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.     

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.     

Indole-3-Carbinol,a Phytochemical Aryl Hydrocarbon Receptor-Ligand,Induces the mRNA Overexpression of UBE2L3 and Cell Proliferation Arrest

Cervical cancer (CC) is one of the most common cancers in women, and is linked to human papillomavirus (HPV) infection. The virus oncoprotein E6 binds to p53, resulting in its degradation and allowing uncontrolled cell proliferation. Meanwhile, the HPV E7 protein maintains host cell differentiation by targeting retinoblastoma tumor suppressor. The host cell can ubiquitinate E6 and E7 through UBE2L3, whose expression depends on the interaction between the aryl hydrocarbon receptor (AhR) with Xenobiotic Responsive Elements (XREs) located in the UBE2L3 gene promoter. In this study, we used cell culture to determine the effect of indole-3-carbinol (I3C) over cellular viability, apoptosis, cell proliferation, and mRNA levels of UBE2L3 and CYP1A1. In addition, patients’ samples were used to determine the mRNA levels of UBE2L3 and CYP1A1 genes. We found that I3C promotes the activation of AhR and decreases cell proliferation, possibly through UBE2L3 mRNA induction, which would result in the ubiquitination of HPV E7. Since there is a strong requirement for selective and cost-effective cancer treatments, natural AhR ligands such as I3C could represent a novel strategy for cancer treatment.     

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.     

Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins

Low-risk type human papillomavirus (HPV) 6 and 11 infection causes recurrent respiratory papillomatosis (RRP) and genital warts. RRP is the most common benign tumor of the larynx in children with frequent relapses. Repeated surgeries are often needed to improve vocal function and prevent life-threatening respiratory obstruction. Currently, there are no effective treatments available to completely eliminate these diseases, largely due to limited knowledge regarding their viral molecular pathogenesis. HPV E6 proteins contribute to cell immortalization by interacting with a variety of cellular proteins, which have been well studied for the high-risk type HPVs related to cancer progression. However, the functions of low-risk HPV E6 proteins are largely unknown. In this study, we report GST-pulldown coupled mass spectrometry analysis with low-risk HPV E6 proteins that identified sterile alpha motif domain containing 9 (SAMD9) as a novel interacting partner. We then confirmed the interaction between HPV-E6 and SAMD9 using co-immunoprecipitation, proximity ligation assay, and confocal immunofluorescence staining. The SAMD9 gene is down-regulated in a variety of neoplasms and deleteriously mutated in normophosphatemic familial tumoral calcinosis. Interestingly, SAMD9 also has antiviral functions against poxvirus. Our study adds to the limited knowledge of the molecular properties of low-risk HPVs and describes new potential functions for the low-risk HPV E6 protein.     

Identification of six putative novel human papillomaviruses (HPV) and characterization of candidate HPV type 87.

Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the candHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.     

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.     

Degradation of p53 by Human Alphapapillomavirus E6 Proteins Shows a Stronger Correlation with Phylogeny than Oncogenicity

Background

Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.

Principal Findings

We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71) were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA) and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.

Conclusions

These data suggest that the alpha-HPV E6 proteins'' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.     

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.     

Protection against human papillomavirus type 16-induced tumors in mice using non-genetically modified lactic acid bacteria displaying E7 antigen at its surface

Human papillomavirus (HPV) is the causative agent of cervical cancer (CxCa) and the most commonly sexually transmitted pathogen worldwide. HPV type 16 (HPV-16) E7 oncoprotein is constitutively produced in CxCa and considered as a good antigen candidate for the development of new therapeutic CxCa vaccines. Here, we report the use of non-genetically modified, E7-expressing lactic acid bacteria (LAB) by using the cell-binding domain from Lactobacillus casei A2 phage lysin as a cell wall anchor. The versatility of this system was validated by investigating E7 stability at the surface of Lactococcus lactis and L. casei, two major species of LAB. Moreover, we demonstrated the successful use of these LAB displaying E7 antigen as a mucosal live vaccine in mice. Altogether, these results show the feasibility of using non-genetically modified LAB for low-cost mucosal immunotherapy against HPV-related CxCa in humans.     

