Human Papillomavirus
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Description

Human papillomavirus (HPV) infection transcends multiple fields of science and medicine. The management of HPV-related disease is demanding and often requires a persistent multimodal approach involving various medical disciplines. In this volume, experts present a comprehensive view of HPV research with an emphasis on clinical presentations, diagnosis, management and vaccine development. The state of the art in molecular biology is provided in addition to discussions on clinical morphology and the utility of dermatoscopy in identifying HPV disease. In a multidisciplinary approach to dermatological, plastic and reconstructive, gynecological, otolaryngological and colorectal management, different treatment strategies are highlighted. Finally, Dr. Neil Christensen discusses viral immunology, and the difficulties and successes in the development of an HPV vaccine.
Bringing together basic science and clinical information on HPV, this book is an excellent resource and reference for all researchers and clinicians who encounter human papillomavirus-related disease.

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Date de parution 13 mars 2014
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EAN13 9783318025279
Langue English
Poids de l'ouvrage 2 Mo

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Human Papillomavirus: Bench to Bedside
Current Problems in Dermatology
Vol. 45
Series Editors
Peter Itin Basel
Gregor B.E. Jemec Roskilde
Human Papillomavirus
Bench to Bedside
Volume Editors
Marigdalia K. Ramírez-Fort Houston, Tex.
Farhan Khan Houston, Tex.
Peter L. Rady Houston, Tex.
Stephen K. Tyring Houston, Tex.
61 figures, 55 in color, and 15 tables, 2014
Current Problems in Dermatology
_______________________ Marigdalia K. Ramírez-Fort Farhan Khan Stephen K. Tyring Center for Clinical Studies Houston, TX (USA)
_______________________ Peter L. Rady Department of Dermatology The University of Texas Health Science Center at Houston Houston, TX (USA)



Library of Congress Cataloging-in-Publication Data
Human papillomavirus (Ramírez-Fort)
Human papillomavirus: bench to bedside / volume editors, Marigdalia K. Ramírez-Fort, Farhan Khan, Peter L. Rady, Stephen K. Tyring.
p. ; cm. –– (Current problems in dermatology, ISSN 1421-5721 ; v. 45)
Includes bibliographical references and index.
ISBN 978-3-318-02526-2 (hard cover: alk. paper) –– ISBN 978-3-318-02527-9 (electronic version)
I. Ramírez-Fort, Marigdalia K., editor of compilation. II. Khan, Farhan, editor of compilation. III. Rady, Peter L., editor of compilation. IV. Tyring, Stephen K. (Stephen Keith), editor of compilation. V. Title. VI. Series: Current problems in dermatology ; v. 45. 1421-5721
[DNLM: 1. Papillomavirus Infections. 2. Papillomaviridae. W1 CU804L v.45 2014 / QZ 200]
RC168.P15
616.9’11––dc23
2014003258
Bibliographic Indices. This publication is listed in bibliographic services, including MEDLINE/Pubmed.
Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
© Copyright 2014 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland)
www.karger.com
Printed in Germany on acid-free and non-aging paper (ISO 9706) by Kraft Druck GmbH, Ettlingen
ISSN 1421-5721
e-ISSN 1662-2944
ISBN 978-3-318-02526-2
e-ISBN 978-3-318-02527-9
Contents
Foreword
Brodell, R.T. (Mississippi/New York)
Introduction
Human Papillomavirus Genomics: Past, Present and Future
Harari, A.; Chen, Z.; Burk, R.D. (Bronx, N.Y.)
Molecular Biology and Pathogenesis
The Biology of Human Papillomaviruses
Nguyen, H.P.; Ramírez-Fort, M.K.; Rady, P.L. (Houston, Tex.)
Viral Oncogenesis
Doan, H.Q. (Galveston, Tex.); Ramírez-Fort, M.K.; Rady, P.L. (Houston, Tex.)
Pathogenesis of Infection by Human Papillomavirus
Brendle, S.A.; Bywaters, S.M.; Christensen, N.D. (Hershey, Pa.)
Host Responses to Infection with Human Papillomavirus
Stanley, M.A.; Sterling, J.C. (Cambridge)
Epidemiology and Clinical Manifestations
The Epidemiology of Human Papillomaviruses
Nyitray, A.G. (Houston, Tex.); Iannacone, M.R. (Brisbane, Qld.)
Cutaneous Human Papillomavirus Infection: Manifestations and Diagnosis
Tschandl, P. (Vienna); Rosendahl, C. (Brisbane, Qld.); Kittler, H. (Vienna)
Genitoanal Human Papillomavirus Infection and Associated Neoplasias
Gross, G. (Rostock)
Epidermodysplasia Verruciformis
Burger, B.; Itin, P.H. (Basel)
Human Papillomavirus Infections of the Oral Mucosa and Upper Respiratory Tract
Nguyen, H.P.; McNiece, K.L. (Houston, Tex.); Duong, A.A. (Austin, Tex.); Khan, F. (Houston, Tex.)
Human Papillomavirus and Immunosuppression
Wieland, U. (Cologne); Kreuter, A. (Oberhausen); Pfister, H. (Cologne)
Laboratory Diagnosis
Laboratory Diagnosis of Human Papillomavirus Infection
Ikenberg, H. (Frankfurt)
Treatment and Prevention
Management of Cutaneous Human Papillomavirus Infection: Pharmacotherapies
Ramírez-Fort, M.K.; Au, S.-C. (Boston, Mass.); Javed, S.A. (Houston, Tex.); Loo, D.S. (Boston, Mass.)
Management of Cutaneous Human Papillomavirus Infection: Surgery
Ramírez-Fort, M.K. (Boston, Mass.); Sam, H.; Manders, E.K. (Pittsburgh, Pa.)
Management of Cutaneous Human Papillomavirus Infection in Immunocompromised Patients
Varada, S.; Posnick, M.; Alessa, D. (Boston, Mass.); Ramírez-Fort, M.K. (Boston, Mass./Houston, Tex.)
Management of Human Papillomavirus-Related Gynecological Malignancies
Heinzelmann-Schwarz, V.A.; Kind, A.B.; Jacob, F. (Basel)
Management of Human Papillomavirus-Related Anal and Colon Cancer
Yen Moore, A. (Arlington, Tex./Dallas, Tex./Galveston, Tex.); Tong, L.X. (Los Angeles, Calif.); Moore, T. (Arlington, Tex.)
Management of Human Papillomavirus-Related Head and Neck Cancer
Coughlin, A.M.; Qiu, S.; Underbrink, M.P. (Galveston, Tex.)
Vaccines and Immunization against Human Papillomavirus
Christensen, N.D.; Budgeon, L.R. (Hershey, Pa.)
Author Index
Subject Index
Foreword
It’s about time! Finally, information that is scattered throughout the worldwide web and in thousands of books and journal articles from a range of clinical and basic science disciplines is pulled together into one tome. This innovative book is truly the definitive treatise on human papillomaviruses (HPV). The clinical chapters include the latest information on the manifestations of HPV disease, diagnosis, and management. Included are current therapeutic guidelines, and prevention of warts in a manner that emphasizes the efficacy and side effects of different treatment modalities. It is organized in a manner so that specific information can be easily and quickly accessed. The research chapters are understandable by both the clinician and the researcher and provide a comprehensive view of the state of HPV science with an emphasis on vaccine development.
Most importantly for the first time, basic science and clinical information on HPV are presented together in the same book. Research in the 21st century is increasingly focused on translational efforts that require an interchange of ideas between researchers and clinicians. Successful grant applications take the latest information from the bench and focus it on bedside applications. At the same time, deficiencies in both diagnostic and therapeutic approaches must be identified to plug gaps in knowledge by targeting research to solve clinical problems. I am certain a broad swath of bench researchers, clinicians from the fields of dermatology, gynecology, surgery, and primary care fields will find this book to be a helpful reference whether read cover to cover or used when questions arise in their work. With just a little luck, the cross-pollination of research and clinical ideas will stimulate creative thinking that will stimulate creative advances in this field. It’s about time!

