ICTV Virus Taxonomy Profile: Papillomaviridae.

The Papillomaviridae is a family of small, non-enveloped viruses with double-stranded DNA genomes of 5 748 to 8 607 bp. Their classification is based on pairwise nucleotide sequence identity across the L1 open reading frame. Members of the Papillomaviridae primarily infect mucosal and keratinised epithelia, and have been isolated from fish, reptiles, birds and mammals. Despite a long co-evolutionary history with their hosts, some papillomaviruses are pathogens of their natural host species. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Papillomaviridae, which is available at http://www.ictv.global/report/papillomaviridae.

Recombination of human papillomavirus-16 and host DNA in exfoliated cervical cells: a pilot study of L1 gene methylation and chromosomal integration as biomarkers of carcinogenic progression.

Human papillomavirus-16 DNA replicates in productive infections in circular form, but is found in most carcinomas integrated into the host cell DNA. Because this transition is essential for carcinogenesis, detailed research is desirable and may help to triage patients with abnormal Pap smears. Previous studies addressed the chromosomal integration of HPV-16 DNA in biopsies of tumors by an indirect biomarker, methylation of the viral L1 gene and by reverse ligation polymerase chain reaction (rliPCR). The pilot study reported here asked whether these techniques can be targeted successfully at DNA prepared from exfoliated cervical cells. Abnormal Pap smears of 21 patients that were positive for HPV-16 were analyzed for (i and ii) methylation of the L1 gene after bisulfite modification and PCR amplification by direct sequencing and indirectly in cloned DNA and (iii) recombination with chromosomal DNA by rliPCR. Four of these 21 patients contained highly methylated L1 DNA, which was integrated in three of the samples with sufficient DNA for rliPCR analysis. Seven patients contained sporadically methylated L1 DNA, which was integrated in two and episomal in three samples with sufficient DNA. Ten patients contained only unmethylated DNA, which was episomal in six but possibly integrated in two samples. It is concluded that HPV-16 is found integrated chromosomally in a fraction of precancerous infections, and with higher frequency in methylated than in low or unmethylated samples. Since L1 gene methylation indicates integration, it has the potential to be used as a clinical marker of cancer progression.

Cholinergic signaling through nicotinic acetylcholine receptors stimulates the proliferation of cervical cancer cells: an explanation for the molecular role of tobacco smoking in cervical carcinogenesis?

We have analyzed the expression of mRNAs encoding nicotinic acetylcholine receptors (nAChRs) in CaSki, SiHa and HeLa cell lines, which are derived from two squamous and one adenocarcinoma of the cervix, respectively. We detected with reverse transcription-polymerase chain reaction mRNAs for ten of the 16 nAChR subunits, namely strong signals for alpha-5, alpha-7, alpha-9, beta-1 and epsilon, and weak signals for alpha-4, beta-2, beta-4, gamma and delta. We confirmed the translation of alpha-5 and beta-1, corresponding to the two strongest RNA signals, in SiHa and HeLa cells by Western blotting, and the localization of these proteins to the plasma membrane by immunofluorescence. The beta-1 subunit was detected membrane-associated in normal and neoplastic squamous epithelia of the cervix in situ, but appeared to be absent from the underlying mesenchyme and even from adjacent columnar epithelia. These observations suggest that normal and neoplastic cervical squamous epithelial cells express several combinations of the pentameric nAChRs. We also measured that the proliferation of SiHa and HeLa cells is stimulated by nicotine. This indicates that cholinergic signaling under normal physiological conditions and stimulated by nicotine in tobacco users affects epithelial homeostasis and neoplastic progression at the cervix in a way similar to the known effects on epithelia of the mouth, the airways and the lung. Since tobacco smoking is established as a risk factor in cervical carcinogenesis, and since nicotine and its derivatives become concentrated in cervical mucus, nAChR-dependent signaling is apparently an important molecular cofactor of human papillomavirus-dependent cervical carcinogenesis.

Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation.

Penile carcinomas are frequently associated with high risk human papillomavirus (HPV) types. Because little is known about the molecular biology of this association, we investigated three properties of HPV genomes in penile carcinomas from Brazilian patients: (i) HPV DNA methylation, (ii) junctions between HPV and cellular DNA and (iii) genomic variation. In cervical carcinogenesis, recombination between HPV and chromosomal DNA is frequent and likely necessary for progression, and DNA hypermethylation-specifically of the L1 gene-is a biomarker for cancerous progression. The same mechanisms apparently occur during penile carcinogenesis, because 95 HPV-16 molecules derived from 19 penile lesions had 58% of the CpGs in L1 and 22% in the 5' part of the long control region methylated, more than the percentages found in cervical carcinomas. In addition, 2 out of 3 HPV-18 infections, all present in double infections with HPV-16, showed L1 specific methylation typical of malignant cervical lesions. In 11 out of 15 HPV-16 lesions, we confirmed chromosomal integration by reverse ligation inverted PCR, while 4 samples had concatemeric integrations or episomes. Nine of 17 penile carcinomas contained HPV-16 AA variants, and 8 E variants. As AA variants are relatively rare in Brazilian cohorts of asymptomatic women, the high prevalence in penile carcinomas may indicate a higher risk of progression of AA lesions, as suspected for cervical infections. Our observations of frequent viral DNA methylation, chromosomal integration and the prevalence of high risk variants suggest that HPV-dependent carcinogenesis of the penis and cervix follows similar etiological and epidemiological parameters.

NMR and mass spectrometry studies of putative interactions of cell cycle proteins pRb and CDK6 with cell differentiation proteins MyoD and ID-2.

Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.

The clinical importance of the nomenclature, evolution and taxonomy of human papillomaviruses.

Human papillomaviruses (HPVs) are formally described by isolation of their circular double-stranded DNA genomes and establishment and comparison of the nucleotide sequence of these genomes. Alternatives such as serological diagnosis and maintenance of HPVs in culture are neither clinically useful nor consistently feasible. Novel HPV isolates have traditionally been described as "types". The analysis of specific HPV types is of medical importance, because HPV types typically induce type-specific lesions, i.e. they may be specific for cutaneous or mucosal epithelia, or give rise to benign warts or malignant carcinomas. Recently, it was formally decided that papillomaviruses are a virus family separate from the polyomaviruses. Within the papillomavirus family, closely or remotely related types form species or genera. These formal agreements were important as they brought the taxonomy of papillomaviruses in line with that of other viruses, bacteria and higher organisms, although their impact on medical practice and terminology used in clinical studies is limited. Notably, however, HPV types that are closely related (i.e. form "species") are associated with similar lesions. Confusion of the terms "type" and "subtype" should be avoided, as the latter term refers to some specific but rare taxonomic assemblages. In contrast to many RNA viruses, HPV types evolve very slowly, and diverged since the origin of humans only by about 2%. These divergent isolates are called "variants". HPVs evolved together with humankind and Homo sapiens was never without HPVs, and consequently never without warts and cervical cancer. Variants of the same HPV type may have different pathogenicity and may account for part of the worldwide disparities in the occurrence of genital cancers.

Analysis of the regulation of viral transcription.

Despite the small genomes and number of genes of papillomaviruses, regulation of their transcription is very complex and governed by numerous transcription factors, cis-responsive elements, and epigenetic phenomena. This chapter describes the strategies of how one can approach a systematic analysis of these factors, elements, and mechanisms. From the numerous different techniques useful for studying transcription, we describe in detail three selected protocols of approaches that have been relevant in shaping our knowledge of human papillomavirus transcription. These are DNAse I protection ("footprinting") for location of transcription-factor binding sites, electrophoretic mobility shifts ("gelshifts") for analysis of bound transcription factors, and bisulfite sequencing for analysis of DNA methylation as a prerequisite for epigenetic transcriptional regulation.

