain-containing host proteins thatregulate cell polarity and signal transduction,including hDLG, hScribble, and MAGI(reviewed by 18). In addition, the E6 proteincan modulate G protein signaling by degradingGAP proteins (19) and can transactivate thecatalytic subunit of the telomerase gene (hTert)(20). These properties are undoubtedly impor-tant for oncogenesis driven by the HR HPVs.In vitro, E6 alone can immortalize human mam-mary cells. In collaboration with the HR HPVE7 protein, which destabilizes the pRB family

Fig. 4. HPV-11 DNA amplification and capsid antigen detection. Sections of a formalin-fixed laryngeal papil-loma were subjected to in situ analyses. Left panel: fluorescence in situ hybridization (FISH) with nick-translatedDNA probes (green) to detect amplified viral DNA in the differentiated strata. Nuclear DNA is stained blue withDAPI. Right panel: L1 antigen detection by immunohistochemistry (reddish stain) in the same papilloma from adifferent area. Most regions were negative for L1. In situ analyses were performed by Dr. Hsu-Kun Wang andMs. Eun Young Kho from the Chow ⁄Broker laboratory.

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of pocket proteins, E6 immortalizes primaryhuman foreskin or cervical keratinocytes (5, 21).However, the function of the E6 protein in theproductive phase of the viral infection is notunderstood because significant E6 or E7mutants cannot be stably maintained in trans-fected PHKs (22–25). As the LR HPV E6 pro-teins do not share the same properties, acommon ability between the HR and LR HPVE6 is most likely to be involved. In this regard,both HR and LR HPV E6 can repress p53-dependent transcription regulatory functions byinhibiting p300 acetylation of p53 (26–28 andreferences therein). Our recent experiments inorganotypic cultures of PHKs (see Part IV) har-boring an HPV-18 mutant genome unable toencode a full-length E6 protein demonstratethat the mutant is severely affected in viralDNA amplification. High levels of p53, which isknown to be induced by E7 protein activity,accumulate in numerous cells (29), implicatingp53 in suppressing viral DNA amplification.This observation is consistent with transientreplication assays where ectopic p53 repressesamplification of human and bovine papilloma-virus origin-containing plasmids by ectopicallyexpressed homologous E1 and E2 replicationproteins (30–32). However, these observationsdo not rule out the possible roles of other E6-targeted host proteins. The mechanisms of thisaspect of E6 function remain to be investigated.

E7 – The maintenance mode of HPV DNA rep-lication takes place in basal cells that cycle peri-odically and in the TA keratinocytes that cycledaily. Viral DNA amplification occurs only in asubset of post-mitotic differentiated keratino-cytes (reviewed by 13). As viral DNA replica-tion depends heavily on the cellular DNAreplication machinery, the virus must promotethe reestablishment of such a permissive milieu.This is the function of the E7 protein (of about98 amino acids in length). Both HR and LRHPV E7 proteins can promote S-phase reentryby differentiated keratinocytes in a squamousepithelium developed in vitro (9, 33–35). Inter-estingly, unlike the cycling cells where the majortumor suppressor pRB (retinoblastoma suscep-tibility protein) controls the cell cycle entry, inthe stratified squamous epithelia, including thelarynx, cervix, and foreskin, the pocket proteinp130, which is related to pRB, is primarily

responsible for maintaining the homeostasis ofdifferentiated cells, preventing them from reen-tering the S phase. The E7 protein destabilizesp130 (10, 36) and promotes S-phase reentry.The HR HPV E7 proteins can additionallydestabilize pRB. If E7 is inadvertently over-expressed in undifferentiated cycling cells, E7bypasses the growth stimuli normally neededfor activating cyclin-dependent kinases (cdks)cdk4 or cdk6 by D type cyclins to phosphorylateand to inactivate pRB in order to promoteS-phase entry. Moreover, ectopic expression ofHR HPV E6 and E7 can each destabilize thechromosomes. The ability to destabilize twomajor tumor suppressors, p53 and pRB, as wellas other cellular proteins, largely accounts forthe oncogenic potential of the HR HPVs(reviewed by 21; see the article by Pim andBanks).

E1 and E2 proteins, the replication origin, and themechanisms of viral DNA replication – To supportPV DNA replication, the virus encodes two pro-teins: the dimeric E2 origin-binding protein andthe E1 replicative DNA helicase, which assem-bles into a dihexameric complex (reviewed by37). All other replication enzymes and proteinsare supplied by the host cells. E1 is the onlyenzyme encoded by papillomaviruses, making itdifficult to identify selective inhibitors of HPVreplication. E2 is also required for proper plas-mid partitioning in dividing cells to establishpersistence (38–42 and references therein). Bybinding to the E2BS at the origin, which over-laps the P1 (E6) promoter, the E2 protein canalso regulate transcription (see HPV-associatedcancers).The E1 and E2 mRNAs are derived from the

same primary transcripts. The E1 protein istranslated from a very low-abundance message(43). In the HR HPV, the E1 mRNA is initiatedfrom the E6 promoter and E6 intragenic splicingappears to be important for the translation ofboth the E1 and E2 proteins (44). E1 is altera-tively encoded by an unspliced messenger RNAthat extends from the P3 promoter through theearly poly-A site. The E2 protein cannot betranslated from this transcript, as the 3¢ end ofthe E1 ORF substantially overlaps the 5¢ end ofthe E2 ORF. Rather, the full-length E2 proteinis translated from a spliced mRNA that joinsalternative splice donors in the E1 ORF to an



acceptor about 100 bases upstream of the E2ORF. This E1 intragenic splice leads to a frame-shift and premature termination of the E1 pep-tide, thereby increasing the distance to the E2initiation codon to allow efficient translation re-initiation (43, 45).The three closely spaced E2 protein-binding

sites in the replication origin (ori) bind threedimers of E2 (thus six monomers of E2).Together, these induce a toroidal loop in thesupercoiled DNA (46). The extra twist in theDNA creates torsional stress that is relieved bydenaturation of the AT-rich sequence in the ori-gin, likely facilitating recruitment and loadingof the E1 proteins to form a dihexamer, whichoccurs with the aid of heat shock proteinsHsp70 and Hsp40 (47). The E1 dihexamer is avery active bidirectional helicase on supercoiledDNA substrates when analyzed in the presenceof the single-stranded DNA binding protein, to-poisomerase I, and an ATP regenerating system(47). E1 recruits DNA polymerase a ⁄primaseand the single-stranded DNA-binding proteinRPA to initiate replication. E1 is requiredthroughout initiation and elongation, whereasE2 is only required for the initial recruitment ofthe E1 complex to the ori (reviewed by 48).As E1 is such a potent helicase, its activity

must be controlled so as not to unwind viralDNA without concomitant viral DNA replica-tion. Efficient nuclear import requires the pres-ence of a bipartite nuclear localization sequence(NLS) that is activated by phosphorylation ofserine residues by mitogen-activated proteinkinases (MAPKs) (Erk1 ⁄2 and Jnk). However,the default position of E1 is in the cytoplasm,attributable to a dominant nuclear exportsequence (NES). The NES is only inactivated bycdks recruited by an adjacent cyclin-bindingsite. Most of these regulatory motifs are locatedwithin a span about 45 amino acids situatednear the amino terminus, while the MAPK-binding motifs are located near the carboxyl ter-minus (49, 50). Thus, the unwinding of the viralDNA is tightly coupled to the cell cycle.Alternative RNA splicing or alternative pro-