Robert T. Brodell, MD
Professor and Chair, Department of Dermatology
Professor of Pathology
University of Mississippi Medical Center
Jackson, Mississippi
Instructor in Dermatology
University of Rochester School of Medicine and Dentistry
Rochester, New York
Introduction
Ramírez-Fort MK, Khan F, Rady PL, Tyring SK (eds): Human Papillomavirus: Bench to Bedside. Curr Probl Dermatol. Basel, Karger, 2014, vol 45, pp 1-18 (DOI: 10.1159/000355952)
______________________
Human Papillomavirus Genomics: Past, Present and Future
Ariana Harari a Zigui Chen b Robert D. Burk a - d
Departments of a Microbiology and Immunology, b Pediatrics, c Obstetrics, Gynecology and Women’s Health, and d Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, N.Y., USA
______________________
Abstract
Human papillomaviruses (HPV) are a group of divergent DNA viruses, of which a select few evolutionarily related HPVs have emerged to be highly oncogenic and of significant medical importance. Essentially all cases of cervical cancer, as well as a subset of other anogenital and oral cancers are caused by this limited set of HPV types. At present, over 150 HPV types have been identified and may be classified into genera, species and types based upon comparison of the viral genome. Established nucleotide phylogenies sort the highly pathogenic HPV types to the genus Alphapapillomavirus (α-PV). A species group includes viral types with 60-70% genomic nucleotide similarity that share a most-recent common ancestor; for example the species group’s alpha-9 (HPV16-related) and alpha-7 (HPV18-related), contain the majority of known oncogenic HPV types. Genomes from the same HPV type with 1-10% nucleotide differences designate HPV variant lineages. The established nucleotide variations observed in extant HPV genomes have been fixed through evolutionary processes prior to human population expansion and global dissemination. To characterize viral types and variants associated with pathology for clinical applications (e.g. screening), molecular epidemiological studies have proven essential for identifying links between HPV natural history and carcinogenicity. This chapter presents a historical account of HPV genomics in the context of major discoveries and advances over the past 2 thousand years.
© 2014 S. Karger AG, Basel
Papillomaviruses (PVs) are ubiquitous, highly diverse DNA viruses that have been isolated from all 4 classes of Tetrapoda , including most mammals as well as birds, turtles and snakes [ 1 - 3 ] ; their origin predates the existence of modern humans [ 4 - 7 ]. Observational accounts of warts from ancient Greece and Rome describe condylomatous lesions on the skin and genitals, and it was presumed that genital warts were associated with promiscuous sexual behavior. Warts in general were surmised to be transmissible [ 8 ] . Manifestations of animal PVs have historically been documented in myths and paintings, particularly of the ‘jackalope’. This animal does not exist, but likely represents a case of mistaken identity as a result of a PV infection that produced cornified growths (i.e. horns) on jackrabbits. Historical records and myths provide evidence for the antiquity of PVs and the diseases they cause. In 1842, the Italian physician Dr. Rigoni-Stern was the first to hypothesize that cervical cancer might be linked with sexual behavior. He observed that cervical cancer frequency was disproportionally higher in prostitutes and married women than nuns and virgins, implying that the causative agent was likely sexually transmitted [ 9 , 10 ] . Rigoni-Stern’s observation would be validated almost 150 years later; today, human PVs (HPVs) are known to be among the most commonly sexually transmitted infections, and infections by specific high-risk (HR) HPV types are known to be the etiological agents of cervical cancer [ 11 - 14 ].
In 1911, Francis Peyton Rous famously demonstrated that filtered tumor cell extract obtained from chicken carcinomas and transplanted to naïve chickens promoted sarcoma growth of a virulent nature, identifying the first oncogenic virus, i.e. Rous sarcoma virus. Almost 25 years later, Dr. Rous and Dr. Richard Shope identified the first PV, known as Shope PV or cottontail rabbit PV, from warty growths on cottontail rabbits. They went on to demonstrate that transmission of cottontail rabbit PV exhibited neoplastic potential in domestic rabbits [ 15 , 16 ] . Approximately 75 years later, Prof. zur Hausen was awarded a Nobel Prize for the discovery of HPVs causing cervical cancer [ 17 , 18 ] . The discovery that HPVs are major contributors to cancer and represent highly adaptive, carcinogenic viral pathogens causing essentially all of cervical cancer and approximately 20% of head and neck cancers has energized the research and medical communities [ 14 ] ( fig. 1 ).
Cervical cancer ranks 3rd amongst cancers affecting women worldwide and 2nd in developing countries [ 14 ] . Women in developing countries account for 85% of the global incidence of cervical cancer. Incidence rates are nearly double in developing compared to developed countries, 17.8 and 9.0%, respectively. This difference is thought to be largely due to the implementation of early diagnostic screening methods, which have reduced the risk of cervical cancer associated with persistent HPV infection. Yet, cervical cancer is still responsible for 275,000 deaths/year [ 14 ].
HPV infection by any of the 150 identified HPV types is not sufficient to cause cancer. All genital oncogenic types belong to the genus Alphapapillomavirus, which is currently comprised of 62 known HPV genotypes that infect the mucosal epithelium. Further classification beyond the genus level is required to distinguish the 13- 15 HR HPV types that are associated with oncogenic risk. It is well established that phylogenetic analyses cluster HPV taxa by host cell tropism (e.g. skin vs. genitalia), degree of oncogenic potential and morphology of clinical lesions [ 1 , 19 ]. Progression to cancer is rare. The malignant potential of specific HPVs likely results from niche adaptation (i.e. evolution of an organism/virus to a specific biological/anatomical ecosystem on the body) of PVs, as cancer is undesirable for both the virus and its host. The majority of HPV infections are cleared within 6-10 months. Persistent infections with HR HPVs are a critical risk factor for the development of HPV-associated precancer and cancer [ 20 - 22 ] . Delineating the differences intrinsic to these HR HPV genotypes, compared to the majority of HPV types that lack oncogenic potential, will help to elucidate the genetic basis of such carcinogenic properties, thus contributing to a better understanding of the biological mechanisms exploited by the virus to facilitate cancer development. Epidemiology studies provide a platform for obtaining viral isolates used to investigate the dynamic relationship of HPV genotype differences and clinical disease. The recent explosion in DNA sequencing technologies will continue to revolutionize methods of HPV detection [ 23 ] contributing to a better understanding of HPV biology and the development of new therapies against HPV-associated cancers.

Fig. 1. History of HPV, cervix cancer and technological advances. The timeline displays the landmarks in PV biology and clinical discovery. The color of the boxes serves to distinguish events and/or discoveries relating to basic PV scientific advancements in red, biological discoveries/technology innovations in blue, or epidemiology/public health in green. RSV = Rous sarcoma virus; BPV = bovine PV; IPV = International Papillomavirus meeting; pRb = retinoblastoma protein; IARC = International Agency for Research on Cancer; FDA = Food and Drug Administration; GAVI = Global Alliance for Vaccines and Immunization.
Human Papillomavirus Genome Characteristics
HPV is a circular, nonenveloped, double-stranded DNA virus approximately 8 kb in size that infects basal keratinocytes. Upon infection, the virus exists as an autonomous episome in the host cell nucleus. The viral life cycle is mediated by a series of virus-host interactions, which govern viral transcription, virion production and eventual clearance in the majority of infections [ 24 , 25 ] ( fig. 2 ).
The structure and function of the HPV genome are conserved throughout the Papillomaviridae and are broadly divided into 3 general components. (1) The early gene region, denoted by ‘E’, consists of 6 open reading frames (ORFs): E6, E7, E1, E2, E4, and E5. The early genes E1, E2, E6 and E7 are generated as a polycistronic transcript. Several additional early ORFs E3, E5 and E8 have also been identified, but their expression is not uniformly observed throughout the Papillomaviridae. Viral transcripts can undergo extensive alternative splicing, contributing to the intricate balance between viral and host-regulated transcription [for a review, see 26 ] . The early genes code for nonstructural proteins that function in viral replication, adaptation of the cellular milieu for viral activities, trans- activation of viral transcription and cellular transformation and proliferation. (2) The late gene region, denoted by ‘L’, consists of the L2 and L1 ORFs. L1 and L2 encode the structural proteins, the major and minor capsid proteins, respectively. The L2 ORF encodes for group-specific epitopes whereas the L1 ORF contains type-specific protein domains. (3) The upstream regulatory region (URR) is the noncoding region between the end of the L1 ORF and the E6 start codon, comprising approximately 10% of the genome. The URR contains DNA recognition sites for both viral and host transcription factors and regulates early gene transcription, viral amplification and cellular tropism. The URR contains a keratino-cyte-specific enhancer region proximal to the early gene promoter (p97), which highlights the significance of host cell tropism to viral gene expression and life cycle. A smaller noncoding region located between the E5 stop and L2 start codons harbors a highly conserved early polyadenylation signal required for gene expression from the early promoter, including alternatively spliced early transcripts and their gene products [ 26 ] . Both the early gene region and the URR display variability, useful in assessing genetic heterogeneity [ 27 ] . Increased understanding of the inherent genomic differences between HPV species that contribute to viral function is predicated on biochemical techniques such as DNA sequencing and polymerase chain reaction (PCR) which have contributed greatly to understanding the molecular pathogenesis of HPV. Great strides have been made since the inception of clinical HPV molecular biology in the 1970s [ 7 , 28 - 30 ].

