Nucleotide sequence and genome organization of human papillomavirus type 5

Human papillomavirus (HPV) type 5 is associated with benign and malignant lesions of the disease epidermodysplasia verruciformis (EV). Because of the strong correlation between the presence of HPV-5 and malignant progression in these patients, we have elucidated the nucleotide sequence of the HPV-5 genome. The size of the HPV-5 genome is 7746 nucleotides and its organization is similar to that of other papillomaviruses. The HPV-5 genome exhibits extensive sequence homology with another EV-associated papillomavirus, HPV-8, although HPV-5 appears to contain at least one additional open reading frame.     

A subtype of human papillomavirus 5 (HPV-5b) and its subgenomic segment amplified in a carcinoma: nucleotide sequences and genomic organizations.

A subtype of human papillomavirus 5 (HPV-5b) is closely associated with carcinomas in the disease epidermodysplasia verruciformis (EV). The complete genome was cloned from virus particles in benign lesions of a patient with EV and sequenced: it was 7779 nucleotides long and consisted of six open reading frames (ORFs) (E6, E7, E1, E2, E4, and E5) in the early region, three ORFs (L2, L3, and L1) in the late region, and a noncoding region, all existing on one DNA strand. The 40% segment of the HPV-5b genome specifically amplified in carcinomas was cloned from a primary carcinoma of the same EV patient and sequenced: it was 3143 nucleotides long and corresponded to a segment of the original HPV-5b genome containing the entire sequences of E6, E7, and the noncoding region and portions of E1 and L1. Compared to the whole genomic DNA, no mutations were detected in this probable malignancy-associated viral subgenomic segment cloned from carcinoma. These results suggest that amplification of the viral segment containing E6, E7, and the noncoding region may play a role in the malignant conversion of HPV-5b-infected benign lesions and that mutations in these genes or regions are not necessarily required.     

Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene E6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3' part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs "CCAAC" (nt 57-73) and M29 (nt 84-112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P(175). In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.     

A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan

There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.     

Differences in transforming activity and coded amino acid sequence among E6 genes of several papillomaviruses associated with epidermodysplasia verruciformis.

Using the polymerase chain reaction technique, we cloned and sequenced DNA fragments containing the E6 genes of the epidermodysplasia verruciformis (EV)-associated HPVs 5, 8, 14, 20, 21, 25, and 47, of which only the sequences of HPVs 5, 8, and 47 have previously been reported. Based on the deduced amino acid sequence homology (57.3 to 83.0%), these HPVs could be divided into two clusters: HPVs 5, 8, and 47, and HPVs 14, 20, 21, and 25. The E6 genes of three HPVs from each cluster were examined for transforming activity toward a cultured rat fibroblast cell line, 3Y1, using the retrovirus-mediated gene transfer technique, and all were found to induce morphological transformation. However, the E6 genes of the first cluster were more potent than those of the second. Since HPVs 5 and 8 are the most frequently found HPVs in malignant lesions of EV patients, the observed in vitro transforming activities of the E6 genes may reflect their oncogenic potential in humans.     

Structural analysis of human papillomavirus type 6c isolates from condyloma acuminatum and juvenile-onset and adult-onset laryngeal papillomata

The human papillomavirus type 6c (HPV-6c) genome was molecularly cloned from biopsy specimens of a juvenile-onset and an adult-onset respiratory-tract papillomata and a condyloma acuminatum of the cervix. To determine if the genital-tract isolate and respiratory-tract isolates contain divergent sequences that may account for a difference in tissue trophism or for a difference in the age of onset of the disease, fine-structure mapping, heteroduplex analysis by electron microscopy, and nucleotide sequencing were used to examine the sequence relationship among these HPV-6c isolates. No differences were found in the digestion among these HPV-6c isolates. No differences were found in the digestion patterns with 23 restriction enzymes. Heteroduplex analysis among the three genomes demonstrated that they were colinear without apparent deletions or rearrangements and had greater than 90% sequence identity. In heteroduplex analyses with a different subtype (HPV-6e) that was molecularly cloned from a genital wart, the genomes were colinear with greater than 90% sequence identity over 90% of their length. The most divergent region had 75–80% sequence identity and was localized to the part of the genome containing the E5a and E5b open reading frames (ORFs). Comparison of the sequence of 1430 nucleotides in this region for two of the HPV-6c isolates did not identify any differences between them. Comparison with the published sequences of HPV-6b identified deletions/insertions and base changes with approximately 75% sequence identity, and comparison with HPV-11 identified only six base changes. Conservation of sequences in the E4-E5 region and similarity in the restriction enzyme maps demonstrated that HPV-6c and HPV-11 are independent isolates of the same HPV-6 subtype.     

