Evolution and taxonomic classification of human papillomavirus 16 (HPV16)-related variant genomes: HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67

Background

Human papillomavirus 16 (HPV16) species group (alpha-9) of the Alphapapillomavirus genus contains HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. These HPVs account for 75% of invasive cervical cancers worldwide. Viral variants of these HPVs differ in evolutionary history and pathogenicity. Moreover, a comprehensive nomenclature system for HPV variants is lacking, limiting comparisons between studies.

Methods

DNA from cervical samples previously characterized for HPV type were obtained from multiple geographic regions to screen for novel variants. The complete 8 kb genomes of 120 variants representing the major and minor lineages of the HPV16-related alpha-9 HPV types were sequenced to capture maximum viral heterogeneity. Viral evolution was characterized by constructing phylogenic trees based on complete genomes using multiple algorithms. Maximal and viral region specific divergence was calculated by global and pairwise alignments. Variant lineages were classified and named using an alphanumeric system; the prototype genome was assigned to the A lineage for all types.

Results

The range of genome-genome sequence heterogeneity varied from 0.6% for HPV35 to 2.2% for HPV52 and included 1.4% for HPV31, 1.1% for HPV33, 1.7% for HPV58 and 1.1% for HPV67. Nucleotide differences of approximately 1.0% - 10.0% and 0.5%–1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Each gene/region differs in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) /noncoding region 2 (NCR2) >upstream regulatory region (URR)> E6/E7 > E2/L2 > E1/L1.

Conclusions

These data define maximum viral genomic heterogeneity of HPV16-related alpha-9 HPV variants. The proposed nomenclature system facilitates the comparison of variants across epidemiological studies. Sequence diversity and phylogenies of this clinically important group of HPVs provides the basis for further studies of discrete viral evolution, epidemiology, pathogenesis and preventative/therapeutic interventions.     

Evolution and Taxonomic Classification of Alphapapillomavirus 7 Complete Genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70

Background

The species Alphapapillomavirus 7 (alpha-7) contains human papillomavirus genotypes that account for 15% of invasive cervical cancers and are disproportionately associated with adenocarcinoma of the cervix. Complete genome analyses enable identification and nomenclature of variant lineages and sublineages.

Methods

The URR/E6 region was sequenced to screen for novel variants of HPV18, 39, 45, 59, 68, 70, 85 and 97 from 1147 cervical samples obtained from multiple geographic regions that had previously been shown to contain an alpha-7 HPV isolate. To study viral heterogeneity, the complete 8 kb genome of 128 isolates, including 109 sequenced for this analysis, were annotated and analyzed. Viral evolution was characterized by constructing phylogenic trees using maximum-likelihood and Bayesian algorithms. Global and pairwise alignments were used to calculate total and ORF/region nucleotide differences; lineages and sublineages were assigned using an alphanumeric system. The prototype genome was assigned to the A lineage or A1 sublineage.

Results

The genomic diversity of alpha-7 HPV types ranged from 1.1% to 6.7% nucleotide sequence differences; the extent of genome-genome pairwise intratype heterogeneity was 1.1% for HPV39, 1.3% for HPV59, 1.5% for HPV45, 1.6% for HPV70, 2.1% for HPV18, and 6.7% for HPV68. ME180 (previously a subtype of HPV68) was designated as the representative genome for HPV68 sublineage C1. Each ORF/region differed in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) / noncoding region 2 (NCR2) > upstream regulatory region (URR) > E6 / E7 > E2 / L2 > E1 / L1.

Conclusions

These data provide estimates of the maximum viral genomic heterogeneity of alpha-7 HPV type variants. The proposed taxonomic system facilitates the comparison of variants across epidemiological and molecular studies. Sequence diversity, geographic distribution and phylogenetic topology of this clinically important group of HPVs suggest an independent evolutionary history for each type.     