Fig. 2. Schematic of the HPV-16R genome. The HPV-16 genome displayed shows the general organization of the HPV open reading frames (ORFs) and regulatory regions. Early (E) genes and related transcriptional regulatory motifs are shown in green; late (L) genes and related transcriptional regulatory motifs are shown in blue; the 2 noncoding regions are shown in gray; the smaller region between E5 and L2 is termed the noncoding region (NCR), and the larger region between L1 and E6 is known as the upstream regulatory region (URR). The URR contains regulatory elements including the viral DNA origin of replication (Ori) depicted by a yellow oval and the early gene promoter p97 depicted by a green arrow. The blue arrow in the E7 ORF depicts late gene expression regulated by the late promoter p670. Polyadenylation sites termed early (pAE) or late (pAL) are shown as green or blue triangles, respectively. Arrowheads at the tip of each ORF depict if they overlap. The thick solid line represents genomic regions commonly overexpressed in cancer, the thin black line represents genomic regions frequently disrupted by integration, and the dashed line represents the genomic region rarely expressed in cancer.
The viral proteins E1 and E2 function in viral genome replication and are dependent on the host DNA polymerase and replication machinery. E1 functions as an ATP-dependent helicase capable of melting double-stranded DNA for strand separation, prior to DNA polymerization. The E2 ORF encodes for a viral-DNA binding transcription factor. E1 and E2 proteins form heterodimers at the viral origin of replication to initiate bidirectional genome synthesis. Recently, E1 and E2 have been shown to function in the early induction of the DNA damage response pathway contributing to a permissive environment for viral genome amplification [ 31 ] . E1 can induce beaks in the host double-stranded DNA that activates the ataxia-telangiectasia DNA damage response pathway, signaling cell cycle arrest [ 31 ] . The URR contains 4 highly conserved E2 binding sites that differentially regulate viral replication and early gene transcription [ 32 , 33 ] . E2-dependent downregulation of early promoter activity maintains low-copy numbers of viral genomes prior to differentiation dependent activation of the late promoter and genome amplification. In high-grade neoplastic lesions/cancer the E2 ORF can be disrupted by viral integration into the host genome. Integration results in the loss of E2-dependent early promoter regulation, a ramification of which can be overexpression of E6 and E7 [ 24 , 25 , 34 ] . The viral proteins E6 and E7 function as oncogenes in the HR α-HPVs. HR HPV E6 and E7 proteins disrupt cell cycle regulation in upper epithelial cells (stratum spinosum), which normally exit the cell cycle to terminally differentiate. Virally mediated inactivation of key cell cycle regulators, the known tumor suppressors, p53 and pRb (by E6 and E7, respectively), are distinct among certain α-PV types, although not specific to those causing cancer [ 35 ] . The E5 ORF encodes a transmembrane protein that probably contributes to cell signaling [ 36 ] . The E5 ORF as defined by the presence of a true start and stop codon is found in select members of the human α-PVs: the HR HPV species, the α-10 species and other PVs including bovine PV 2 implicated in urinary cancer (cattle). At the nucleotide level, E5 is not highly conserved. The E5 protein is associated with late gene viral life cycle events and interacts with epidermal growth factor and platelet-derived growth factor to influence cellular proliferation [ 37 ] . The integrity of the E5 ORF provides another example of the differences revealed by studying HPV genotypes and phylogeny [ 38 ] . The late genes L1 and L2 function in virion maturation and orchestrate virion self-assembly that packages the genome for release in the upper epithelia. During late stages of precancer/cancer, L1 and L2 proteins are not expressed. This initially made early studies on HPV identification difficult and required the use of extracted DNA from warts to be obtained for analysis. Virus-like particles made from HR HPV L1 readily self-assemble and induce a neutralizing antibody response. This is the basis for the two prophylactic vaccines currently available.
Viral Identification and Classification: Phylogeny and Taxonomy
HPV genome heterogeneity was apparent during the initial studies examining viral isolates obtained from dysplastic lesions (cutaneous or genital). By the late 1970s, an unknown viral agent was known to be the causative agent implicated in the development of multiple wart types, condylomata acuminata (genital warts) and possibly cervical cancer. Attempts to identify the viral agent from lesions were hampered by the fact that DNA probes often did not cross-react with DNA extracts obtained from different wart types, implicating the presence of diverse viruses [ 39 ] . Furthermore, early attempts to study the infectious nature of the virus obtained from warts was restricted by the highly specific host epithelial-cell specificity required for a productive HPV life cycle [ 30 , 40 ] . Understanding the delicate balance of HPV-host interactions proved fundamental to addressing the clinical significance of HPV-related disease.
HPVs are extremely ancient viruses, and related PVs have been identified in most nonhuman primates, including humans’ most recent common ancestors [ 41 - 43 ]. Sequence and phylogenetic analysis of nonhuman primate PVs isolated from the cervi-covaginal area revealed genomic similarity to the α-HPVs. Several nonhuman primate PVs identified from rhesus macaques (Macaca mulatta) , cynomolgus macaques (Macaca fascicularis) , and olive baboons (Papio hamadryas anubis) comprise the α-12 species. These phylogenetically related α-PV types can induce epithelial dysplasia of varying degrees, resembling the high-grade cervical intraepithelial neoplasia (CIN) associated with persistent HR mucosal-HPV infections, specifically within the α-9 species, which includes HPV-16 [ 5 , 6 , 44 ] . Experimental transmission of M. fascicularis PV-3 from a naturally infected female to naïve female macaques was associated with the development of CIN [ 45 ] . These nonhuman primate PVs are more similar to the α-9 species, than more distantly related HPV types, such as the α-10 species, suggesting that the mechanisms governing cellular transformation result from a common ancestral trait that predates human/primate divergence.
Diversification of HPVs occurred prior to the emergence of Homo sapiens approximately 200,000-150,000 years ago [ 46 ] . The extensive diversification and demographic range of HPVs reflect human dispersal and population expansion [ 4 ], and may intuitively suggest that HPVs have had to undergo a high rate of mutations to adapt to such diverse hosts; however, this is not the case. Rates of nucleotide mutations are remarkably low in the virus, observed at a rate of approximately 10 -8 -10 -7 nucleotides/year [ 47 ].
HPV characterization in population-based studies affords the unique opportunity to study hominid evolution through a viral lens to better understand virus-host interactions as they pertain to immune system surveillance and cancer progression [ 5 , 6 , 38 ] . The unique coupling of the PV-host-dependent life cycle has selected for specialized viral adaptation to specific host niches, coincident with the ability of the virus to evade a host immune response. These features have enabled PVs to capitalize on their hosts, and exploit global diversity to thrive throughout evolutionary time. Genital HPV infections are commonly dependent on sexual transmission. This exemplifies one way the virus invests in its existence; they have hijacked the most fundamental aspect of host species success, reproductive fitness. After infection, viral gene expression is tightly regulated such that it is dependent on host epithelial differentiation for near exclusive activation of either the early or late gene promoters driving gene expression. Such adaptation tactics suggests a commensal virus-host relationship, yet HPV-dependent malignancies defy this viewpoint. Molecular epidemiological studies assessing the phylogenetic association of HPVs based on oncogenic risk support specific biological and pathological traits distinct to HPV genera, species and types [ 38 ]. HPV-16 is unique in its ability to establish persistence that is highly associated with neoplastic progression, both of the cervix and in head and neck carcinogenesis. The α-HPVs exhibit agreement between (clinical) natural history studies and HPV phylogeny and taxonomy, providing evidence to support that carcinogenicity is an evolved trait most probably related to niche adaptation [ 38 , 48 - 50 ].
Human Papillomavirus Classification
Methods to culture HPV in vitro or produce infectious virus through xenotropic models are not efficient, and do not provide a robust method for identification and characterization of HPV types [ 51 ] . Furthermore, a lack of a robust, consistent antibody response in infected individuals limits the use of antibody titer and serology for HPV taxonomy [ 51 ]. Historically, HPV has been identified from biopsies of warts or lesions and classified by comparison to known types by restriction endonuclease cleavage patterns and/or DNA-DNA or DNA-RNA blot hybridization. Such methods had innate flaws for characterizing PVs; specifically there was no quantifiable means of comparison amongst HPV types from different lesions, the virus titer in lesions was not available, and cross-hybridization was difficult to explain except for association by anatomical site [ 30 , 40 ] . The appreciation for a genomic, DNA-sequence-based classification system was agreed upon by the PV research community by the late 1980s. This system has relied on the rapid advances in DNA technologies from PCR to the Next-Gen sequencing era upon us.
Today, PCR-based amplification of DNA obtained from clinical samples is common. As the realization that phylogeny and genotyping methods validated one another, the notion that only a limited set of HPV types were associated with cancer spurred the need for highly specific assays that could discriminate HPV types. Consensus PCR primers targeting highly conserved regions within the L1 ORF, such as the MY09/11 PCR assay or the GP5+/GP6+ PCR assays, are typically used for HPV identification [ 52 ] . A review of HPV detection methods has recently been published [ 53 ] . In addition, sequencing of the PCR amplicons for alignment to known HPV type(s) facilitates classification of genotype by nucleotide identity and identification of novel types.
PVs belong to the family Papillomaviridae and were given this status by recognition of a genome-based, DNA sequence system for classification [ 54 ] . DNA sequencing provides a quantifiable means to catalog nucleotide heterogeneity, affording the classification and taxonomy of HPV to genera, species, types and variant lineages [ 27 , 55 ] . Classification of HPV genera and species based on DNA sequence was recognized by the PV Working Group at the 14th International Papillomavirus conference in Quebec in 1995, later adapted by the International Committee on the Taxonomy of Viruses [ 1 , 19 , 51 , 56 ]. Variant lineage classification is a more recent development within the PV community that will become increasingly more relevant as high-resolution techniques, such as next-generation sequencing, generate a plethora of PV sequencing data that need to be coherently analyzed, named and correlated with phenotype and geographic locations [ 55 ].
Human Papillomavirus Genome ‘Typing’
The L1 gene is highly conserved and provides the basis for HPV genotyping. An HPV ‘type’ is designated when the nucleotide sequence of the L1 ORF from the cloned viral genome is more than 10% dissimilar to all known types [ 1 ].
Nucleotide differences across HPV genotypes are correlated with viral lineages based on evolution without significant, if any, recombination, and these correlated changes are the result of lineage fixation [ 57 ] . Whole genome sequencing established L1 sequence identity as representative of complete genome variation due to its high conservation [ 1 , 50 ] . Nucleotide sequences are used to build phylogenetic trees used for HPV taxonomy. Phylogenetic analysis suggests the underlying relationship between the biological observations identified within this heterogeneous group of viruses: including host cell tropism (mucosal or cutaneous), carcinogenic risk and associated pathology [ 38 ] . Currently, over 150 HPV types have been formally identified and predominantly cluster to 3 main genera. (1) Alphapapillomavirus are primarily isolated from the genital, mucosal epithelium and are the overwhelming cause of anogenital cancer; (2) Betapapillomavirus are primarily isolated from skin lesions. The β-PVs include HPV types frequently associated with the rare genetic disorder epider-modysplasia verruciformis (EV), which predisposes individuals to develop HPV-associated cutaneous, scaly wart-like squamous cell carcinomas. Many β-HPV types were originally identified in isolates obtained from EV patients, previously termed HPV EV types and include HPV-5 and HPV-8, both members of the β-1 species, identified in approximately 90% of EV-related cutaneous squamous cell carcinomas. Less prevalent EV types extend to the β-2 species (HPV-38) and β-3 species (HPV-49). These types are also found associated with malignancy in immunocompromised hosts, and are less prevalent in the general population [ 58 ] . (3) Gammapapillomavirus are primarily isolated from cutaneous epithelia, some from cutaneous lesions histo-logically defined by the presence of homogeneous intracytoplasmic inclusion bodies [ 1 , 59 ] . Both Gammapapillomaviruses and Betapapillomaviruses have been identified in oral samples suggesting they have an expanded tropism including the oral cavity [ 60 ] ( fig. 3 ). Thus, the tropism of these later genera has to be reconsidered in light of the new information. This also demonstrates that not testing a specific anatomical site (e.g. the oral cavity) for HPV (using methods to detect the gamut of types) does not mean the virus is not there.
α-HPV Phylogenetic Association with Cancer Risk
Phylogenetic analysis based on the sequence of the HPV L1 ORF has been the standard for genomic analysis and type classification [ 1 , 19 , 54 ] . Observations of phylogenetic incongruence within the α-PVs are shown by comparison of trees built utilizing either the early or late regions of the genome. This results in differences regarding the monophyletic origin of the oncogenic α-HPV types. Trees generated from the early genes or the complete genome cluster α-PVs by associated oncogenic risk as a mono-phyletic clade. Phylogeny based on late gene regions (L1 and L2) does not support a monophyletic origin for the 5 oncogenic α-HPV species (α5, α6, α7, α9, α11). This phylogenetic incongruence is suggestive of genomic distinctions within the α-PVs that are exemplified by examining differences inherent to the species α9 (HPV-16-related) and α7 (HPV-18-related) that differ in biology and pathological outcome. HPV-16 and HPV-18 are highly prevalent, oncogenic types implicated in 70% of cervix cancer, and as members of different species groups they exhibit different biological niches and cellular tropisms, and manifest as precancerous and/or cancerous lesions differently. HPV-16 targets the cutaneous squamous epithelia abundant in the ectocervix and predominantly causes cervical squamous cell carcinoma that evolves through differing grades of squamous intraepithelial neoplasia observed by histological/cytological screens [ 61 ] . HPV-16 also infects the squamous epithelia of the oropharynx and is identified in oropharynx cancer (targeting the regions of the throat including the base of the tongue, the soft palate and the tonsils). Additionally, HPV-16 is implicated as causal agent for cancers of the vulva, vagina, penis and anus [ 18 ]. Conversely, HPV-18 disproportionately targets glandular, mucin-secreting columnar epithelial cells found primarily in the endocervix, and is overrepresented in the development of cervical adenocarcinoma [ 62 , 63 ].