Assessment of the HPV DNA methylation status in cervical lesions.

The genomes of the human papillomaviruses HPV-16 and HPV-18 undergo increased CpG methylation during the progression of cervical neoplasia, possibly in response to increased recombination between viral and cellular DNA in high-grade lesions. This behavior makes HPV DNA methylation a useful biomarker of carcinogenic progression of HPV infections. The first step in detecting DNA methylation involves modification by bisulfite, which converts cytosine residues into uracil, but leaves 5-methylcytosine residues unaffected. A combination of this reaction with PCR and DNA sequencing permits to evaluate the methylation status of the sample DNA. This chapter describes the basic protocol to measure HPV-16 and HPV-18 CpG methylation by direct sequencing of the PCR products and discusses the value of modified strategies including DNA cloning, amplification with methylation-specific primers, and real-time PCR with TaqMan probes.

The nicotinic acetylcholine receptor-mediated reciprocal effects of the tobacco nitrosamine NNK and SLURP-1 on human mammary epithelial cells.

Recent research has demonstrated that the nicotinergic signaling network of mammary epithelium can both mediate the physiological control of normal breast epithelial cells (BECs) and exhibit tumor-promoting effects on malignant BECs. Therefore, mammary nicotinic acetylcholine (ACh) receptors (nAChRs) may become a specific target for novel anti-breast cancer therapies. Toward this goal, we investigated the difference in the ACh receptor repertoires between normal and malignant BECs, determined effects of nicotinic ligands on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-dependent activation of ERK1/2 and tumorigenic transformation of MCF10A cells, and characterized reciprocal effects of NNK and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor related protein-1)-1 on the expression of nAChR subunits and several oncogenes and tumor-suppressing genes in BECs. Both the non-malignant MCF10A and malignant MCF7 breast cells expressed α3, α5, α7, α9, α10, ß1, ß2, γ, δ and ε nAChR subunits and M(1), M(3), M(4) and M(5) muscarinic receptor subtypes. The malignancy was associated with expression of α1, α4 and ß4 nAChR subunits and M(2) subtype. Malignant transformation of BECs was also associated with overexpression of α7-, and α9-made nAChRs. NNK upregulated ERK1/2 phosphorylation, stimulated expression of the gene encoding the tumor-promoter HGF, downregulated expression of the tumor suppressor gene CDKN2A, and induced tumorigenic transformation of MCF10A cells. Compared to the canonical nAChR antagonists, SLURP-1 showed the highest ability to abolish the nAChR-mediated effects of NNK in both cell-signaling and cell-transformation assays and reversed many effects of NNK on gene expression. SLURP-1 also markedly upregulated the tumor suppressor genes CDKN2B, RUNX3 and TP73. Altogether, the obtained results provided new insight into the molecular mechanisms of nAChR-mediated oncogenic effects of NNK on BECs and demonstrated the ability to abolish or reverse these effects by SLURP-1.

Gene expression of genital human papillomaviruses and considerations on potential antiviral approaches.

Genital human papillomaviruses (HPVs) are carcinogenic to humans and are associated with most cases of cervical cancer, genital and laryngeal warts, and certain cutaneous neoplastic lesions. Five of the more than 50 known genital HPV types, HPV-6, -11, -16, -18 and -31, have become the models to study gene expression. The comparison of the studies of these five viruses and analyses of the genomic sequences of those genital HPV types that have not been transcriptionally studied make it likely that genital HPVs share most strategies for regulating their transcription. These strategies are quite different from those of unrelated human and animal papillomaviruses. Among these common properties are (i) a specific promoter structure allowing for fine-tuned negative feedback, (ii) a transcriptional enhancer that is specific for epithelial cells, (iii) regulation by progesterone and glucocorticoid hormones, (iv) silencers, whose principal function appears to be transcriptional repression in the basal layer of infected epithelia, (v) specifically positioned nucleosomes that mediate the functions of some enhancer and the silencer factors, (vi) nuclear matrix attachment regions that can, under different conditions, repress or stimulate transcription, and (vii) as yet poorly understood late promoters positioned very remote from the late genes. Most of these properties are controlled by cellular proteins that, due to their simultaneous importance for cellular processes, may not be useful as HPV-specific drug targets. It should be possible, however, to target complex cis-responsive elements unique to these HPV genomes by nucleotide sequence-specific molecules, such as antisense RNA, polyamides and artificial transcription factors. The application of small molecule-based drugs may be restricted to target proteins encoded by the HPV DNA, such as the replication factor E1 and the transcription/replication factor E2.