moter usage also generates transcripts that canencode E1MÊ2C (joining together the amino-terminal portion of E1 and the C-terminal por-tion of E2), E1MaÊ4 (51), or E2C (also calledE8Ê2C). The 5¢ exon spanning the short E8ORF contains the initiation codon for the E2C

product (52–54). Of the many forms of E2-related proteins, only the full-length E2 proteincan support viral ori-dependent replication, asits amino terminus interacts with the E1 protein(55). Competition between full-length E2 pro-tein and E2-related proteins that contain theE2C dimerization and DNA-binding domaincan modulate viral replication and transcription(51, 54, 56). In addition, the E8 domain can alsorecruit co-repressors and is indeed a potent tran-scription repressor (54, 57).As will be discussed in Part VI of this intro-

duction, in a newly developed model system inwhich organotypic cultures of PHKs harboringHPV-18 are used to study the reproductive pro-gram, the HPV-18 genome amplifies to a highcopy number in the mid- and upper spinouscells. High titers of infectious virions are repro-ducibly generated. Detailed in situ analyses ofthe squamous epithelium demonstrated thatviral DNA amplification lags behind the S phasewhen host DNA replication occurs and initiatesin cells with high levels of cytoplasmic cyclin B,a signature of G2 arrest. Concomitant with viralDNA amplification, the E7 activity is lost, asevidenced by the reappearance of p130, disap-pearance of E7-induced PCNA, and inability toreenter into another round of the S phase. Theprogram then switches to late gene expression.The progeny DNA is packaged into virions inthe superficial cells, while particle maturationtakes place in the stratum corneum where theoxidizing environment allows the disulfidecross-linking of the L1 capsid proteins (29).

E1Ê4 – The E1Ê4 protein is the most diver-gent protein in sequence and length among thedifferent papillomavirus types. The mRNA isinitiated from the P3 promoter and is splicedfrom the E1 ORF to a site in the E4 ORF. Thus,the encoded protein is comprised of the aminoterminal several amino acids of the E1 ORFwith the rest of the protein encoded by the E4ORF. Both the protein and the mRNA are themost abundant among all viral products. E4ORF exactly overlaps the central hinge regionof the E2 protein (reviewed by 13). In benignlesions, this protein is primarily detected in theupper strata in cells containing high copies ofviral DNA and the L1 antigen (reviewed by 58).This expression profile reflects that E1Ê4 isadditionally present as the first ORF in a

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bicistronic late message, which also encodes theL1 or the L2 protein (reviewed by 13). Ectopicexpression of the E1Ê4 protein causes the col-lapse of the cytokeratin intermediate filamentsin submerged monolayer cultures, although thiswas not observed in differentiated cells in thesquamous epithelium (59, 60). Ectopic E1Ê4can sequester cyclin B ⁄ cdk1 to the cytokeratin,causing G2 arrest (reviewed by 61). However, inthe raft culture model with a fully productiveHPV-18 program (see Part VI), the elevatedcytoplasmic cyclin B1 was located in the lowerand mid-spinous cells where viral DNA amplifi-cation initiated, whereas the E1Ê4 protein wasdetected in the upper spinous cells coincidentalwith high viral DNA, but not with cyclin B.These results suggest that E1Ê4 alone is notresponsible for the cytoplasmic accumulation ofcyclin B1 to cause G2 arrest. The dramatic up-regulation of E4 protein in the upper stratacould have been the consequence of elevatedviral DNA templates for the transcription ofmonocistronic or bicistronic E1Ê4 mRNAs.Indeed, the expression of HPV E7 alone in dif-ferentiated cells causes not only S-phase reentrybut also a prolonged G2 phase (N.S. Banerjee,T.R. Broker, and L.T. Chow, unpublishedresults). The possible involvement of the E1Ê4protein of CRPV and several HPV types inDNA amplification has also been directly inves-tigated by mutational analyses in rabbit skinand in organotypic culture of immortalized celllines, respectively. The results were not consis-tent. The function of the highly abundantE1Ê4 protein remains to be elucidated.

E5 – The E5 ORF is located immediatelyupstream of the early poly-A site and is presentin all early region mRNAs. Two reports indi-cate, respectively, that E5 can be translatedfrom an mRNA which also encodes the E2 orthe E1Ê4 protein (62, 63). E5 is a small, multi-functional membrane protein, predominantlylocalized to the endoplasmic reticulum (64 andreferences therein). It interacts with the 16-kDavacuolar-ATPase and prevents the acidificationof early endosomes, thereby altering the traf-ficking, turnover, and signal transduction ofepidermal growth factor receptor (EGFR) andrelated receptor tyrosine kinases, hence modu-lating cell growth (65–67). Thus, E5 may havean important role in establishing and expanding

the infected basal ⁄parabasal cell populationduring the tissue repair phase after the wound-ing during which the virus first gains entry. Invitro, E5 increases the immortalization efficiencyof HR HPV E6 and E7. Moreover, EGFR andits activities are elevated in laryngeal papillomasand in cultured papilloma cells (68). However,the role of E5 in the viral life cycle is not under-stood, as genetic dissection in organotypic cul-tures has not yielded clear answers (69, 70).Considering that efficient E1 protein nuclearimport depends on phosphorylation by MAP-Ks, which are downstream effectors of receptortyrosine kinase signal transduction, E5 isexpected play a major role in the efficiency ofviral DNA amplification, and it indeed does inthe new model system in which organotypic cul-tures are developed from PHKs (J-H. Yu, T.R.Broker, and L.T. Chow, unpublished observa-tion). Along with E6 and E7, E5 can down-regu-late host immune responses to viral infection.Therefore, E5 would be a contributing factor inthe early stages of viral oncogenesis, althoughE5 is not expressed in HPV cancers (21). Atransgenic mouse system also indicated a role ofE5 in cervical carcinogenesis (71).

The late capsid genes L2 and L1 – Papillomavi-ruses switch to late transcription by (i) dramati-cally increasing the number of template copiesof DNA as a result of vegetative amplification,(ii) diminishing or eliminating the activity ofthe P1 and P2 promoters, leading to up-regula-tion of the P3 promoter, (iii) suppressing theutilization of the early polyadenylation sitesuch that transcripts from the P3 promoter areelongated through to the late poly-A site; and(iv) gaining stability of late RNAs, possiblybecause specific RNA instability factors are lostin terminally differentiated keratinocytes. TheL1 protein is primarily encoded by the E1Ê4-L1 bicistronic mRNA, although minor mRNAspecies also exist, which have other 5¢ ends,implicating additional promoters (reviewed by13). A long transcript spanning E1Ê4, E5, L2,and L1 could be a splicing intermediate for theE1Ê4-L1 mRNA or could be the mRNA forthe L2 protein.L1 forms pentameric capsomeres that com-

prise the major portion of the icosahedral vir-ion, while a single copy of the L2 protein isaxially embedded into each of the 72 pentamers



and helps establish shape and stability (72).Some post-translational modifications to L1such as limited glycosylation apparently occur,but their function remains unknown. Papil-lomavirus virions do not have a membraneenvelope. Because the L1 capsid proteins arecross-linked by disulfide bonds, they are notori-ously stable to environmental extremes (29,73-75). Virus-like particles (VLPs) comprised ofthe L1 protein alone form the basis for currentanti-HPV vaccines, as described below.The amino-terminal domain of the L2 proteinhas a furin cleavage site that is highly conservedamong PV types. During de novo infection,virion binding to extracellular matrices inducesa conformational change, exposing this site,and proteolysis is essential to binding to a co-receptor on the cell surface and to particleuptake into the cell (76, for a review, see 77).Presumably, the reducing atmosphere of the liv-ing cell breaks the disulfide cross-links of the L1protein, and the acidic environment of theendosome dissociates the capsid, releasing thechromatinized viral genome into the cytoplasmwhere it traffics to the nucleus.