Fig. 3. HPV phylogenetic tree clustering the majority of HPVs into 3 genera, Alphapapillomavirus, Betapapillomavirus and Gammapapillomavirus. A maximum likelihood tree was constructed using RAxML version 7.2.8.27 [ 80 ] and an alignment of the nucleotide sequences of the L1 ORF of 120 published HPV types. HPV species groups were generally classified according to the classification system for PVs by Bernard et al. [ 19 ] . All HR mucosal HPV types cluster within the genus Alphapapillomavirus, highlighted in red. The clades in blue represent mucosal HPV types with low risk or no risk of cervical cancer. The scale bar represents a nucleotide change of 0.2 per site.
Comparison of phylogenetic trees generated from either early or late gene sequences results in different phylogenies for distinguishing higher-level taxa [ 48 ] ; the clades defining species groups remain intact. Yet, the nodes defining a clades’ most recent common ancestor appear differently depending on the genes used for the phylogenetic tree construction, reflecting a degree of phylogenetic incongruence. This hints at the occurrence of early selection events likely driven by ecological niche adaptation such that early and late genes are regulated through distinct promoters that are strictly governed by the availability of host proteins differentially expressed within the stratified epithelia [ 25 , 48 ] . Incongruence may also result from disproportionate selective pressures on the two genomic regions. Incongruence does not likely result from an early viral recombination event, as similar genome characteristics are maintained across diverse hosts throughout evolutionary time, such as humans and nonhuman primates, supporting the clonal nature of viral expansion as opposed to recombination [ 38 , 48 ].
Variant Lineages: Model for Recent Human Papillomavirus Speciation
Classification below the species level is not formally recognized by the International Committee on the Taxonomy of Viruses [ 19 , 55 ] . Evidence from large epidemiological studies identifying HPV genomic heterogeneity and associated pathologies supports the need for distinction of taxa below the HPV type level and will likely be updated soon [ 55 ] . The term HPV subtype has become obsolete. It previously referred to isolates that exhibited 2-10% differences in nucleotide identity compared to its closest known type and/or differences in restriction enzyme cleavage patterns [ 1 ]. As better systems for identification and classification of HPVs emerged, subtypes are now considered variant lineages.
Variant Lineages and Sublineages: Updates on Current Nomenclature Guidelines
HPV variant lineages represent viral isolates that exhibit genomic nucleotide differences of 1-10%, as compared to their prototype, or reference genome [ 5 , 6 , 27 , 50 , 55 , 64 ] . Viral variants share a most recent common ancestor that is unique to the specific HPV type. HPV variants are common and may differ in the risk for development of cervical precancer (CIN2/3) or cancer. Initial studies examining HPV-16 and HPV-18 intratypic variation aimed to discern the contribution of variant lineages to geographic differences in virus distribution [ 5 , 6 , 65 ] . Variants of both HPV-16 and HPV-18 were initially classified by sequencing the URR, as this noncoding region exhibits greater nucleotide variability than protein-coding regions and is a valuable tool for identifying stable nucleotide polymorphisms, the basis for HPV classification. However, unlike type identification that is sufficiently designated by sequencing the L1 ORF, complete genome sequencing is required to identify HPV variants since the distribution of differences is not evenly spaced across the genome. In addition, a variant nomenclature system based on the complete genome permits the identification and quantification of nucleotide polymorphisms using different regions of the genome [ 55 ] . Evidence supports that certain genomic regions exhibit greater heterogeneity than others.
HPV replication depends on host DNA replication machinery; proofreading capabilities by host DNA polymerases maintain a low rate of mutation within the viral genome at approximately 10 -8 -10 -7 nucleotide substitutions/year [ 4 ]. Intratypic HPV genetic variation results from random nucleotide polymorphisms or insertions/deletions (indels) acquired through genetic drift or natural selection that become fixed over time. Stable acquisition over time, of these nucleotide changes, eventually leads to PV speciation through a process termed lineage fixation [ 57 ] and is further supported by a lack of evidence for viral recombination events [ 49 ] . Furthermore, the stable acquisition of polymorphisms among isolates of the same HPV type (sharing a most recent common ancestor) eventually leads to type speciation, characterized by genomic nucleotide identity that is less than 90% and occurs over millions of years [ 46 ] . To this end, evidence of prehistoric human population bottlenecks is reflected through inter- and intratypic PV genomic diversity. Distinction below the species level (PV type) is common within the PV research community, and is useful for physicians, researchers and epidemiologists investigating HPV variants for association with geographical host population and viral genetic changes that confer variable phenotypic outcomes, including varying pathological manifestations such as cancer. At present, guidelines for variant HPV lineage classification are beginning to be formally established [ 55 ]. A formal classification and nomenclature system to describe intratypic HPV variants, at the lineage or sublineage level, will undoubtedly become increasingly useful as the future of HPV genomics expands to include data obtained from next-generation sequencing and metagenomic studies, and will facilitate a better system for cataloguing genotype-phenotype changes.
Variant Lineages and Sublineages: Identification and Clinical Relevance
Variant lineage classification is based on isolates of a known type that have had their complete genome sequenced and reveal genetic heterogeneity of at least 1% and less than 10%, based on multiple sequence alignments to the prototype (first characterized genome of a given type). Parameters for variant classification have recently been established by phylogenetic analysis on isolates from the α 9 -species (including HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, HPV-58 and HPV-67), types from the α-7 species and two types from the α-10 species, HPV6 and HPV11 [ 27 , 55 , 64 ]. A divergence rate of 1% conservatively designates variant lineages. Similarly, pairwise nucleotide identity differences in the range of 0.5-1% define a type sublineage. Nomenclature for lineage and sublineage is based on an alphanumeric system wherein the prototype ‘reference’ genome is always designated with an ‘A’. If sub-lineages for the given type are present, the prototype reference sequence is designated as ‘A1’ ( table 1 ).
Table 1. HPV taxa definition and nomenclature overview