Lactobacillus gasseri LGV03 isolated from the cervico-vagina of HPV-cleared women modulates epithelial innate immune responses and suppresses the growth of HPV-positive human cervical cancer cells

Persistent human papillomavirus (HPV) infections is necessary for the development of cervical cancers. An increasing number of retrospective studies have found the depletion of Lactobacillus microbiota in the cervico-vagina facilitate HPV infection and might be involved in viral persistence and cancer development. However, there have been no reports confirming the immunomodulatory effects of Lactobacillus microbiota isolated from cervico-vaginal samples of HPV clearance in women. Using cervico-vaginal samples from HPV persistent infection and clearance in women, this study investigated the local immune properties in cervical mucosa. As expected, type I interferons, such as IFN-α and IFN-β, and TLR3 globally downregulated in HPV+ persistence group. Luminex cytokine/chemokine panel analysis revealed that L. jannaschii LJV03, L. vaginalis LVV03, L. reuteri LRV03, and L. gasseri LGV03 isolated from cervicovaginal samples of HPV clearance in women altered the host's epithelial immune response, particularly L. gasseri LGV03. Furthermore, L. gasseri LGV03 enhanced the poly (I:C)-induced production of IFN by modulating the IRF3 pathway and attenuating poly (I:C)-induced production of proinflammatory mediators by regulating the NF-κB pathway in Ect1/E6E7 cells, indicating that L. gasseri LGV03 keeps the innate system alert to potential pathogens and reduces the inflammatory effects during persistent pathogen infection. L. gasseri LGV03 also markedly inhibited the proliferation of Ect1/E6E7 cells in a zebrafish xenograft model, which may be attributed to an increased immune response mediated by L. gasseri LGV03.     

Identification of a Novel Human Papillomavirus by Metagenomic Analysis of Samples from Patients with Febrile Respiratory Illness

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.     

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.     

Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.     

White Lupin (Lupinus albus) response to phosphorus stress: evidence for complex regulation of LaSAP1

Proteoid roots are a unique adaptation that allow white lupin (Lupinus albus L. var Ultra) to survive under extreme phosphorus (P) deficient conditions. The cascade of events that signals P-deficiency induced gene expression in proteoid roots remains unknown. Through promoter::GUS analysis we showed that expression of acid phosphatase (LaSAP1) in P-deficient proteoid roots depends on DNA located from ?465 bp to ?345 bp 5′ of the ATG start codon and that the P1BS (PHR1 Binding Site) element, located at ?160 bp, also contributes regulatory control. DNA located within the ?414 bp to ?250 bp region of the LaSAP1 promoter was bound by nuclear proteins isolated from P-sufficient normal roots in electrophoretic mobility shift assays (EMSA), suggesting negative regulation. Competition experiments were performed with unlabeled oligonucleotides to further delineate the region of the LaSAP1 promoter bound by P-sufficient normal root nuclear proteins to a motif spanning ?361 bp to ?346 bp. The promoter motif characterized through EMSA spanning ?361 bp to ?345 bp was used as “bait” in a yeast one-hybrid (Y1H) experiment and 31 putative DNA binding proteins were isolated. Taken together, our results increase understanding of P-deficiency signaling by identifying regulatory regions and putative regulatory proteins for LaSAP1 expression.     

Bap31 is a novel target of the human papillomavirus E5 protein

The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.     

Bovine papillomavirus type 1 3'' early region transformation and plasmid maintenance functions.

Human papillomavirus (HPV) 8 induces skin tumors which are at high risk for malignant conversion. The nucleotide sequence of HPV8 has been determined and compared to sequences of papillomaviruses with different oncogenic potential. The general organization of the HPV8 genome is similar to that of other types. Highly conserved, genus-specific sequences were found in open reading frames (ORFs) E1, E2, and L1. In ORFs E6, E7, and L2, HPV8 is more distantly related, but it was possible to differentiate subgenera in which HPV8 belonged to the HPV1-cottontail rabbit papillomavirus group. Sequences within ORF E4 and part of ORF L2 are rather type specific. HPV8 stands out by several unique features: the considerably reduced size of the noncoding region (397 base pairs), with a seemingly low potential for forming complex secondary structures; a cluster of putative promoter elements in the 3' half of ORF E1; an RNA polymerase III promoter-like sequence close to the C terminus of ORF E2; and of particular interest, the homology between the putative protein encoded by ORF E4 and the Epstein-Barr virus nuclear antigen 2 protein, which may reflect similar mechanisms in virus-mediated transformation.