PART IV. NATURAL HISTORY OF

PAPILLOMAVIRUS INFECTIONS

HPV infections and interactions with the hostcells need to be considered in four distinctphases: (i) latent, persistent infections depen-dent on long-term maintenance of the viral gen-ome as autonomous plasmids. Such infectionscan remain subclinical for years, or they canbecome activated, particularly as a result ofwounding or immunosuppression. (ii) Activa-tion of viral gene expression leads to a wartylesion that can but does not necessarily result inextensive replicative amplification of the viralDNA and its packaging into infectious particles.Most benign HPV lesions of the mucosal epithe-lia, especially laryngeal papillomas, producevery few virions as inferred from the sparsecells positive for the capsid antigen (Fig. 4). (iii)Persistent infection by the HR HPV types can ata low frequency undergo neoplastic progressionto high-grade dysplasias and (iv) to carcinomas,where the viral DNA is often integrated intohost chromosomes. This review will focusprimarily on the HPVs of the mucosa

comprising the upper respiratory tract and ano-genital tract as they have been most intensivelystudied.

Latent infections

Latent infections have been difficult to examinebecause the viral DNA and RNA are scarce,and cells in culture exhibit a proliferative‘wounding’ phenotype that activates latentinfection. PCR detection of the DNA and RT-PCR analysis of the transcripts have, nonethe-less, determined that HPV can be harbored incells ⁄ tissues completely subclinically at andbeyond the apparently normal margins of manyactive, visible lesions and in patients in remis-sion (12, 78). There is also good evidence that asignificant fraction of the population carrieslatent HPV DNA in their upper airway withno history of papillomatous disease (79, 80).Latency is the most likely outcome of HPVinfection when considered on a per-cell basis,because individuals with HPV-induced diseasessuch as RRP have a relatively small number oflesions but extensive latent infection throughoutthe airway. When the basal cells divide, theHPV must replicate to keep up the low copynumber in these cells. To replicate viral DNA,the E1 and E2 replication proteins are required,and the E6 and E7 proteins are almost certainlyneeded as well. Notably, E6 and E7 mutantgenomes cannot be stably maintained in trans-fected human keratinocytes (22–25). Once thebasal cells have withdrawn from the cell cycleand returned to quiescence, there is no need forthe early viral gene expression. These extremelylow levels of viral gene expression may well beone of the viral strategies to minimize evidenceof its presence, to ‘fly below the radar’ of thehost immune system.

Active infections

The basal and parabasal cells maintain low viralDNA copy number, requiring minimal expres-sion of viral early proteins when the cells peri-odically cycle. In an active infection, theparabasal cycling compartment expands tomany cell layers before the daughter cells per-manently exit the cell cycle to differentiate intospinous cells. However, when the daughter cellshave withdrawn from the cell cycle to undergo

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squamous differentiation, it becomes imperativefor the virus to restore the host replicationmachinery to enable viral DNA amplificationand progeny virus production. In these lesions,not only does the viral DNA amplify in a subsetof differentiated cells, but also the host DNAreplicates to become tetraploid (9, 29, 81),accounting for the enlarged nuclei in a subset ofspinous cells typical of papillomas, condylo-mata and low-grade squamous intraepitheliallesions (SIL). Although the induction of hostDNA replication proteins such as the PCNA bythe E7 protein in the differentiated strata of asquamous epithelium is an infallible indicator ofHPV infections (8, 9, 29), only some of thePCNA-positive cells replicate host and viralDNA (see Figs 3 and 4) (8–10, 29, 35) This isattributable to the sequestration of cyclinE ⁄ cdk2 by its inhibitors p27kip1 and p21cip1that are constitutively transcribed in the differ-entiated cells, but with varied protein stabilities(81–85). Moreover, benign lesions do not neces-sarily harbor many cells with amplified viralDNA or productive of the L1 capsid protein(see Figs 3 and 4). Such a modulated virus–hostinteraction contributes to the long-term persis-tence in the hosts without arousing the immunesystem to eliminate infected cells and hencevirus from the host. This interpretation is con-sistent with highly elevated viral activities inimmune-suppressed patients and in infectedhuman xenografts implanted in immune-com-promised mice (86, for reviews, see 87, 88).

Neoplastic progression

Given the strategy of the viral productive pro-gram, which depends on the viral oncoproteinsto disrupt host cell cycle regulatory proteins,one can appreciate that persistent over-expres-sion of the HR HPV oncogenes in the basalcompartment can lead to excessive cell cyclingand accumulation of deleterious host genemutations selected for survival and growth.Thus, persistent infections with any of the HRHPV genotypes pose a small but very real riskof progression that can result in moderate andhigh-grade dysplasias in which the zone ofcycling cells further expands to comprise themajority or even the entire thickness of thesquamous epithelium, with little or no differenti-ation in the upper strata. The potential to

support viral DNA amplification and virionproduction is correspondingly reduced. There isevidence that integration of viral DNA into thehost chromosomes often occurs in preneoplasticlesions and these lesions progress faster thanthose containing entirely extrachromosomalHPV DNA (89).

HPV-associated anogenital cancers

Further neoplastic progression can lead toinvasive and metastatic carcinomas. In suchlesions, the viral genome is often integratedinto host chromosomes. HPV-18 has beenreported to integrate preferentially near c-myc(90). The most prominent example is the co-amplification and translocation of HPV-18DNA adjacent to the c-myc gene in the HeLacells derived from a cervical adenocarcinoma(91). The viral integration site is invariablylocated in and disrupts the E1 or E2 gene andis often accompanied by deletion of some ofthe downstream viral sequences, encompassingthe E5 gene and early poly-A addition site (3).The consistent deletion of the DNA-bindingdomain of the E2 gene in HPV cancers resultsin the loss of negative feedback regulation ofthe P1 promoter. Accordingly, the E6 and E7RNA transcripts are over-expressed. Becauseof the downstream deletion of the viral earlypoly-A site, the viral mRNA is chimeric, andtranscription extends into the flanking hostDNA, where a particularly stable cellular poly-A sequence is hijacked (92). In the most rapidlygrowing tumor cells, the levels of E6 and E7transcription remain unchecked and invariablyhighly elevated. Analyses of a cervical cancercell line, an oropharyngeal cancer, and epider-mal keratinocytes immortalized in vitro byHPV-16 or HPV-18 (93) unexpectedly revealedthat only a single copy of the papillomaviralgenome or isogenic HPV copies arising fromthe loss of heterozygosity were transcriptionallyactive, no matter how many copies of the viralgenome were integrated or at how many dif-ferent host chromosomal loci (94). In cases oftandemly integrated viral genomes, only thedownstream 3¢ border copy, which was inter-rupted in the E2 gene, was transcriptionallyactive, while the upstream intact copies weresilenced by DNA methylation, as were all othernon-transcribed copies at other loci. Such



selection invariably resulted in cells lackingexpression of a functional E2 or E2-relatedprotein.What triggers the neoplastic progression of

benign lesions harboring HR HPVs? The earlyregion gene expression is up-regulated as a resultof wounding and healing or following local orsystemic immunosuppression. Such over-expres-sion can begin to exert real and permanent dam-age through the combination of excessive cellcycling and interference with the DNA damagecontrol functions of p53, resulting in theaccumulation of mutations or chromosomalaberrations and pushing the cells towardimmortalization and transformation. Indeed,continuous expression of the viral oncoproteinsE6 and E7 is necessary to maintain the trans-formed phenotype, as ectopic E2 expression incervical carcinoma cell lines that express the HRHPV oncogenes shuts off the P1 promoter,resulting in senescence or apoptosis (95–97).