Detection by antibody probes of human papillomavirus type 6 E5 proteins in respiratory papillomata

We have demonstrated the expression of proteins arising from the E5a and E5b open reading frames (ORFs) of human papillomavirus type 6c (HPV-6c) in respiratory tract papillomata. Recombinant plasmids were constructed to express the ORFs in the bacterial vectors pATH and pRIT2T. Fusion proteins were purified and injected into rabbits to produce polyclonal antibodies. Characterized antibodies generated against these fusion proteins were used in immunoperoxidase assays to identify the presence and distribution of HPV-6 E5 proteins in biopsy specimens of respiratory tract papillomata. The results showed that the E5a and E5b proteins were distributed throughout the thickness of the epithelium in the papillomata but not in the basal layer. The proteins were found in nuclei and in the cytoplasm of koilocytotic cells. Positive reactivity with a similar distribution in the epithelium and subcellular location was obtained in papillomata induced by other HPV-6 subtypes. This cross-reactivity was not unexpected, since nucleotide and amino acid (aa) sequence comparisons between HPV-6c and -6e demonstrated 79% sequence identity with 15 aa substitutions in the 91 aa of E5a. The E5b ORF of HPV-6c has the potential to encode a protein of 74 aa that differed at 28 positions compared with the 72 aa of HPV-6e.     

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.     

Molecular Cloning and Nucleotide Sequence Analysis of a Novel Human Papillomavirus (Type 82) Associated with Vaginal Intraepithelial Neoplasia

The genome of a novel human papillomavirus (HPV-82) was cloned from a vaginal intraepithelial neoplasia grade I. In our series of 291 biopsy specimens, HPV-82 was identified in one case each of cervical intraepithelial neoplasia grade II and grade III by blot hybridization. The histological localization of HPV-82 DNA in the three lesions was confirmed by in situ hybridization. The results indicated that HPV-82 is an etiologic agent for vaginal and cervical intraepithelial neoplasia. By nucleotide sequence similarity of L1 open reading frame (ORF), HPV-82 was closely related to HPV-26, -51, and -69. To know the precise relationship between the HPVs, we determined the complete sequence of HPV-82, as well as that of HPV-69. Sequencing revealed that the four HPVs had no initiation codon in the E5 ORF and had extensive nucleotide sequence similarities in all ORFs. In addition, they exhibited unique frame position patterns for ORFs, different from those of the other genital HPVs.     

Echovirus 5: infectious transcripts and complete nucleotide sequence from uncloned cDNA

Echovirus 5 (EV5) may be isolated from various neurological and exanthematic diseases. To determine the relationship of EV5 to other enteroviruses and for studies of its interactions with the target cell, the complete nucleotide sequence of EV5 was determined. Three overlapping fragments, collectively representing the complete genome, were amplified with RT-PCR and sequenced. Analysis of the EV5 sequence revealed a typical enterovirus-like organization of the genome. To verify that the cDNA generated sequence was derived from infectious viruses, complete EV5 genomes were amplified in one amplicon by long distance PCR. Transfection of in vitro transcribed RNA from these amplicons into cell cultures resulted in replicating EV5. Comparison of the overall nucleotide and amino acid sequences demonstrates that EV5 can be regarded as a coxsackievirus B-like enterovirus. Variable sequences between EV5 and the well characterized coxsackievirus B3 (CVB3) are for the most part observed for amino acid residues that correspond to exposed sequences in the CVB3 capsid. This observation indicates that the reported EV5 strain recently diverged from group B coxsackieviruses.     

Molecular cloning and nucleotide sequence analysis of a novel human papillomavirus (Type 82) associated with vaginal intraepithelial neoplasia

The genome of a novel human papillomavirus (HPV-82) was cloned from a vaginal intraepithelial neoplasia grade I. In our series of 291 biopsy specimens, HPV-82 was identified in one case each of cervical intraepithelial neoplasia grade II and grade III by blot hybridization. The histological localization of HPV-82 DNA in the three lesions was confirmed by in situ hybridization. The results indicated that HPV-82 is an etiologic agent for vaginal and cervical intraepithelial neoplasia. By nucleotide sequence similarity of L1 open reading frame (ORF), HPV-82 was closely related to HPV-26, -51, and -69. To know the precise relationship between the HPVs, we determined the complete sequence of HPV-82, as well as that of HPV-69. Sequencing revealed that the four HPVs had no initiation codon in the E5 ORF and had extensive nucleotide sequence similarities in all ORFs. In addition, they exhibited unique frame position patterns for ORFs, different from those of the other genital HPVs.     

Genetic heterogeneity among human papillomaviruses (HPV) associated with epidermodysplasia verruciformis: evidence for multiple allelic forms of HPV5 and HPV8 E6 genes.