Past, present, and future of HPV research: highlights from the 19th International Papillomavirus Conference-HPV2001.

This manuscript summarizes the papers presented at the 19th International Papillomavirus Conference, held in Florianopolis, Brazil, September 1-7, 2001, divided in four main areas: Clinical diagnosis and screening, Epidemiology, Biology and Immunology. It provides an overview of what we know about the biology and life cycle of these viruses, their interaction with human and animal hosts, and the diseases that they cause. Highlights derive from the analysis of more than 500 papers presented at the Conference.

Methylation of human papillomavirus 16, 18, 31, and 45 L2 and L1 genes and the cellular DAPK gene: Considerations for use as biomarkers of the progression of cervical neoplasia.

During progression of cervical cancer, human papillomavirus genomes and cellular tumor suppressor genes can become methylated. Toward a better understanding of these biomarkers, we studied 104 samples with HPV16, 18, 31, and 45 representing five pathological categories from asymptomatic infection to cancer. We grouped all samples by HPV type and pathology and measured the overall methylation of informative amplicons of HPV late genes and the cellular DAPK gene. Methylation of all four HPV types as well as of the DAPK gene is lowest in asymptomatic infection and increases successively in all four pathological categories during progression to cancer. 27 out of 28 cancer samples showed methylation both in the L2/L1 genes as well as in DAPK, but a much lower fraction in all other pathological categories. We discuss the problem to develop diagnostic tests based on complex methylation patterns that make it difficult to classify amplicons as "methylated" or "unmethylated".

Taxonomy and phylogeny of papillomaviruses: an overview and recent developments.

For more than 30 years, papillomaviruses are standing in the center of medical and molecular interest as they cause several important cancers in humans. Research of the sheer unlimited number of different papillomavirus genomes, their host specificity and slow mutation rate is an important a branch of these efforts and has led to fascinating insight into the phylogeny of a virus family that can be traced back for several 100 million years.

Regulatory elements in the viral genome.

The small DNA genomes of papillomaviruses contain a surprisingly large number of regulatory or cis-responsive elements, which regulate replication and transcription of the virus, and control details like specificity for certain epithelial cells, specificity for layers in squamous epithelia, feedback mechanisms and coupling between host cell physiology and virus biology. Most of these elements occur in the long control region, while others are located elsewhere in the genome. Many papillomaviruses show a similar composition of cis-responsive elements, although these are scattered and do not occur as long segments of sequence similarity. This review summarizes our knowledge of the regulatory elements in several well-studied Alphapapillomavirus types, and indicates some similarities to other papillomavirus genera, whose properties are yet poorly understood.

Reciprocal effects of NNK and SLURP-1 on oncogene expression in target epithelial cells.