PART V. HPV INFECTIONS IN THE H&N

REGION AND LUNGS

Recurrent respiratory papillomatosis

Infections – Recurrent respiratory papillomato-sis and associated lesions in the upper airwayare extremely difficult to treat, not only becauseof their location, but also of their tendency torecurrent regrowth (98) (see also the articles bySyrjanen, by Smith, and by Larson and Der-kay). The primary treatment modality is surgi-cal removal under general anesthesia, mostnotably with the microdebrider and with lasers.Typically the seemingly normal tissues sur-rounding overt papillomas as well as more dis-tant tissues in the airway are latently infectedwith HPV (11, 12), and papillomas can regrowbecause of activation of viral gene expression inthe surgical margins or nearby squamous tissuesduring the wound healing phase, quickly rees-tablishing the obstructive papillomas. In addi-tion, papillomas of the upper esophagus can beseen in RRP patients. Whether this is due tomigration of HPV-infected cells from a primarylesion in or near the larynx or is a direct infec-tion remains unknown.Adult-onset RRP is probably caused by a new

infection associated with oral sex. In contrast,

juvenile-onset RRP is attributed to verticaltransmission from mothers with genital condy-lomata, possibly during passage through thebirth canal. Intriguingly, trophoblasts, whichhelp establish the placenta early in pregnancy,can be infected with and support HPV replica-tion in vitro (99). Indeed, HPV DNA has beenfound in placental tissues and in umbilical cordblood (100, 101), possibly leading to trans-placental infections. HPV has also been foundin near-term amniotic fluid (102), and somebabies do have laryngeal lesions at birth.Moreover, some children delivered by cesareansection go on to develop RRP. Thus, some oraland airway infections are established in utero.

Modification of normal squamous epithelial biologyin HPV-induced papillomas – While HPV expres-sion and replication are dependent on cellularfactors that are characteristic of squamous epi-thelium, some of these factors are altered inactive HPV infections. Studies of recurrentrespiratory papilloma tissues and primary cellsderived from those papillomas have helped elu-cidate alterations that occur in benign HPV-induced lesions. Globally, the transcriptionalprofile of the tissues when analyzed on geneexpression arrays is significantly altered, with apattern of induction or suppression of manygenes that is quite characteristic of carcinomas,although the lesions are benign (103). The papil-lomas are characterized by abnormalities indifferentiation that lead to an expansion ofbasal-like cells and accumulation of spinouscells, with reduced cell loss at the tissue surface(104). Although involucrin is expressed in thesuprabasal cells, the switch from basal cell-liketo differentiation-specific keratins occurs in onlya small subset of cells. Most cells maintainbasal-cell markers, and the terminal differentia-tion marker (pro)filaggrin is essentially absent.This perturbation of squamous differentiationin RRP might be one reason why respiratorypapillomas produce very little virus relative togenital condylomas.HPV6 ⁄11-infected papilloma cells have signifi-

cant alterations in signal transduction pathways.They over-express surface EGFR because ofrecycling of the receptor following ligand bind-ing and internalization, and have enhanced sen-sitivity to EGF so that the threshold for Erk

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activation is at least 10-fold lower than in nor-mal cells (68). Other signal transduction altera-tions, many linked to the constitutive activationof the EGFR in vivo, include activation of phos-phoinositol-3-kinase, p38 MAP kinase, andNuclear Factor kappa B (NFjB), and elevatedlevels of the signaling intermediate Rac1, a smallGTPase that regulates many signaling pathways(105–108). These alterations contribute to theabnormal phenotype of papilloma cells in vitro(109), andWoodworth et al. (110) have reportedthat EGFR signaling is required for the forma-tion of HPV-induced papillomas in a transgenicanimal model.EGFR signaling through Rac1 also mediates

expression of the enzyme cyclooxygenase 2(COX-2) and its downstream product prosta-glandin E2 (PGE2) in papilloma cells (107, 108).COX-2 is not only normally induced duringinflammation and wound healing, but is alsoexpressed in benign tumors and many carcino-mas. COX-2 expression can inhibit epithelialdifferentiation and apoptosis, and inhibition ofCOX-2 in laryngeal papilloma cells with theselective inhibitor celecoxib suppressed cellgrowth and enhanced spontaneous apoptosisin vitro (107). COX-2 can be induced in normallaryngeal cells by the expression of the HPV-11E7 protein, and treatment of papilloma cellswith celecoxib reduces HPV-11 transcription(R. Wu, A. Abramson and B. Steinberg, unpub-lished data). Thus, virally induced alterations insignal transduction culminating in COX-2expression might be an important contributorto papillomavirus formation. However, we haverecent evidence (A. Lucs, R. Wu, N. Jamal, A.Abramson and B. Steinberg, unpublished data)showing that COX-2 is constitutively expressedin clinically normal airway tissues of respiratorypapilloma patients. Thus, constitutive expres-sion of COX-2 might also be a risk factor in thedevelopment of disease following HPV infec-tion.

Pulmonary involvement – The HPV-driven laryn-geal papillomas or oropharyngeal cancers canbe found extending down the airway into thetrachea, bronchi, and most rarely in the lungs ina subset of patients. The risk of extension intothe trachea and lower airway is significantlyincreased if a tracheotomy is performed to cre-ate an alternative airway when the narrow

laryngeal region is largely or fully obstructedwith papillomas (111). The spread might becaused by cell migration. Papilloma cells canblock the natural clearing action of ciliated air-way lining in removing secreted mucus andmicrobial infections. On the other hand, itmight be due to the activation of latent HPVinfection in response to wound healing or due tothe squamous metaplasia induced by tracheot-omy, as similar amounts of viral DNA can bedetected in the trachea and in the larynx.Approximately 5% of juvenile-onset laryngealpapillomatosis cases eventually progress to lunginvolvement (RRP Foundation Patient DataAnalysis: http://www.RRPF.org). These tend tobe patients who had early and severe laryngealpapillomas. Such expansion of the lesions usu-ally occurs over a span of 10–20 years. In thelung, even LR HPV types can also cause neopla-sias and cancers. Pulmonary involvement isdetermined by CT scan and biopsy. HPV lesionsin the lungs are especially dangerous becausethey cannot be accessed readily by surgery andthey have so far proved recalcitrant to adjuvantpharmacotherapies. Without the opportunityfor periodic debulking, pulmonary papillomascan become obstructive for exchange of air,leading ultimately to necrosis and cavitation ofthe lung tissue, and to death.