Characterization of Two Novel Gammapapillomaviruses,HPV179 and HPV184, Isolated from Common Warts of a Renal-Transplant Recipient

Gammapapillomavirus (Gamma-PV) is a diverse and rapidly expanding PV-genus, currently consisting of 76 fully characterized human papillomavirus (HPV) types. In this study, DNA genomes of two novel HPV types, HPV179 and HPV184, obtained from two distinct facial verrucae vulgares specimens of a 64 year-old renal-transplant recipient, were fully cloned, sequenced and characterized. HPV179 and HPV184 genomes comprise 7,228-bp and 7,324-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2); the non-coding region is typically positioned between L1 and E6 genes. Phylogenetic analysis of the L1 nucleotide sequence placed both novel types within the Gamma-PV genus: HPV179 was classified as a novel member of species Gamma-15, additionally containing HPV135 and HPV146, while HPV184 was classified as a single member of a novel species Gamma-25. HPV179 and HPV184 type-specific quantitative real-time PCRs were further developed and used in combination with human beta-globin gene quantitative real-time PCR to determine the prevalence and viral load of the novel types in the patient’s facial warts and several follow-up skin specimens, and in a representative collection, a total of 569 samples, of HPV-associated benign and malignant neoplasms, hair follicles and anal and oral mucosa specimens obtained from immunocompetent individuals. HPV179 and HPV184 viral loads in patients’ facial warts were estimated to be 2,463 and 3,200 genome copies per single cell, respectively, suggesting their active role in the development of common warts in organ-transplant recipients. In addition, in this particular patient, both novel types had established a persistent infection of the skin for more than four years. Among immunocompetent individuals, HPV179 was further detected in low-copy numbers in a few skin specimens, indicating its cutaneous tissue tropism, while HPV184 was further detected in low-copy numbers in one mucosal and a few skin specimens, suggesting its dual tissue tropism.     

The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb.     

Prevalence of human papillomavirus (HPV) type 16 variants and rare HPV types in the central Amazon region

Infection by human papillomavirus (HPV) is one of the primary causes of mortality by cancer in northern Brazil. Sexually active women from Manaus, Amazonas, without cytological alterations and women with pre-malignant and malignant cytological alterations were examined for HPV virus, identified via PCR and sequencing. The target region for this study was part of the L1 capsid gene of HPV. Twenty-three samples that were PCR-positive were sequenced. Analysis of 336 bp demonstrated a high incidence of high-risk HPV types in the population of Manaus, identified as HPVs 16, 33, 58, 66, 68. HPV type 16 was the most prevalent, presenting two variants similar to the Asian-American (AA) and East-Asian type (As) variants. A rare HPV type 13 related to "Heck's disease" was also detected. This preliminary provides important information about the HPV circulating in Amazonas State.     

Genetic diversity and phylogenetic analysis of HPV 16 & 18 variants isolated from cervical specimens of women in Saudi Arabia

Human papillomavirus (HPV) are well known to be associated with the development of cervical cancer. HPV16 and HPV 18 are known as high-risk types and reported to be predominantly associated with cervical cancer. The prevalence and genetic diversity of HPV have been well documented globally but, in the Kingdom of Saudi Arabia, data on HPV genetic diversity are lacking. In this study, we have analyzed the genetic diversity of both HPV16 and HPV18 based on their L1 gene sequence because L1 gene is a major capsid protein gene and has been utilized to develop a prophylactic vaccine. In January 2011–2012, a total of forty samples from cervical specimens of women in Saudi Arabia were collected. The association of HPV16, HPV18 was detected by polymerase chain reaction, sequenced and submitted to GenBank. The sequences identity matrix and the phylogenetic relationship were analyzed with selected HPVs. The highest sequence identity (99.5%) for HPV16 and (99.3%) for HPV was observed with selected HPVs. The phylogenetic analysis results showed that HPVs from Saudi Arabia formed a closed cluster with African, Asian, East Asian as well as American HPVs distributed into multiple linages from various geographical locations. The results provided the valuable information about genetic diversity, but there is an urgent need to generate full genome sequence information which will provide a clearer picture of the genetic diversity and evolution of HPVs in Saudi Arabia. In conclusion, the generated data will be highly beneficial for developing molecular diagnostic tools, analyzing and correlating the epidemiological data to determine the risk of cervical cancer and finally to develop a vaccine for Saudi Arabian population.     