In order to get some insight into modifications of human papillomavirus (HPV) genomes which could play a role in tumor progression in epidermodysplasia verruciformis (EV), we studied three EV patients infected by HPV5 and one by HPV8, with cancers containing mostly or only episomal viral genomes with a deletion. The mutants were compared with the full-length genomes present in the benign lesions of each patient. Deletions affected the L1 and/or L2 open reading frames (ORFs), and extended in the 5' end of the long control region in two cancers. The isolates studied showed a polymorphism of restriction endonuclease cleavage sites and variations in the nucleotide sequence of the E6 ORF and the regions flanking the deletions. However, except for one patient infected by two distinct HPV5 variants, no difference was observed in the nucleotide sequence of isolates cloned from the benign lesions and the cancer of the same patient. This may suggest that point mutations are not involved in tumor progression. Comparison of nucleotide sequence data revealed an unexpectedly high number of nucleotide substitutions among the four HPV5 variants and the HPV8 variant, as compared with HPV5 and HPV8 published sequences. Changes involved 49 of the 457 nucleotides of HPV5 E6 ORF and 14 of the 465 nucleotides of HPV8 E6 ORF. This corresponds to amino acid substitutions affecting 17 of the 157 amino acids of HPV5 E6 proteins and 7 of the 155 amino acids of HPV8 E6 proteins. Half of the substitutions represent nonconservative changes. The variants showing the highest degree of sequence variation were detected in additional EV patients by PCR. This points to the existence of a set of HPV5 and HPV8 stable variants, encoding for multiple allelic forms of the transforming E6 gene.     

Nucleotide sequence of human papillomavirus (HPV) type 41: an unusual HPV type without a typical E2 binding site consensus sequence.

The complete nucleotide sequence of human papillomavirus type 41 (HPV-41) has been determined. HPV-41 was originally isolated from a facial wart, but its DNA has subsequently been detected in some skin carcinomas and premalignant keratoses (Grimmel et al., Int. J. Cancer, 1988, 41, 5-9; de Villiers, Grimmel and Neumann, unpublished results). The analysis of the cloned HPV-41 nucleic acid reveals that its genome organisation is characteristic as for other papillomavirus types. Yet, the analysis indicates at the same time that this virus is most distantly related to all other types of human-pathogenic papillomaviruses sequenced thus far and appears to identify HPV-41 as the first member of a new subgroup of HPV. The overall nucleotide homology to other sequenced HPV types is below 50%. The closest other HPV type is represented by HPV-18, sharing 49% identical nucleotides. The typical E2 binding sequence ACCN6GGT, found in all papillomaviruses analyzed to date, does not occur in the URR of the HPV-41 genome. Modified E2 binding sequences, as described for BPV 1 (Li et al., Genes Dev. 1989, 3, 510-526), are located in the domain proximal to the E6 ORF. These are ACCN6GTT, AACN6GGT and the two perfect palindromic sequences AACGAATTCGTT.     

Human papillomavirus type 17 DNA in skin carcinoma tissue of a patient with epidermodysplasia verruciformis

Epidermodysplasia verruciformis (EV) is a serious skin disease caused by certain types of human papillomavirus (HPV), because the flat wart-like lesions of EV very frequently change to malignant squamous cell carcinoma. The relation between HPV and skin carcinoma was examined by studies on an EV patient who had a squamous cell carcinoma. HPV-17 was isolated from EV lesions of this patient. With HPV-17 DNA as a probe, cellular DNA prepared from the carcinoma tissue was analyzed by Southern blot hybridization. Results showed that cells contained about 100 copies of monomeric and oligomeric extrachromosomal HPV DNA. These results suggest that HPV-17 is involved in skin carcinogenesis in EV.     

Detection of HPV-2 and identification of novel mutations by whole genome sequencing from biopsies of two patients with multiple cutaneous horns.

BACKGROUND: Human papillomavirus-2 (HPV-2) is generally associated with common warts. The association of cutaneous horns with HPV-2 infection has never been reported. OBJECTIVES: To identify the papillomavirus (PV) type(s) involved in cutaneous horns and analysis the genomes of these viruses. STUDY DESIGN: We screened biopsies from two patients with multiple cutaneous horns using PV type-specific PCR assays, and sequenced the whole genomes of the viruses by a PCR-by-PCR strategy. Sequence comparison with the reference genome and its closely related PVs in the same phylogenetic group was performed to identify sequence variation across the genome(s) of newly detected PV(s). RESULTS: Two strains of HPV-2 were identified from the biopsies of two patients respectively. No double or multiple infections were detected. Novel mutations were found in the HPV-2 genome, located both in the coding and non-coding regions. Amino acid changes occurred only in E1 and E7 ORFs. The two strains also shared several mutations at the same positions. CONCLUSIONS: Each patient was infected with a single strain of HPV-2 that developed unique mutations; HPV-2 may play a role in the onset and development of cutaneous horns; amino acid changes in functionally significant viral proteins may confer differential pathogenic risks.     

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.