Oral and ocular infections

HPV-13 and HPV-32 have been found in oralfocal epithelial hyperplasia (i.e. Heck’s dis-ease) (112). HPV-6 and HPV-11 among othergenotypes can infect the ocular conjunctivaand induce papillomas that are cosmeticallydisfiguring and can grow sufficiently toobstruct vision (113) (see the article bySyrjanen).

Head and neck cancers and lung cancers

From a substantial fraction of oral and otherH&N lesions, papillomavirus DNA can beisolated, PCR-amplified, and genotyped. Over-all, about 20% of H&N cancers are believedto be a result of HPV-mediated oncogenesis(see the article by Smith). Most of thesecases are oropharyngeal and tonsillar cancers.Interestingly, HPV-associated cancers have agenerally better long-term prognosis than



HPV-negative oral cancers. Thus, screeningfor HPV in oral lesions has become a newstandard of diagnosis when confronted withH&N cancers, as this information can guidethe choice of therapies.Lung cancers that are very rare sequelae to

upper airway papillomas and oropharyngealcancers have been reported, almost alwayscaused by HPV-16 or HPV-11 (cf. 114). In addi-tion, HPV-16 has also been detected in lung can-cers in patients without RRP or oropharyngealcancers. The viral DNA was detected by PCRand the copy number was very low (below 1genome equivalent ⁄ cell). To provide unambigu-ous determination that HPV is causative andnot a passive passenger in the lung tumors, teststhat are more specific are essential. One is therecovery and characterization of spliced HPVmessenger RNA from tissue extracts usingreverse transcription-PCR (RT-PCR). A moredefinitive approach is to perform in situ hybrid-ization to demonstrate that HPV E6 and E7mRNAs are expressed in the cancer tissues.Through the 1980s and 1990s, this type of assaywas instrumental in ascertaining that HPVs arethe causative agents for the wide spectrum ofanogenital lesions (e.g. 1–3, 115).In summary, HPV infections of the H&N

region are becoming recognized as a veryimportant cause of morbidity and mortality.Considerable further investigation is urgentlyneeded. Upon generating definitive linkage ofHPV infections to diseases other than in theanogenital mucosa, a far greater appreciationof the capabilities and consequences of HPVinfections will alter current medical standardsfor diagnosis and clinical care. Perhaps mostnotably, such information is likely to changepublic perceptions that HPV is primarily asexually transmitted disease and should moti-vate the necessary funding for new researchand for development of effective therapeuticapproaches.

PART VI. ORGANOTYPIC EPITHELIAL

CULTURES AS EXPERIMENTALMODELS

TO STUDY HPV PATHOBIOLOGY

Several key capabilities necessary to investigateHPV infections in a meaningful and realisticexperimental setting have been: (i) a steady-state

model with which to generate stratified,differentiating epithelium; (ii) efficient ways tointroduce the viral sequences to study theirregulation and interaction with the host in thesquamous epithelium; (iii) efficient ways tointroduce the complete viral genome and reca-pitulate the productive program and to performgenetic analyses; and (d) infectious virions toinfect keratinocytes and verify the productiveprogram. All these studies of course requireprobe technologies to evaluate viral and hostcell gene expression in the context of the dif-ferentiating squamous epithelium (29, 116).Several probe technologies are illustrated inFigs 3 and 4.

Organotypic epithelial (raft) cultures

The initial conundrum for tissue culture modelsis to generate adequate amounts of non-cycling, differentiating cells, virtually a contra-diction in capabilities. The possibilities offeredby organotypic epithelial tissue cultures werefirst suggested by Broker and Botchan (117).The key capability was the raft culture systeminitially developed to produce tissues for skingrafts (118) in which healthy primary humankeratinocytes from a patient in need of surgicalrepair are grown on a dermal equivalent con-sisting of collagen matrix and human fibro-blasts. Initially, the keratinocytes are grownsubmerged in an appropriate culture mediumwhere the cells divide and spread laterallyacross the collagen surface in a manner akin towound healing until they are nearly confluent.The assembly is then ‘lifted’ to and maintainedat the liquid medium–air interface such thatthe upper surface of the epithelial cells isexposed to the atmosphere in the cell incuba-tor, while the dermal equivalent is kept moistand supplied with nutrients and various growthfactors secreted by the embedded fibroblasts.Continuous capillary movement of mediathrough the collagen is driven by the evapora-tion of water from the upper surface of thedeveloping epithelium. The keratinocytes beginto divide asymmetrically, establishing a cyclingbasal stratum and suprabasal, post-mitotic dif-ferentiated spinous strata. Within 10 days, afull thickness squamous epithelium is devel-oped, from basal, lower and upper spinousstrata to superficial granular strata covered by

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the stratum corneum (reviewed by 119).Because of the short duration, the TA stratumis not reestablished as in native squamous epi-thelia. These cultures can be sustained forabout two weeks, after which the spinous stratabecome progressively thinner, as the cell divi-sion from the basal stratum diminishes orceases, while the terminal differentiation andprogrammed cell death continue to occur nor-mally at the superficial stratum. Very similarcultures are achieved by placing pieces of epi-thelial tissue biopsy directly on the collagen–fibroblast matrix. Cycling epithelial cellsmigrate across the surface and then stratifyand differentiate into a squamous epithelium.Notably, the type of epithelium that emerges isentirely comparable with the morphology ofthe source of the biopsy (120). For example,Fig. 5 is an explanted laryngeal papillomagrown as an organotypic raft. Thus, the firstexperimental challenge was met: the productionof squamous epithelial tissue cultures.The Chow–Broker laboratory and that of

Laimonis Laimins independently developed or-ganotypic raft culture systems to recapitulate

the HPV productive phases (121, 122). Virionswere produced in the outgrowth of an explant-ed HPV-11 condyloma or in an HPV-31-con-taining cell line derived from a dysplasticpatient specimen. The ‘raft’ tissue model hasbecome a standard for experimental studies ofHPVs.

Introduction of subgenomic HPV sequences into PHKs

to investigate virus–host interactions

The next challenge was to introduce sub-geno-mic HPV DNA into the keratinocytes destinedfor organotypic culturing. It is difficult to per-form DNA transfections of PHKs because theefficiency is low and many cells do not survivethe common protocols such as calcium-mediatedtransfection or electroporation. As an alterna-tive, retrovirus-mediated gene transfer (e.g. withMuLV or lentivirus vectors) has proven very effi-cient and has been used extensively for geneticanalysis of HPV transcription regulatorysequences and E7 genes. For example, the HPV-18 and HPV-11 URR-P1 promoters linked to ab-galactosidase reporter have been transducedinto the PHKs and the reporter expression exam-ined in submerged PHKs and in raft cultures.The URR-P1 was active in submerged cultures,which resembles the wound healing state. How-ever, in the squamous epithelium, the reporteractivity was only detected in the differentiatingepithelium unless histone deacetylases wereinhibited, demonstrating the changing activity ofthe URR in different milieu. Mutagenic analysesreveal that the binding sites for transcription fac-tors AP1, Oct, and Sp1 act synergistically andthat mutation of any of the three significantlyreduces the promoter activity (123–126). HPV-18 URR-P1 promoter-driven HPV-18 E7 or theLTR-driven HPV-11 E7 or HPV-1 E7 can eachpromote S-phase reentry by a subset of the spi-nous cells without sacrificing squamous differen-tiation while inducing a mildly dysplastichistology. Mutagenic analyses have also beenperformed to identify motifs critical to E7 activ-ity. Indeed, these experiments have revealedp130 to be the E7-targeted pocket protein in dif-ferentiated strata (9, 10, 35, 126). On the otherhand, when the HR HPV E6 and E7 oncogeneswere under the control of the constitutive LTRpromoter and then introduced into PHKs byacute retrovirus infection, the resulting raft