Identification of a Novel Human Papillomavirus,Type HPV199, Isolated from a Nasopharynx and Anal Canal,and Complete Genomic Characterization of Papillomavirus Species Gamma-12

The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.     

高危型HPV 复制相关蛋白在宫颈组织中的研究

人乳头瘤病毒(Human Papillomavirus,HPV)复制机制的研究,对复制与宫颈病变的相关性可提供重要的依据。HPV是有衣壳包裹的小型环状双链DNA病毒。其基因组可分早期及晚期蛋白编码区和一个调控区。高危型HPV通过E1、E2蛋白及Ori序列启动复制。高危型HPV在宫颈上皮细胞中的复制是分化依赖型的,在高危型HPV感染的成熟的宫颈表皮细胞中,HPV E7蛋白使细胞再次进入增殖分裂期,HPV-DNA得以复制,但同时E7蛋白亦会诱发宿主细胞染色体不稳定,增加癌变风险。由此推理,高危型HPV的复制与宫颈癌的发病有一定相关性。目前有学者对高危型HPV的复制机制,复制与致癌的关系方面正开展相关研究。     

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.     

HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.

The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.     

Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis

Human papillomavirus (HPV) is small, double-stranded DNA virus that infects mucosal and cutaneous epithelial tissue. HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or lowrisk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign warts and lesions. The expression of E6 and E7 is normally controlled during the normal viral life cycle when viral DNA replicates extrachromosomally. HPV E6 and E7 oncoproteins are overexpressed when the viral genome integrates into the host DNA. Deregulated overexpression of E6 and E7 oncoproteins can cause several changes in cellular pathways and functions leading to malignant transformation of cells and tumorigenesis. In this review, we focus on several cellular mechanisms and pathways that are altered in the presence of E6 and E7, the target proteins of E6 and E7 inside the host cell and how they contribute to the development of the transformed phenotype.     

DNA sequence and genome organization of genital human papillomavirus type 6b.

The complete nucleotide sequence of the circular double-stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.     

Genomic instability of the host cell induced by the human papillomavirus replication machinery

Development of invasive cervical cancer upon infection by 'high-risk' human papillomavirus (HPV) in humans is a stepwise process in which some of the initially episomal 'high-risk' type of HPVs (HR-HPVs) integrate randomly into the host cell genome. We show that HPV replication proteins E1 and E2 are capable of inducing overamplification of the genomic locus where HPV origin has been integrated. Clonal analysis of the cells in which the replication from integrated HPV origin was induced showed excision, rearrangement and de novo integration of the HPV containing and flanking cellular sequences. These data suggest that papillomavirus replication machinery is capable of inducing genomic changes of the host cell that may facilitate the formation of the HPV-dependent cancer cell.     

E4 Antibodies Facilitate Detection and Type-Assignment of Active HPV Infection in Cervical Disease

High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58) were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247)) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination.     

Structures of a human papillomavirus (HPV) E6 polypeptide bound to MAGUK proteins: mechanisms of targeting tumor suppressors by a high-risk HPV oncoprotein

Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.     

The promoter of a novel human papillomavirus (HPV77) associated with skin cancer displays UV responsiveness, which is mediated through a consensus p53 binding sequence.

An aetiological role has been proposed for human papillomavirus (HPV) in skin carcinogenesis within the immunosuppressed patient population. To examine this possibility, we have focused on an HPV type that, to date, has been identified only in the cutaneous lesions of renal transplant recipients despite a high degree of sequence homology with other HPVs commonly found in warts in the general population. We report that the non-coding region of this virus, HPV type 77, contains a consensus binding site for the tumour suppressor protein p53, and we show by gel-retardation analysis that this sequence does indeed bind p53. Furthermore, using reporter gene assays, we demonstrate that HPV77 promoter activity is stimulated by UV radiation and that this response is mediated through the p53 binding site. This is the first report of a p53-dependent positive response element within a viral genome. Our results suggest a possible novel mechanism by which specific types of HPV might act as cofactors with UV radiation in cutaneous transformation.