Variant lineages from many clinically relevant α-HPVs are known. Variants have been characterized for the majority of the HR mucosal HPV types: HPV-16 [ 5 , 65 , 66 ], HPV-18 [ 6 , 46 , 64 ], HPV types 31, 33, 35, 52, 58, and 67 [ 27 ] ; other α-HPV variants that have been identified include members of the α-4 species, i.e. HPV-2, HPV-27 and HPV-57 [ 67 ], and two types from the low-risk α-10 species, specifically HPV-6 and HPV-11 [ 55 ], associated with genital warts have been extensively examined.
Phylogenetic analysis of HPV-16 variation revealed variants reflective of human dispersal out of Africa and divergence into the 3 major human races, Africans, Caucasians and Asians. Initial phylogenetic studies identified 4 major HPV-16 variants representative of host geographic origin. The major HPV-16 variants were broadly termed ‘European’ now lineage ‘A’ or ‘non-European’ now lineages ‘B, C and D’. The non-European lineages are more heterogeneous and included 2 African HPV-16 lineages, African-1 and African-2 (B and C, respectively), and Asian-American variants (lineage D) [ 5 , 57 , 62 , 68 ] . These variants display phylogenetic congruence with host ethnicity and geographic origin [ 5 ] . However, with the recent update to nomenclature the designation of variant lineage simply (for the case of the HPV-16 example) as non-European or European is misleading and overlooks details within the viral genomes ( fig. 4 ). Previously termed HPV-16 variants have thus been renamed according to the updated variant nomenclature guidelines, whereby European variants are classified as the ‘A’ lineage, further resolved to 4 sublineages (A1, A2, A3, A4). The HPV-16 non-European variants are now recognized as 3 distinct lineages with the appropriate sub-lineage designation: HPV16-B (previously Af-1) contains sublineages B1 and B2; HPV-16C (previously Af-2); HPV-16D (previously NA1, AA1, AA2) contains sublineages D1, D2 and D3. This update helps to resolve and term the viral distinctions below the type level and is important since many of the nucleotide changes are correlated within taxa ( fig. 4 ).

Fig. 4. HPV-16 variant tree topology and pairwise comparisons of individual complete genomes. A maximum likelihood tree was inferred from a global alignment of 13 complete nucleotide sequence genomes of HPV-16 using RAxML version 7.2.8 [ 80 ] . Distinct variant lineages (i.e. termed A/B/C/D) and sublineages (e.g. termed A1/A2/A3/A4) were classified according to the topology and nucleotide sequence differences from >1% to <10%, and >0.5% to <1%, respectively [ 19 , 27 ]. The percent nucleotide sequence differences were calculated for each isolate compared to all other isolates based on the nucleotide sequences (complete genome) and are shown in the panel to the right of the phylogeny. Values for comparison from an isolate are connected by lines, and the comparison to self is indicated by the 0% difference point. Symbols and colored lines are used to distinguish each isolate. The scale bar at the bottom of the tree represents a nucleotide change of 0.002 per site. Multiple isolates for the HPV-16 A4 sublineage are shown to highlight the intrasublineage relationship note the clustering in the right hand graph that depicts nucleotide sequence differences.
The Future of Human Papillomavirus Genomics
Biomedical Technology Advancements and Biomarker Prediction
Global epidemiological studies now permit access to vast numbers of clinical HPV samples to better understand the natural history of HPV infections [ 50 ] and the biological and clinical ramifications [ 50 , 69 ] . The risk associated with a persistent HR infection, such as HPV-16, is significantly associated with neoplastic progression. However, determinants of viral persistence and clearance are not well understood. Recent evidence suggests differences in the duration of persistence for different HPV types and/or lineages associated with clinical outcomes [ 50 , 70 , 71 ] . Understanding of HPV genomics and classification will help further characterize host/viral components involved in viral persistence and/or clearance [ 72 , 73 ] . This may contribute to the design of new treatments and therapeutics targeting those most at risk for cancer. On a broader scale, it may also help delineate which infections require treatment versus those that naturally regress, having implications on the financial burden associated with cancer prevention and treatment globally.
Rapid technology advances during the past decade have, and will continue to, advance our understanding of PV genomic heterogeneity that contributes to pathological clinical outcomes. Next-generation sequencing provides increased resolution for the detection and classification of viral DNA, at the single molecule level, and will enhance methods for identification of new HPV taxa that may have previously fallen under the limit of detection by current methods [ 74 ] . This will facilitate a more complete view of Papillomaviridae diversity and should contribute to an improved understanding of the mechanisms of HPV-associated malignancy [ 75 , 76 ] . Increased use of novel genotyping methods will improve the efficiency of identifying HPV DNA from an array of samples obtained by large cohort studies. The explosion in data generated by next-generation sequencing techniques reinforces the need for current, widely accepted methods to characterize the results. This is highlighted by recent reports describing an extensive array of novel Betapapillomavirus and Gammapapillomavirus types, which are currently difficult to classify given the extensive diversity of these types and the lack of a simple method for their characterization in a standard clinical assay.
New Codes in the HPV Genome
The appreciation of an additional ‘epigenetic code’ (i.e. CpG sites that can be methylated) in the HPV genome has energized new and future studies on understanding the significance of information. HPV epigenetics is a burgeoning field [for a review, see 77 ]. Accounts of CpG methylation of viral DNA, predominately from HPV-16 and HPV-18 were initiated at the turn of the 21st century and identified regions of viral CpG methylation by methylation-specific restriction endonuclease maps. Different cleavage patterns from viral isolates obtained from women with different stages of HPV-associated CIN have been observed. The onset of technological advances within the past 2 decades has generated methods for more accurately identifying and quantitating DNA methylation. CpG sites are highly conserved amongst HPV species. CpG sites appear throughout the genome and exhibit varied methylation states and likely play a physiological role in the viral life cycle. Large epidemiological studies that aim to elucidate the oncogenic properties of HPV have demonstrated that methylation at specific CpG sites within the viral genome is predictive of clinical outcome, such as precancer (CIN2/3) or cancer [ 77 - 79 ] . Specifically, CpG sites found within the HPV-16 L1 ORF are highly predictive of CIN2/3 [ 78 ] . Interestingly, the α-7 species contain higher numbers of CpG sites relative to the α 9 -species. The development of high-resolution methods for HPV identification and classification should lead to the discovery of additional coded information in the HPV genome beyond nucleotide, amino acid, CpG and DNA-binding protein regions.
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Robert D. Burk Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, NY 10461 (USA) E- Mail robert.burk@einstein.yu.edu
Molecular Biology and Pathogenesis
Ramírez-Fort MK, Khan F, Rady PL, Tyring SK (eds): Human Papillomavirus: Bench to Bedside. Curr Probl Dermatol. Basel, Karger, 2014, vol 45, pp 19-32 (DOI: 10.1159/000355959)
______________________
The Biology of Human Papillomaviruses
Harrison P. Nguyen a Marigdalia K. Ramírez-Fort b Peter L. Rady c
a Baylor College of Medicine, b Center for Clinical Studies, and c Department of Dermatology, University of Texas Health Science Center at Houston, Houston, Tex., USA
______________________
Abstract
Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that cause lesions in cutaneous and mucosal tissue and are responsible for carcinomas of the cervix, vagina, vulva and penis. HPVs sort into 5 genera with a total of approximately 150 species that have been sequenced. Its genome is comprised of an early (E) region encoding the viral regulatory proteins, a late (L) region encoding the viral structural proteins and a noncoding region that is essential to the viral life cycle. For infection to occur, the virus must access the basal epidermal layer where, following endocytosis and viral capsid disassembly, the L2 protein mediates viral genome transfer to the nuclei of mitotic kera-tinocytes. The viral genome is maintained in episomal form during the normal life cycle and replicates in synchrony with the host cell DNA under the mediation of E1, E2, E4 and E5 viral proteins. In most high-grade cervical neoplasms, however, the viral DNA is integrated into the host genome through the disruption of the E2 open reading frame. The oncoproteins E6 and E7, which were previously suppressed by E2, are then free to inhibit the Rb and p53 tumor suppressor pathways. The viral life cycle concludes with the packaging of the viral genome and virus release, which entails the E2-me-diated recruitment of L2 to regions of replication, the expression of L1 and the assembly of the ico-sahedral capsid in the nucleus. Overall, the complex biology of HPV continues to be an important area of research with substantial implications for public health.
© 2014 S. Karger AG, Basel
Introduction
Human papillomaviruses (HPVs) comprise a family of small DNA tumor viruses that require host cell machinery to replicate their circular, double-stranded genome. HPVs display a notable affinity for infecting human stratified squamous epithelia at distinct anatomical locations. Nearly every site in the body that contains stratified squamous epithelia has been shown to harbor an HPV infection, including the anogenital regions, aerodigestive tract, nonanogenital skin and even the eye. HPVs are responsible for causing both malignant and benign tumors in cutaneous and mucosal tissue. A subset of ‘high-risk’ HPV types have been associated with greater than 95% of all cervical cancers and over 50% of all vaginal, vulvar and penile cancers. The abundance of viral types and the variety in clinical manifestation are testament to the complex mechanism of the virus (addressed in the book’s chapter ‘Epidemiology and clinical manifestations’) and to the difficulty in controlling HPV-associated disease.
Virion and Genome Structure
HPVs were once grouped with polyomaviruses and were classified as a single family, the Papovaviridae. Both papillomaviruses and polyomaviruses possess a double-stranded, circular genome, an icosahedral capsid of 72 pentamers, a nonenveloped virion, the nucleus as a site of virion assembly and replication, and the capability to transform host cells [ 1 ] . However, the application of genome sequencing revealed that the papillomavirus genome and its unidirectional transcription mechanism mostly contrasted with the arrangement of genes and the bidirectional transcription found in other papovaviruses. These significant differences led the International Committee on the Taxonomy of Viruses to designate Papillomaviridae as a separate family [ 2 ].
HPV particles are 52-55 nm in diameter and consist of an approximately 8,000-bp circular genome with a GC content of approximately 42% [ 3 ] . The viral DNA is contained within a spherical capsid that consists of 2 virally encoded structural proteins: an approximately 55-kDa major protein (L1) that accounts for 80% of total viral protein and an approximately 75-kDa minor protein (L2) [ 4 , 5 ] . Both proteins are essential for efficient viral infection.
While their DNA is double-stranded, all of the open reading frames (ORFs) are located on only one strand of the viral DNA, and these ORFs are well conserved among HPV types [ 5 , 6 ] . The coding strand is comprised of 8-10 translational ORFs and is classified into 2 regions, the early (E) and the late (L) region. The E region encodes the viral regulatory proteins E1, E2, E4, E5, E6 and E7, which are required for viral DNA replication and are expressed in both productively and nonproductively infected cells. The L region contains genes that encode for the structural proteins L1 and L2, and these are only expressed in productively infected cells [ 5 , 6 ] . An approximately 1-kbp non-coding region - also referred to as the long control region or the upstream regulatory region - separates the early and late regions. The noncoding region contains the origin of DNA replication as well as several motifs that likely bind transcription factors that are involved in regulating downstream protein expression [ 7 ] . As a result, the noncoding region plays an indirect role in both gene expression and viral protein production.
Human Papillomavirus Types
The emergence of polymerase chain reaction simplified the amplification, sequencing, and comparison of viral DNA and exposed the need for a classification system for HPV types. At the 1995 International Papillomavirus Workshop, it was agreed that HPVs would be considered to be different types if they possess >10% dissimilarity in the L1 region, the most highly conserved gene. Based on this classification system, HPVs are classified into 5 genera: α, β, γ, μ, and ν. Moreover, other genes also display major homology among HPVs, which has led to the hypothesis that papillomaviruses emerged from point mutations in various locations of the viral genome and have evolved concurrently with humans [ 8 , 9 ].
To date, approximately 150 HPV types have been sequenced ( table 1 ) [ 2 , 9 ]. For clinical purposes, HPVs can be broadly grouped into types that preferentially infect cutaneous tissue and types that generally occupy mucosal surfaces. Computer-assisted construction of phylogenetic trees indicates that the genomic classification system is mostly consistent with the clinical groupings that are based on which tissue type the viruses infect (cutaneous or genital-mucosal) and what type of tumors are produced (benign or malignant) [ 10 , 11 ] . The different strategies of transmission and propagation within the epithelium as well as their interactions with the host immune system likely account for the diversity of epithelial disease that is associated with HPV [ 12 , 13 ] . Twelve types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58 and -59), which all belong to the genus α, are designated as category 1 types since they are considered to be high-risk cancer-causing species [ 13 ] . The genus α also contains low-risk HPVs that cause flat warts and common warts (HPV-2, -3, -10 and -57) as well as genital condylomas (HPV-6 and -11) ( table 1 ) [ 2 ] . Strains from the genera β, γ, μ and ν are considered to be low risk since the papillomas and verrucae that result from infection generally do not progress to cancer [ 3 ] . However, in immunocompromised patients [i.e. severe combined immunodeficiency, epidermodysplasia verruciformis (EV), and organ transplant recipients], these low-risk types can cause debilitating presentations and can result in the appearance of neoplastic precursors (Bowen’s disease and actinic keratosis) and the development of nonmelanoma skin cancers at sun-exposed sites. Pathogenesis in these individuals is related to the compromise of both the infected keratinocyte and the immune system [ 14 , 15 ] . A notable difference between high- and low-risk types is that the former has evolved an ability to drive cell proliferation in the basal and parabasal cell layers, often for many years [ 16 , 17 ] . This level of persistence is not required for viral production and is not observed during low-risk type infection in healthy individuals [ 18 - 21 ].
The biology of disease for HPV in cervical carcinogenesis is relatively well understood, but their life cycle organization at other epithelial sites - such as the endocervix, the penis, the oropharynx and the anus - is still poorly understood [ 22 ] . Deregulation of viral gene expression occurs to varying extents at the different sites of high-risk HPV infection, with the squamocolumnar junction (i.e. epithelial reserve cells that lie immediately underneath the columnar epithelium of the endocervix) being the most prone to neoplasia formation ( fig. 1 ) [ 23 ] . However, it is important to note that only a minority of individuals who are infected with high-risk HPV develops anogenital cancer [ 24 ] . Since carcinogenic infections tend to include a long latency period, it is believed that host genetic changes and predisposing host factors greatly influence tumor development [ 13 ] . With the growing public awareness of the carcinogenic capabilities of some HPV types, several preventative measures have been developed and have entered the market, including a bivalent vaccine that protects against types 16 and 18 and a tetravalent vaccine that immunizes against types 6, 11, 16 and 18.
Table 1. HPV types reported and cloned
Genus and species
Type
Associated clinical disease