Laryngeal Papilloma

Papilloma Explant

Fig. 5. Explanted laryngeal papilloma tissues cul-tured as organotypic rafts. Top panel: histology of alaryngeal papilloma revealed by hematoxylin andeosin staining. Bottom panel: histology of explantedpapilloma tissues from the same lesion grown as anorganotypic raft culture. Explants and photographswere produced by Dr. Delf Schmidt-Grimmingerfrom the Chow ⁄Broker laboratory.



cultures resembled high-grade dysplasias (33).Similarly, raft cultures display severely dysplas-tic histology comparable with high-grade SIL orcarcinoma when HR HPV oncogene-immortal-ized keratinocytes or HPV-transformed cervicalcarcinoma cell lines are used. There was limitedor no differentiation (10, 93, 127–129). Collec-tively, these experiments demonstrate that ele-vated HPV oncogene expression drivesneoplastic progression.

Introduction of the HPV genome into keratinocytes to

recapitulate the productive program in raft cultures

Traditionally, HPV genomes have been excisedfrom recombinant plasmids and transfected intoPHKs (reviewed by 130, 131). However, thetransfection efficiency of non-supercoiled DNAis low compared with that of supercoiled DNA.Furthermore, without the proper negativesupercoiling, such DNAs are poor templates forRNA transcription and DNA replication. Thus,it is necessary to select for and expand the trans-fected cells using a drug resistance marker geneexpressed from a cotransfected plasmid. Suchselection can take weeks if not months and thusthe ability of the HR HPV oncoproteins toextend the life span and to immortalize thePHKs comes into play. LR HPV or HR HPVmutants that cannot persist cannot be studied.As an alternative approach, selected immortal-ized epithelial cell lines were used as the recipient(reviewed by 132; see also 133). However, raftcultures developed from extensively passaged orimmortalized epithelial cells have not supporteda highly productive program of the introducedviral DNA, compromising the ability to performgenetic analyses, probably because of alteredgrowth and differentiation properties.Recently, a new method has been developed in

which HPV genomic plasmid is generated inPHKs from transfected supercoiled recombinantplasmids (29). The entire HPV genome linearizedin a non-critical position in the URR and thenflanked by bacteriophage P1 LoxP recombina-tion sites was cloned into a bacterial plasmidcontaining a neomycin drug-selectable gene.When cotransfected into PHKs with a secondplasmid that expresses the phage P1 Crerecombinase targeted to the nucleus (nlsCre), theCre-mediated excision recombination generatestwo plasmids, the HPV genome and the cloning

vectors, each harboring a single residual LoxPsite of 34 base pairs. After a 4-day G418 drugselection, about 30% of the PHKs survive theselection and uniformly harbor HPV genomes.When these cells are placed on the dermal equiv-alent and grown as raft cultures, the HPV-18genomes replicate as autonomous plasmids inthe nuclei of the keratinocytes and amplify tohigh copy numbers in a large proportion of themid- and upper spinous cells. Viral capsidgenes are expressed in the superficial cells, andthe newly replicated viral DNA is packagedinto virions, which complete their maturationin the stratum corneum. These patterns areidentical to those in benign lesions, and theunchecked high productivity most closelyapproximates what is seen in benign lesionsfrom HIV ⁄AIDS patients and in HPV-infectedhuman xenografts in immune-deficient mice(86). Importantly, using detailed time coursestudies coupled with metabolic labeling, theentire productive program was elucidated, asalready discussed in Part III.In this new experimental model system, the

progeny HPV particles are highly infectious.For the first time, the HPVs can be passagedin raft cultures of naıve PHKs and the entireproductive program repeated with exceptionalefficiency. Moreover, only a high multiplicityof infection (MOI) of naıve PHKs can elicit aproductive infection. Below a certain thresholdof MOI, the virus expresses its early genes,persists in the infected cells, and promotes cel-lular DNA replication in the differentiatedcells, but very few cells are able to supportviral DNA amplification and virion produc-tion. The reason remains to be elucidated.Such relatively non-productive infections areoften observed in benign lesions in immune-competent patients.As this method of introducing HPV genome

into PHKs bypasses the need for immortaliza-tion functions of the HR HPV, one should inprinciple be able to analyze mutant genomesincapable of immortalization and also LR HPVgenotypes, which do not extend the lifespan ofPHKs. Indeed, an HPV-18 E6 mutant genomewas successfully studied in raft cultures and theresults show that the E6 protein is critical forefficient viral DNA amplification. Site-directedmutations of additional HPV genes can also besimilarly studied.

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PART VII. TRANSLATIONAL

APPLICABILITY OF BASIC HPV

RESEARCH

Clinical diagnosis

Detection of cervical infections can be achievedin multiple ways: (i) when the tissue is exposedto dilute acetic acid ⁄vinegar, regions of activeHPV infection of the mucosa will briefly turn acloudy white (‘aceto-whitening’); this test is uti-lized in low-resource settings. (ii) The Papanico-laou smear of exocervical and endocervical cellsis the current ‘gold standard’ for the diagnosisof active HPV infections, based on a scale ofcytological abnormalities. The optimalapproach uses liquid-based cytology, as exfoli-ated cells are less clumped and easier to visualizeand characterize by light microscopy. The Papsmear has been one of the most successful diag-nostic tests in all of medicine. It has reduceddeaths resulting from cervical cancer to just20% of the pre-Pap mortality in populations ofsexually active women who are screened at leastevery few years, starting several years after sex-ual debut through to older ages (e.g. 60 or so ifthe cytology remains normal). (iii) If cytology isabnormal, the appropriate follow-up calls forcolposcopically directed biopsies and pathologi-cal evaluation according to diagnostic criteriaestablished by the ‘Bethesda’ consensus, rangingfrom low- to high-grade squamous intraepitheli-al lesions, to CIS and to invasive cancer. (iv)The biopsy can be examined for the inductionof host S-phase biomarkers such as PCNA,MCM7, and KI67 (8, 134) across the spectrumof HPV diseases or for the accumulation ofp16ink4 in cases of HR HPV-induced cancers(135 and references therein). p16ink4a inhibitsthe assembly of cdk 4 or 6 ⁄ cyclin D, which nor-mally phosphorylates and inactivates pRB asthe initial step to promote the G1–S transition.Upon pRB destabilization by the HR HPV E7,the lack of feedback control then leads toincreased p16ink4a protein. Indeed, p16ink4ahas been immunohistochemically detected in avariety of HPV-associated high-grade lesions.(v) Screening for high expression of HR viralE6-E7 transcripts in cervico-vaginal lavagesusing RT-PCR. (vi) Testing for HR HPV DNAin Pap samples or cervico-vaginal lavages usingone of several molecular probe technologies,

some of which can identify specific HPV geno-types as well as coinfections with multiple types,a common occurrence (136). A combination ofPap smear and HR HPV DNA typing has beenused effectively to identify preneoplastic lesions,while reducing the false-negative incidence ofthe Pap smear alone and increasing the intervalsbetween Pap smear screenings (137).Aceto-whitening is not used to detect H&N

infections. Rather, initial diagnosis is based onthe presence of exophytic papillomas or evi-dence of possible malignancy, confirmed by thehistology of biopsies. As only a subset of H&Ncancers is caused by HPV, and not all benignlesions are exophytic, determination of possibleHPV-associated disease is dependent on detect-ing HPV DNA or (preferably) RNA in biopsiesor exfoliated cells, supplemented with analysesof biomarkers, as just described.These molecular tools for clinical diagnosis

have evolved from decades of basic scientificassessment of HPV biology and viral interac-tions with the host cells in naturally infected tis-sue samples. They are excellent examples ofclinical importance of basic research in supportof translational research. The medical and epi-demiologic fields concerned with papillomavirusdiseases are in a degree of flux with regard tochoices among the many new diagnosticoptions. Efforts to produce even better molecu-lar diagnostic tests that are fast, sensitive andspecific, predictive of disease outcomes, andaffordable in developing countries remain highpriorities.