AIMS: To elucidate how the nicotinic acetylcholine receptors expressed on bronchial and oral epithelial cells targeted by the tobacco nitrosamine (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) (NNK) facilitate carcinogenic transformation. MAIN METHODS: Since NNK-dependent transformation can be abolished by the nicotinergic secreted mammalian Ly-6/urokinase plasminogen activator receptor related protein-1 (SLURP-1), we compared effects of NNK and recombinant (r)SLURP-1 on the expression of genes related to tumorigenesis in human immortalized bronchial and oral epithelial cell lines BEP2D and Het-1A, respectively. KEY FINDINGS: NNK stimulated expression of oncogenic genes, including MYB and PIK3CA in BEP2D, ETS1, NRAS and SRC in Het-1A, and AKT1, KIT and RB1 in both cell types, which could be abolished in the presence of rSLURP-1. Other cancer-related genes whose upregulation by NNK was abolishable by rSLURP-1 were the growth factors EGF in BEP2D cells and HGF in Het-1A cells, and the transcription factors CDKN2A and STAT3 (Het-1A only). NNK also upregulated the anti-apoptotic BCL2 (Het-1A) and downregulated the pro-apoptotic TNF (Het-1A), BAX and CASP8 (BEP2D), all of which could be abolished, in part, by rSLURP-1. NNK decreased expression of the CTNNB1 gene encoding the intercellular adhesion molecule ß-catenin (BEP2D), as well as tumor suppressors CDKN3 and FOXD3 in BEP2D cells and SERPINB5 in Het-1A cells. These pro-oncogenic effects of NNK were abolished by rSLURP-1 that also upregulated RUNX3. SIGNIFICANCE: The obtained results identified target genes for both NNK and SLURP-1 and shed light on the molecular mechanism of their reciprocal effects on tumorigenic transformation of bronchial and oral epithelial cells.

New associations of the genetic polymorphisms in nicotinic receptor genes with the risk of lung cancer.

AIMS: Previous studies revealed association of lung cancer risk with single nucleotide polymorphisms (SNPs) in chromosome 15q25 region containing CHRNA5-CHRNA3-CHRNB4 nicotinic acetylcholine receptor (nAChR) subunit gene cluster. The genetic variations in other lung nAChRs remained unknown. In this study, we perform case-control analysis of CHRNA9 and CHRNA3 genes using 340 non-small cell lung cancer cases and 435 controls. MAIN METHODS: All exons, 3'UTR, intron 1 and parts of other introns surrounding exons 2-5 of CHRNA9 gene as well as exons 2, 3 of CHRNA3 gene and parts of surrounding intronic regions were sequenced. The study was controlled for gender, age and ethnicity related differences. Each SNP in analyzed groups was assessed by allele frequency, genotype distribution and haplotype analysis. KEY FINDINGS: The case-control analysis revealed that an increased risk is associated with two SNPs in CHRNA9, rs56159866 and rs6819385, and one in CHRNA3, rs8040868. The risk was reduced for three SNPs in CHRNA9, rs55998310, rs56291234, and newly discovered rs182073550, and also in carriers of the haplotype NP_060051.2 containing ancestral N442 variant of α9. SIGNIFICANCE: The nonsynonymous substitutions can produce receptors exhibiting unique ligand-binding and downstream signaling characteristics, synonymous as well all intronic SNPs may affect protein production at the transcriptional and/or translational levels, or just manifest association with cancer by genetic linkage to other alleles. Elucidation of the mechanisms by which individual genetic variations in α9 affect predisposition to lung cancer may lead to development of personalized approaches to cancer prevention and treatment as well as protection against tobacco consumption.

Muscarinic cholinergic signaling in cervical cancer cells affects cell motility via ERK1/2 signaling.

AIMS: The etiology of cervical cancer depends primarily on infection with human papillomaviruses, but tobacco smoking is the most important behavioral risk factor for this cancer. Therefore, we have previously confirmed involvement of nicotinic acetylcholine receptors (nAChRs) in cervical cancer biology. In order to comprehensively evaluate the role of cholinergic signaling in cervical cells, we have addressed additional participation of muscarinic acetylcholine receptors (mAChRs). MAIN METHODS: We have studied the expression of mAChRs and cholinergic system components by reverse transcription PCR and Western blots, the motility of cervical cancer cells in cell culture, and the signaling from mAChRs via the ERK1/2 signaling pathway. KEY FINDINGS: The cervical cancer cells HeLa, SiHa and CaSki express four of the five mAChRs, M1, M3, M4, and M5, and the acetylcholine (ACh) synthesizing and degrading enzymes choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), and vesicular ACh transporter (VAChT). mAChR-dependent signaling induces cervical cell motility, which requires ERK1/2 activation, and could be abrogated by mAChR antagonists. SIGNIFICANCE: The epidemiological finding that tobacco smoke raises the prevalence of cervical cancer has led to analysis of the cholinergic signaling in cervical biology and carcinogenesis. Cervical cancer cells express several nAChRs and mAChRs, whose activation leads to changes of cellular properties such as increased motility and proliferation that favor a carcinogenic phenotype. The signaling involves intracellular phosphorylation cascades including ERK1/2.