α 1
HPV-32
Benign lesions of oral or genital mucosa

HPV-42

α 2
HPV-3
Low-risk cutaneous and, less frequently, mucosal lesions

HPV-10


HPV-28


HPV-29


HPV-78


HPV-94


HPV-117

α 3
HPV-61
Low-risk mucosal lesions

HPV-72


HPV-81


HPV-83


HPV-84


HPV-62


HPV-86


HPV-87


HPV-89


HPV-102


HPV-114

α 4
HPV-2
Common skin warts and benign genital lesions in children

HPV-27


HPV-57

α 5
HPV-26


HPV-51


HPV-69


HPV-82

α 6
HPV-30
Cervical intraepithelial neoplasia, cervical squamous cell carcinoma

HPV-53


HPV-56


HPV-66

α7
HPV-18
Cervical intraepithelial neoplasia, cervical squamous cell carcinoma, anogenital warts

HPV-39


HPV-45


HPV-59


HPV-68


HPV-70


HPV-85


HPV-97

α 8
HPV-7
Low-risk mucosal and cutaneous lesions; often found in HIV-infected patients

HPV-40


HPV-43


HPV-91

α 9
HPV-16
High-risk, malignant mucosal lesions

HPV-31


HPV-33


HPV-35


HPV-52


HPV-58


HPV-67

α10
HPV-6
Mostly associated with benign mucosal regions; HPV-6 in verrucous carcinoma

HPV-11


HPV-13


HPV-44


HPV-74

α11
HPV-34
High-risk anogenital warts

HPV-73

α13
HPV-54
Low-risk mucosal lesions
α14
HPV-71
Low-risk mucosal lesions

HPV-90


HPV-106

β1*
HPV-5
Most frequently found in cutaneous lesions and commonly associated with lesions in EV or immunosuppressed patients

HPV-8


HPV-12


HPV-14


HPV-19


HPV-20


HPV-21


HPV-25


HPV-36


HPV-47


HPV-93


HPV-98


HPV-99


HPV-105


HPV-118


HPV-124

β 2 *
HPV-9
Most frequently found in cutaneous lesions and commonly associated with lesions in EV or immunosuppressed patients

HPV-15


HPV-17


HPV-22


HPV-23


HPV-37


HPV-38


HPV-100


HPV-104


HPV-107


HPV-110


HPV-111


HPV-113


HPV-120


HPV-122

β 3 *
HPV-49
Skin lesions in immunosuppressed patients

HPV-75


HPV-76


HPV-115

β 4 *
HPV-92
Premalignant and malignant cutaneous lesions
β 5 *
HPV-96
Premalignant and malignant cutaneous lesions
γ1*
HPV-4
Cutaneous lesions; histologically distinct homogeneous intracytoplasmic inclusion bodies

HPV-65


HPV-95

γ2*
HPV-48
Anogenital warts
γ3*
HPV-50
Cutaneous lesions in EV patients
γ4*
HPV-60
Plantar epidermoid cyst
γ5*
HPV-88
Cutaneous lesions
γ6
HPV-101
Cervical intraepithelial neoplasia

HPV-103


HPV-108

γ7*
HPV-109
Squamous cell carcinoma

HPV-123

γ8*
HPV-112
Condyloma

HPV-119

γ9*
HPV-116
Low-grade cervical lesion
γ10*
HPV-121

μ1
HPV-1
Plantar and palmar warts
μ2
HPV-63
Cutaneous warts (rare) and multiple plantar punctate keratoses
1
HPV-41
Cutaneous plane warts
The asterisk indicates absence of the E5 ORF; the cross indicates absence of the E6 ORF. EV = Epider-modysplasia verruciformis.
Viral Life Cycle
HPVs appear to infect only human cells and, in particular, demonstrate an affinity for stratified squamous epithelium. For infection to occur, HPVs must gain access to the basal lamina, which allows the viruses to infect the mitotically active basal keratinocytes ( fig. 1 ). Transmission of cutaneous HPVs can result from contact with the skin of an infected individual or from some form of environmental exposure. Transmission of genital HPVs requires direct contact, often through sexual activity or perinatal transmission. For proper entry of the virus genome into the nucleus and subsequent lesion formation, active cell division, as observed in wound healing, is likely required, which would indicate that the initial infection must occur in a mitotically active cell or a basal stem cell [ 25 ].