Prophylactic vaccines

One of the greatest sources of excitement inrecent HPV research has been the developmentand clinical validation of prophylactic vaccinesthat prevent primary HPV infection and persis-tence (for a review, see 138). These vaccines arebased on VLPs composed of the L1 major cap-sid protein only. The neutralizing antibodiesrecognize conformational epitopes on the virusparticles, accounting for the need to use VLPsinstead of unassembled L1 monomers. Forthe same reason, the prophylactic vaccines areHPV type-specific, but they do have somelimited cross-reactivity with very closely relatedgenotypes (138). VLPs spontaneously self-



assemble in cells expressing the L1 protein ifpresent in sufficient concentrations, and thisincludes production in the bacterium Escheri-chia coli, yeast Saccharomyces cerevisiae, andeucaryotic insect cells Sf9 when infected withbaculovirus expression vectors. VLPs can alsoself-assemble in vitro, and this property enablesthe dissociation–reassociation to remove tracesof molecular contaminants during manufacture.Notably, the VLPs are devoid of any viralgenomic material or any other DNA or RNAand, as such, they pose absolutely no risk ofinfection. Ironically, the absence of DNA isalso one of their potential limitations. Unlikeviral vaccines based on live, attenuated virusessuch as the Sabin polio vaccine, the HPV vac-cine does not establish a prolonged stimulationto the immune systems. Thus, the VLPs aremixed with an adjuvant to improve the initialresponse. Vaccination requires at least twoinjections, preferably three (with a primaryshot at time 0 followed by a booster at 1 or2 months and again at 6 months), to inducehigh titers of antibodies against the VLPs.Post-vaccination tracking indicates that theinduced serological titers fall from peak valuesbut stabilize at levels well above those seen innatural infections and that they remain protec-tive for many years. What is not yet known is(i) the length of time before titers fall below athreshold necessary to prevent new infections;(ii) whether subsequent natural exposure canelicit a rapid enough memory response to pre-vent establishing new infections; and (iii)whether booster shots would be needed period-ically, at a schedule to be determined throughcareful monitoring of vaccinated populations.The requirement for maintaining native con-

formation adds considerably to the difficulty ofmanufacture, storage, and delivery of vaccines.As the L1-based VLPs are largely HPV type-restricted and expensive to produce, additionalapproaches are being proposed. One is to usecapsomeres assembled from five L1 monomers.The antibodies elicited are also type specific butthe antigen can be produced rather inexpen-sively in bacteria (139–141). Another approachis to develop a cross-reactive vaccine. A regionof L2 is relatively conserved among diverse pap-illomaviruses, and a peptide corresponding tothat region indeed elicits broadly cross-reactiveantibodies in preclinical studies (for a review,

see 142). Both are being explored as possiblenext-generation prophylactic HPV vaccines.

Therapeutic vaccines

The concept is to treat patients with seriousHPV infections such as RRP and other refrac-tory benign lesions and also HPV-associatedcancers with vaccines composed of HPV pro-teins that could stimulate a cell-mediated immu-nity against the cells expressing the viralproteins. Success in optimizing therapeutic vac-cines for papillomavirus infections depends onan understanding of the immunology of thesediseases and the reason(s) why patients withpersistent HPV infections do not mount aneffective immune response (see the article on theimmunology of recurrent respiratory papillo-matosis by Bonagura). One current approachuses DNA-based vaccines to express a detoxi-fied E7 protein, which no longer can bind thepRB family of pocket proteins. This has beenefficacious in preclinical studies. A few clinicaltrials are ongoing (reviewed by 143).

PART VIII. THERAPEUTIC APPROACHES

TO EXISTING HPV INFECTIONS

The most common practice is to removal thelesions using a variety of surgical proceduresincluding cold knife, microdebrider, varioustypes of laser, cryosurgery, and electrocautery,according to the anatomic site. A number ofsmall molecules have also been used either aloneor as adjunctive therapies, as described below.

Small molecule drugs to treat genital warts

Topically delivered potions have been used totreat papillomavirus infections from time imme-morial, just as traditional medicines derivedfrom natural products have been used againstmany other common ailments. Some treatmentshave been partially efficacious and are still inwidespread use and acceptance today to treatanogenital disease. Notably among these arepodophyllotoxin (isolated from the May apple)and a formulated version called podophyllin orCondylox, which have been used to suppressgenital warts (144). One of the major problemsis that podophyllotoxin is anti-mitotic and does

CHOW et al.


not readily allow for the re-epithelialization ofhealthy skin to replace the erosion caused by itsapplication. Imiquimod (Aldara), an immuneresponse modifier, has been used to treat genitallesions with good results (145–147). Neither canbe used to treat RRP because of scarring orpain. Systemic injection of interferon has beenused successfully in some of the patients butrelapse can occur after discontinuation of treat-ment (148). A single study reported that treatingcervical dysplasia patients with celecoxib, aselective COX-2 inhibitor, prevented diseaseprogression and increased regression (149).

Small molecule drugs to treat RRP

In attempts to slow down or to curb post-surgi-cal regrowth of airway papillomas, variousadjunctive pharmacotherapies and immuno-therapies have been attempted. The persistentclinical dilemmas are that (i) none of the agentsused to treat HPV infections, including thosedescribed below, is reliably effective in allpatients and (ii) collateral damage to normalhost epithelium can be considerable. Buildingon the wealth, breadth, and depth of knowledgegained in the past decades of intensive world-wide research on papillomaviruses, selectiveinhibitors of different pathways essential to viralreplication and long-term persistence are beingsought (reviewed by 150). As almost all laryn-geal papillomas are caused by HPV-6 or HPV-11, clinical application of future type-specificinhibitors would be a possibility. However, inalmost all other anatomic sites, HPV type-spe-cific agents are clinically impractical because ofthe large number of possible genotypes respon-sible for the lesions.Vitamin A (all-trans-retinoic acid) treatment

prevents keratinocyte differentiation, which ofcourse then blocks much of the HPV reproduc-tive program. However, this also prevents fullhealing, leaving incompletely differentiatedsquamous epithelia subject to painful sores andpossible microbial infections.Indole-3-carbinol and its derivatives such as