Association of single nucleotide polymorphisms of nicotinic acetylcholine receptor subunits with cervical neoplasia.

AIMS: Cholinergic signaling, particularly in response to non-physiological ligands like nicotine, stimulates carcinogenesis of a variety of tissue types including epithelia of the cervix uteri. Cholinergic signaling is mediated by nicotinic acetylcholine receptors (nAChRs), which are pentamers formed by subsets of 16 nAChR subunits. Recent literature suggests that single nucleotide polymorphisms (SNPs) of some of these subunits, notably alpha5, are risk factors for developing lung cancer in smokers as well as in non-smokers. MAIN METHODS: We have studied the prevalence of four SNPs in the alpha5, alpha9, and beta1 subunits, which are expressed in cervical cells, in 456 patients with cervical cancers, precursor lesions, and healthy controls from two cohorts in Mexico. KEY FINDINGS: A SNP in the alpha9 subunit, the G allele of rs10009228 (alpha9, A>G) shows a significant trend in the combined cohort, indicating that this allele constitutes a risk factor for neoplastic progression. The A allele of the SNP rs16969968 (alpha5, G>A), which correlates with the development of lung cancer, shows a non-significant trend to be associated with cervical lesions. Two other SNPs, rs55633891 (alpha9, C>T) and rs17856697 (beta1, A>G), did not exhibit a significant trend. SIGNIFICANCE: Our study points to a potential risk factor of cervical carcinogenesis with importance for DNA diagnosis and as a target for intervention.

Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments.

We present an expansion of the classification of the family Papillomaviridae, which now contains 29 genera formed by 189 papillomavirus (PV) types isolated from humans (120 types), non-human mammals, birds and reptiles (64, 3 and 2 types, respectively). To accommodate the number of PV genera exceeding the Greek alphabet, the prefix "dyo" is used, continuing after the Omega-PVs with Dyodelta-PVs. The current set of human PVs is contained within five genera, whereas mammalian, avian and reptile PVs are contained within 20, 3 and 1 genera, respectively. We propose standardizations to the names of a number of animal PVs. As prerequisite for a coherent nomenclature of animal PVs, we propose founding a reference center for animal PVs. We discuss that based on emerging species concepts derived from genome sequences, PV types could be promoted to the taxonomic level of species, but we do not recommend implementing this change at the current time.

High-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays.

We have developed microarrays with all eight proteins encoded by 13 different human papillomavirus types associated with anogenital cancer (HPV-16, -18, -31, -33, -35, -45, and -53), genital warts (HPV-6 and -11), or skin lesions (HPV-1, -2, -4, and -5). We analyzed the seroprevalence of antibodies in 546 patients, which had either cervical carcinomas, or precursor lesions, or which were asymptomatic. All patient groups contained sera ranging from high reactivity against multiple HPV proteins to low or no reactivity. Computational analyses showed the E7 proteins of carcinogenic HPV types as significantly more reactive in cancer patients compared to asymptomatic individuals and discriminating between cancer and HSIL or LSIL patients. Antibodies against E4 and E5 had the highest seroprevalence but did not exhibit differential reactivity relative to pathology. Our study introduces a new approach to future evaluation of the overall antigenicity of HPV proteins and cross-reaction between homologous proteins.