Fig. 1. Key events following infection of HPV-16 virions (red pentagons). HPV infection occurs when the infectious virions are able to access the basal lamina, either through a microwound of multilayered stratified epithelia or at an anatomical location in which the squamocolumnar junction is naturally exposed (i.e. cervical transformation zone or endocervix). The different cell layers present in the epithelium are labeled on the left, and the corresponding viral life cycle events are described on the right. Cells in the epidermis expressing cell cycle markers are depicted with red nuclei; mitotic cells above the basal layer result from viral infection and the expression of oncoproteins E6 and E7. Cells shown in green with red nuclei express viral proteins necessary for genome replication following activation of p670 in the upper epithelial layers, and the cells depicted in yellow express the L1 and L2 genes. Cells containing infectious particles are eventually shed from the epithelial surface following nuclear degeneration and the formation of flattened squames.
While it is not certain what receptor(s) mediates HPV cellular entry, heparin sulfate surface proteoglycans and possibly also laminin appear to play a role in initial binding for most HPV types [ 26 , 27 ] . Following initial binding, HPVs bind additional secondary receptors on the basal keratinocytes, which ultimately leads to virus internalization, likely through clathrin-dependent endocytosis and/or caveolin-mediated endocytosis [ 12 ]. α 6 -Integrin has long been thought to be an impactful secondary receptor since antibodies to α 6 are able to prevent virus-like particle binding and cellular uptake [ 28 ] . However, not all HPV types require heparan proteoglycan and the α 6 complex for cellular entry, which illustrates the diversity in the mechanism of infection among HPVs [ 29 ].
Following endocytosis, the viral capsid disassembles in the late endosome, which exposes the viral genome. The minor capsid protein L2 then facilitates the transfer of the genome to the cell nucleus and guides the genome to the transcriptionally active nuclear domain 10 [ 30 ] . L2 interacts with the endoplasmic reticulum protein tSNARE syntaxin 18, which is believed to be important in genome transport to the nucleus [ 31 ] . Without cleavage of L2 at a furin consensus site, endosomal escape does not occur [ 32 ] . The process of infection is relatively slow, taking 12-24 h until viral transcripts can be detected.
The viral genome, which is maintained in episomal form at low copy numbers (approx. 200 copies/cell) during the normal life cycle, replicates in synchrony with the host cell DNA during the S phase [ 13 ] . In the episomal phase, the virus is dependent on the expression of early viral replication proteins, E1 and E2, for the initiation of viral transcription, replication and genome segregation. E2 can form a dimer structure that may be important in its interactions with viral and cellular transcription factors and contains 2 well-conserved domains that aid its recognition and binding of a palindromic motif [AACCg(N 4 )cGGTT] in the noncoding region of the viral genome [ 3 ] . E2 participates in the viral genome segregation during mitosis by its association with the cellular protein Brd4 and critically mediates the anchoring of viral episomes to mitotic chromosomes, which facilitates viral genomic replication along with the cellular DNA during the S phase. In high-risk HPV types, E2 directly associates with the mitotic spindle - independent of Brd4 binding - and it is believed that this is the mechanism for HPV genomic persistence in host cells [ 13 ] . Further studies on the role of E2 in transcriptional regulation will shed light on the emerging role of E2 in mediating novel mechanisms of viral oncogenesis.
The E1 protein functions as an adenosine triphosphate-dependent helicase and appears to be an important mediator of viral replication. E1 must be recruited to the origin by E2, forming an E1-E2 complex. With the dissociation of E2, the E1-E2 complex is transformed into a larger multimeric E1 complex that can distort the replication origin, unwind the DNA and recruit additional E1 molecules. By itself, E1 binds several host proteins necessary for DNA replication - such as replication protein A, DNA polymerase α primase, topoisomerase I, and cyclin E/CDK2 - and thereby recruits the cellular DNA replication initiation machinery to the viral replication origin [ 24 ] . E1 also interacts with several other host cellular proteins, and while these interactions are not well understood, E1 association with the S-phase-specific cyclin E/CDK2 complex appears to be essential to efficient cell cycle-regulated replication [ 33 ].
While E1 and E2 are the important mediators of viral DNA amplification, the E4 and E5 proteins also appear to be key players. Along with E7, the E4 protein is responsible for arresting the cell cycle in G 2 , which facilitates viral genome amplification [ 34 , 35 ] . The manner in which E4 is expressed appears to vary among viral types, but in all types, E4 induces cell cycle arrest by preventing the nuclear accumulation of cell cycle proteins that are required for mitosis. HPV-16 E4 binds to CDK1/cyclin B1 and tethers the complex to cytoplasmic keratins [ 36 ] . E5, a small transmembrane protein principally residing in the endoplasmic reticulum, upregulates epidermal growth factor-mediated receptor signaling and maintains a replication-competent environment in the upper epithelial layers [ 37 ] . It accomplishes this by preventing acidification of endosomes and inhibiting receptor inactivation [ 38 ]. Additionally, HPV-16 E5 is able to bind to the EVER-ZnT1 (zinc transporter-1) complex, which is normally responsible for decreasing the nuclear and nucleolar free zinc concentration, which leads to the downregulation of cellular transcription factors (such as AP-1) stimulated by zinc [ 37 ] . Since a high level of intracellular free zinc is required for AP-1 activity and replication of the HPV-16 genome, the E5-mediated inhibition of the EVER-ZnT1 complex is critical for the HPV life cycle. EV-associated HPVs (i.e. HPV-5 and -8) lack an E5 ORF, and, therefore, it is proposed that the maintenance of low free zinc levels is responsible for preventing the replication of EV-associated HPVs in the general population [ 38 ] . However, EV patients, who possess a mutation in the EVER genes, have unrepressed free zinc concentration and are thus highly susceptible to the EV HPVs [ 39 ].
The final steps in the HPV life cycle - packaging of the viral genome and virus release - involve expression of the L2 minor coat protein, suspension of the cell cycle, and the expression of the L1 major coat protein. Instead of relying on promoter activation that is characteristic of the majority of the HPV life cycle, the virus employs splice site usage mediated by E2 to produce transcripts that terminate at the late polyadenylation site [ 12 ] . HPV-16 E2 regulates late gene expression by causing a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs [ 40 ] . This marks the shift from genome amplification to genome packaging, which chronologically entails the E2-mediated recruitment of L2 to regions of replication, the expression of L1 and the assembly of the icosahedral capsid in the nucleus [ 41 ] . The capsid consists of 360 molecules of L1, which are arranged into 72 pentameric capsomeres, and a smaller, variant number of L2 molecules [ 42 ] . Disulfide bonds form between the L1 proteins, which leads to the production of highly stable infectious virions. Bond formation and virus maturation are facilitated by the oxidizing environment found in the most superficial layer of keratinocytes [ 43 ] . Finally, E4 is hypothesized to be involved in virion release and infectivity in the upper epithelial layers by disrupting keratin structure and preventing the proper assembly of the cornified envelope ( fig. 1 ) [ 44 ].
Genome Integration and Expression
During the normal HPV life cycle, the genome is maintained in episomal form; however, in nearly all high-grade cervical lesions and tumors, the viral DNA is integrated into the host genome [ 45 ] . Integration generally occurs at common fragile sites, and changes in the expression of genes at or near the integration site may participate in oncogenesis [ 46 ] . In addition to its important role in replication, E2 is responsible for repressing the viral oncogenes E6 and E7, and the disruption of the E2 ORF in high-risk types is critical for integration leading to tumorigenesis [ 6 ] . Retention of the E6/E7 region following integration into the host chromosome is accompanied by the dis-ruption of viral sequences encoding E1, E2 and E4. E2 functions as a transactivator (at low E2 levels) or a transrepressor (at high E2 levels) depending on the proximity of the E2 binding sites to the HPV early promoter. HPV-16 has four E2 binding sites that lie upstream of the promoters for E6 and E7 transforming genes, which are initially unable to be expressed due to bound-E2 steric interference ( fig. 2 ). Interestingly, in cervical cancer cell lines that contain continued E2-mediated transcriptional repression of E6 and E7, Rb and p53 tumor suppressor pathways are reactivated, which leads to cellular senescence and cell cycle arrest [ 47 ] . Similarly, the E4 protein inhibits mitosis by preventing the nuclear localization of cyclin B/CDK1 and must be repressed for oncogenesis to occur. In addition to the loss of regulatory proteins, the 3′ end of early transcripts, which can suppress the production of viral mRNA species encoding E6 and E7, is disturbed following integration [ 12 ] . Taken together, these events facilitate the oncogenic expression of E6 and E7.