diindoylylmethane (DIM) have received well-deserved attention because of their ability tosuppress laryngeal papillomas in some patients,possibly by diminishing the production ofpro-estrogenic metabolites in favor of anti-estro-genic derivatives (151, 152). I3C is naturally

conjugated to several possible sugars as a gluco-nate in many cruciferous vegetables. In the acidicenvironment of the stomach, I3C is a very reac-tive compound and, among other possibilities,can dimerize to DIM. The active agent can alsobe chemically prepared as DIM, minimizingunpredictable reactions after ingestion. The pro-posed mode of activity is that I3C andDIM havestructural similarities to estrogen (b-estradiol).The natural elimination pathway for any hydro-phobic steroid hormone involves hydroxylationto increase solubility. b-estradiol is subject toconversion either to 16-a-hydroxy estrone (astrong pro-estrogenic derivative) or 2-hydroxyestrone (an anti-estrogenic derivative). Specula-tion is that the cytochrome P450 hydroxylaseinduced by I3C and DIM to make thesearomatic compounds more aqueous solublehappens collaterally to convert b-estradiol to the2-hydroxyestone and ultimately to suppressmitogenic stimulation to papillomas. ManyRRP patients benefit from I3C or DIM intakeon a daily regimen and, while consistent use canprolong intervals between surgeries, only rarelyis this sufficient for complete remission.Interferon-a generally blocks viral reproduc-

tion and is an integral part of natural anti-viraldefenses in mammals. High-dose systemic injec-tions of IFN-a can suppress RRP regrowthpost-operatively, but at the price of inducingfevers and other significant side effects. Long-term delivery is not well tolerated. Somepatients do experience successful remission oftheir papillomas, but most have a rapid reboundafter termination of IFN treatments.HPMPC (Cidofovir ⁄Vistide) and other acyclic

nucleoside analogs have been used off-label andcompassionately in RRP patients. The pro-drugs are delivered to the tissue by intralesionalinjection, and they require phosphorylationonce inside the cells to become a tri-phosphateequivalent, the immediate substrate for DNA orRNA polymerases. It is not known which kinaseis responsible for their conversion to the tri-phosphate equivalent. On a precautionary note,cidofovir is not a chain terminator after incor-poration (in contrast to most other nucleotideanalogs used to inhibit DNA or RNA polyme-rases). Rather, because it has a 3¢ hydroxylgroup, it can be incorporated within growingpolynucleotides. Its ultimate mechanism of inhi-bition is that polymerases slow down when



encountering acyclic nucleoside phosphonates,as the normal steric orientation of the sugar ringis absent. Incorporated HPMPC apparentlycauses RNA and DNA polymerases to pause orstop. Of particular concern is that the incorpo-rated nucleotide analog can be mutagenicbecause of poor templating. The long-term con-sequences of cidofovir in people treated forRRP remain unknown, and considerable cau-tion in its use should be exercised. Some patientsrespond well to cidofovir and their lesions areminimized over time, but many others have littleor no benefit (153) and may even experiencevocal cord ‘stiffness’, perhaps because healing isdelayed or prevented.Recently, several new agents have entered

clinical trials (Cox II inhibitors), have beenadvanced by RRP patients (artemisinin), or arein use by some ENT surgeons (e.g. intralesionalinjections with the mumps vaccine or Avastin).

Cox II inhibitor (e.g. celecoxib ⁄Celebrex) and othernew agents

As discussed above, the enzyme COX-2 and itsmajor product PGE2 are expressed in HPV-induced respiratory papillomas and promotethe growth and survival of the cells. A pilotclinical trial of celecoxib therapy for RRPshowed striking responses, with 3 ⁄3 patientsfree of disease (A. Abramson, G. Mullooly,B. Steinberg, unpublished data). A large multi-centered double-blinded placebo-controlledstudy of celecoxib therapy has been funded bythe National Institutes of Health and is cur-rently underway. Included in the design of thetrial are a number of studies to address themechanism of celecoxib efficacy in vivo.Artemisinin, artesunate, and related com-

pounds are antimalarial agents derived fromwormwood. They are cytotoxic against HPV-immortalized cells or cervical carcinoma cells,but not against normal cervical cells (154).Delivered orally, they have shown anecdotalbenefits against laryngeal papillomatosis. Themechanism of action may involve selective gen-eration of free radicals and oxidative stress inactive papillomas. Additional research and vali-dation are essential.Mumps vaccine (or the MMR vaccine) injec-

tion directly into papillomas can result inremarkable regression of recalcitrant papillomas

in some patients (155). A presumed mechanismof action is local boosted immune response(because essentially all RRP patients receivedmumps or MMR vaccination as infants) withcollateral suppression of HPV activity.Bevacizumab (Avastin) is an anti-angiogenic

agent that, when injected sublesionally follow-ing angiolytic KTP laser treatment, can preventrapid regrowth of papillomas of the larynx andnearby upper aerodigestive tract sites (156).In summary, adjuvant treatments of RRP

have involved a great deal of trial and error.The described approaches benefit some butdefinitely not all patients. They remain rathernon-specific and appear to be limited byidiosyncracies associated with individualpatients for reasons not understood.

Proscriptions and contraindications – Pharmaceu-tical compounds such as acyclovir and gancyclo-vir have shown efficacy in treating infectionscaused by the herpesviruses because these largeviruses encode a unique thymidine kinase whichis capable of turning these analogs into sub-strates for nucleic acid synthesis. However,HPVs strictly rely on host enzymes to supply thesubstrates and hence these agents would nothave any specificity against HPV lesions. Othertreatments (5-fluorouracil) that have been triedclinically should be avoided as they are likely toblock cellular DNA synthesis or introduceunwanted mutations in cellular genes. In no caseshould ionizing radiation be used to treat anybenign papilloma lesion, as the resulting DNAdamage and strand breaks could promote chro-mosomal DNA instability and viral DNA inte-gration and accelerate neoplastic progression.Such unintended consequences did result fromattempts to treat laryngeal papillomas with radi-ation in the early days of clinical radiology.

CONCLUSIONS

Successful implementation of control measures– prevention, diagnosis, and treatment – ulti-mately comes down to (i) effective communica-tion through various media to the generalpublic, to policy makers, and to fundingsources, (ii) education of health-care providers,(iii) development of inexpensive but reliableproducts for prevention and clinical care, and

CHOW et al.


(iv) a sustainable ‘business plan’ for long-termdelivery. These objectives are especially chal-lenging in underdeveloped nations and regionsbecause of competing health care and economicpriorities and also because socially acceptableapproaches and procedures can differ widelyamong cultures. Future efforts to bring HPV-associated diseases under control must continueto remain highly creative and adaptable, withthe appreciation that the papillomaviral diseasesare among the most promising targets forresearch, development, and delivery of clinicaland public health benefits. With unwaveringmotivation and commitment of resources, suc-cess can and will come in global control andmanagement of the HPV-associated diseases.

We thank Hsu-Kun Wang with assistance in prepar-ing the micrographs. Over the years, much of theresearch in the laboratory of Louise Chow and TomBroker was supported by grants from the US PublicHealth Service ⁄National Institutes of Health ⁄National Cancer Institute: CA36200, CA83679, andCA107338. Bettie Steinberg has been supported by anumber of grants from the National Institute forDeafness and Other Communication Disorders ⁄National Institutes of Health: DC00203, DC007946,and DC008579; and grant AI38029 from the NationalInstitute for Allergy and Infectious Diseases.

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