Fig. 2. Integration of HPV-16 DNA into the host genome. HPV-16 DNA encodes an early (E) region - containing E6, E7, E1, E2, E4 and E5 ORFs - and a late (L) region - with L1 and L2 ORFs. In the nonintegrated form, the HPV genome exists as a circular plasmid (top), and products of the E2 gene repress the expression of the E6 and E7 genes. The nonintegrated episome is observed in latent infections, benign warts, and cervical intraepithelial neoplasia. However, during carcinogenesis, viral DNA is integrated into the host genome (yellow arrow). Part of E2 and L2 as well as E4 and E5 are often deleted following integration. Importantly, the E6 and E7 regions are preserved during linearization of the viral genome (bottom) and are not repressed by E2. Transcription of the E6 and E7 oncogenic regions is modulated by the promoter in the long control region (LCR), leading to the overexpression of the oncoproteins.
While there are certainly many similarities in genome organization among HPVs, it is crucial to recognize there are also many important differences in both protein function and expression patterns across types. For example, the recently discovered γ-HPV types 101, 103 and 108 completely lack an E6 gene [ 48 , 49 ] . This emphasizes the caution that must be taken when applying general principles to all HPV types.
Clearance, Latency and Asymptomatic Infection
Most infections are cleared before deregulated gene expression and the accumulation of secondary genetic errors can occur [ 13 ] . Clearance is regulated by host cell-mediated immune response. Since regression of anogenital warts is accompanied histologically by a CD4+ T-cell-dominated Th1 response, it is hypothesized that the clearance mechanism is modulated by CD4+ T-cell-dependent mechanisms [ 50 ] . If effective cell-mediated immunity is unable to develop, persistent infection and, in the case of oncogenic types, high-grade intraepithelial neoplasia develop.
Although studies in human cells have not fully elucidated the latency and reactivation mechanisms, HPV DNA can induce papilloma formation following infection; undergo spontaneous regression into a quiescent state and subsequently reactivate to induce disease. In many circumstances, the virus can be present and infectious, but the patient may not develop squamous intraepithelial lesions for several months to years. HPV-16 DNA can be detected in cervical biopsies of 5-40% of women who present with no clinical or histological sign of papillomavirus infection [ 51 ]. Similarly, HPV-6 and HPV-11 DNA can be detected in the normal laryngeal epithelium of patients who are in remission from recurrent respiratory papillomatosis [ 52 ]. Viral DNA is present at a much lower copy number under asymptomatic conditions than during infection.
Based on studies conducted on the cottontail rabbit papillomavirus, a model for HPV latency has recently been proposed [ 3 ] . When HPV infects the mitotically active basal keratinocytes, most of the cells differentiate and participate in virion production, which can give rise to the appearance of papillomas. A minority of the infected cells do not differentiate to fill the wound yet maintain HPV episomes, which are detectable by polymerase chain reaction ( fig. 1 ) [ 53 ] . For unknown reasons - likely additional epithelial trauma to the area or hormonal regulation - these latent cells are later reactivated to produce infection. The different host immune responses are responsible for the variation in latency periods and reactivated infection that is clinically observed. For example, the prevalence of oral, anal and cervical HPV infection in HIV+ individuals increases with progressively lower CD4+ levels [ 53 ] . Reactivation of latent HPV results from alterations in cytokine expression within epithelial cells; reduced cytotoxic T lymphocyte reactivity to HPV viral regulatory proteins then leads to undisturbed epithelial proliferation [ 54 ].
Conclusion
The differences among HPV types and the rapid discovery of new species contribute to the complexity of HPV biology. Recent advances in mass spectrometry technology and data processing have catalyzed the identification of interactions between viral and host cellular proteins, leading to insights into the carcinogenic role of papillomaviruses [ 55 ] . Although most research has focused on the study of HPV-16 and HPV-18, it will be important to deepen our understanding of the different risks associated with different high-risk types and to better comprehend the molecular pathways that they subvert. Additionally, there will be a need in the future to understand the mechanisms by which low-risk types can lead to papillomatosis and, occasionally, cancer. The long-term goal of studying the HPV biology is to develop effective targeted antivirals and immunotherapeutics, which will complement current methods of prophylactic disease management.
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32 Richards RM, Lowy DR, Schiller JT, Day PM: Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection. Proc Natl Acad Sci USA 2006;103:1522-1527.
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37 Lazarczyk M, Pons C, Mendoza JA, Cassonnet P, Jacob Y, Favre M: Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses. J Exp Med 2008;205:35-42.
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Harrison P. Nguyen 1 Baylor Plaza Houston, TX 77030 (USA) E- Mail Harrison.p.nguyen@gmail.com
Molecular Biology and Pathogenesis
Ramírez-Fort MK, Khan F, Rady PL, Tyring SK (eds): Human Papillomavirus: Bench to Bedside. Curr Probl Dermatol. Basel, Karger, 2014, vol 45, pp 33-46 (DOI: 10.1159/000355961)
______________________
Viral Oncogenesis
Hung Q. Doan a Marigdalia K. Ramírez-Fort b Peter L. Rady c
a University of Texas Medical Branch, Galveston, Tex., and b Center for Clinical Studies and c University of Texas Medical School, Houston, Tex., USA
______________________
Abstract
Human papillomaviruses (HPVs) are epitheliotropic viruses which cause a variety of lesions at cutaneous and mucosal sites. Lesions range from the benign wart to dysplasia and neoplasia. Importantly, HPV has been shown to be etiological in several malignancies including cervical cancer, other anogenital cancers, oropharyngeal cancers, and cutaneous malignancies in susceptible individuals, causing an estimated 5.2% of virally associated cancers worldwide. HPVs are small, double-stranded DNA viruses of the Papillomaviridae family; to date 150 genotypes have been characterized with approximately one third targeting mucosal sites. Studies of viral oncogenesis have revealed that HPV early genes interact with and modulate mediators of cell growth and cell cycle progression in the host cell. Recent studies have shed light on more novel mechanisms employed by viral oncoproteins including epigenetic modifications, modulating apoptosis pathways, affecting cell morphology and regulating angiogenesis. These recent studies demonstrate that our current understanding is still very limited and will continue to evolve with future research. The purpose of this chapter is to provide a general overview of HPV oncogenesis and to highlight several etiological questions that we hope will be answered by future research.
© 2014 S. Karger AG, Basel
Viruses have been increasingly recognized as causative in several human cancers, with an estimated 12% of human cancers caused by these infectious agents [ 1 ]. Importantly, human papillomavirus (HPV) has been shown to be etiological in several malignancies including cervical cancer, other anogenital cancers, oropharyngeal cancers and nonmelanoma skin cancers in susceptible individuals, causing an estimated 5.2% of virally associated cancers worldwide [ 2 ] . HPVs are small, double-stranded DNA viruses of the Papillomaviridae family. At least 150 genotypes have been characterized with approximately one third targeting mucosal sites [ 3 , 4 ] . Infection with these epitheliotropic viruses most often causes benign proliferations or warts. A large proportion of HPV infections tend to cause self-limited infection and typically clear within 6-12 months [ 5 ] . Although persistent infection occurs in less than 10% of patients, longstanding infection increases the risk for developing precancerous and cancerous lesions [ 6 ] . The specific mechanisms of HPV-associated viral oncogenesis are attributed to the viral proteins which are normally expressed as part of the viral life cycle. These proteins have been termed viral oncoproteins, due to their functional interaction with the intracellular host machinery involved in cell maintenance, cell cycle progression and cell division.
Table 1. Percentage of cancers worldwide attributed to HPV infection, by location
Location
Attributable cases
Percent of all cancers
Cervix
492,800
4.5
Vulva/vagina
16,000
0.2
Penis
10,500
0.1
Oropharynx
6,300
0.1
Mouth
8,200
0.1
All sites
561,200
5.2
Attributable cases are those where HPV was considered the causative agent for the specific cancer and where HPV DNA could be detected in tumor samples. Adapted from Parkin and Bray [ 1 ].
Functional classification of HPV into ‘high-risk’ and ‘low-risk’ genotypes differentiates the cancer transformation properties of these viruses and their propensity to cause virus-associated cancers in humans ( table 1 ). The high-risk genotypes include the α-papillomaviruses HPV-16 and HPV-18 causing approximately 50% of cervical cancers [ 7 ] . Recently, the β-papillomaviruses HPV-5 and -8 were classified as class 2B carcinogens by the International Agency on Research in Cancer Working Group [ 8 ] . This designation supports the findings that HPV-5 and -8 are associated with and possibly etiological in nonmelanoma skin cancers of patients with epidermodysplasia verruciformis (EV), a genetic syndrome associated with an increased risk and prevalence of skin cancers in ultraviolet-exposed areas. While there is a high association between HPV infection and human cancers, it is thought that HPV infection is necessary but not sufficient to cause skin cancers. Moreover, not all HPV-positive lesions progress to malignancies and not all skin cancers contain HPV viral DNA ( table 1 ) [ 7 ].
Common to all Papillomaviridae, the viral genome is composed of approximately 8,000 bp and codes for 8 genes: E1-E7 (for early genes) and L1 and L2 (for late genes). The β-papillomavirus genome does not encode E5. The early genes encode gene products with the E4 protein being a truncation of the E2 protein. Principally the early genes encode proteins involved in replication and maintenance of the viral genome whereas the late genes encode coat proteins necessary for viable virus production [ 2 ].

Fig. 1. Diverse mechanisms employed by papillomaviruses in promoting HPV-mediated oncogenesis. VEGF = Vascular endothelial growth factor; hTERT = human telomerase reverse transcriptase; HIF-1α = hypoxia-inducing factor 1α PLK4 = polo-like kinase 4.
Most of the initial studies on the oncogenic role of HPV infection in the etiology of cervical cancers have come from cell culture and animal studies. These studies found that expression of HPV-16 or HPV-18 genomes was sufficient for organotypic cultures to display oncogenic properties including basilar proliferation, cellular atypia, and increased mitoses [ 9 , 10 ] . Cell cultures that overexpressed viral oncoproteins were sufficient to maintain cellular proliferation; inhibition of these viral oncoproteins led to decreased proliferation in culture [ 11 - 13 ] . Most impressively, transgenic mice globally expressing high-risk HPV-16 E7 oncoprotein developed squamous cell cancers of the skin [ 14 - 17 ] . In these mice, squamous skin cancers developed only after co-treatment with estrogen, indicating that the high-risk E7 oncoprotein participates in but is not sufficient for oncogenesis.
Early studies of HPV viral oncogenes focused largely on high-risk E6 and E7 proteins. Importantly, HPV-16 and -18 E6 was shown to interact with and promote the degradation of cellular p53 [ 18 , 19 ] . High-risk HPV-16 and -18 E7 was also shown to interact with and modulate the retinoblastoma tumor suppressor protein (pRb) [ 20 - 22 ] . These early studies clearly revealed the molecular mechanisms involved in the oncogenesis of high-risk papillomaviruses.

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