Characterization of seven novel human papillomavirus types isolated from cutaneous tissue, but also present in mucosal lesions

The isolation and characterization of papillomavirus DNA from skin and genital warts revealed the existence of distinct human papillomavirus (HPV) types (zur Hausen et al., 1974; Orth et al., 1977). The large number of HPV types associated with mucosal lesions (reviewed by zur Hausen, 2006) and with the hereditary disease epidermodysplasia verruciformis (Orth, 1986) subsequently confirmed the diversity of the virus family Papillomaviridae. The initial rapid identification and characterization of HPV types associated with genital/mucosal lesions focused attention mainly on the large number of HPV types in the genus Alphapapillomavirus (de Villiers et al., 2004a). Availability of DNA-amplification methods facilitated the demonstration of HPV DNA in cutaneous lesions. Several subsequent studies attempted to associate specific HPV types with the aetiology of non-melanoma skin cancer in immunosuppressed and immunocompetent patients (Shamanin et al., 1994, 1996; Berkhout et al., 1995, 2000; de Villiers et al., 1997; Forslund et al., 1999, 2003b, 2007; Asgari et al., 2008; Harwood et al., 2000, 2004), whereas others demonstrated the presence of HPV in and on normal skin (Astori et al., 1998; Antonsson et al., 2000; Hazard et al., 2007; Chen et al., 2008). These studies all resulted in the identification of a vast spectrum of additional putative novel HPV types in the form of PCR-amplified partial open reading frame (ORF) L1 sequences (Shamanin et al., 1996; de Villiers et al., 1997; Astori et al., 1998; Antonsson et al., 2000; Forslund et al., 2003a; Forslund, 2007).

Full-length genomes of a relatively small number of these HPV isolates have been cloned and characterized (Delius etal., 1998; Forslund et al., 2003a; Vasiljevic et al., 2007, 2008). Definition of a papillomavirus type requires the isolation and characterization of a full-length genome in order to verify the genome organization characteristic of papillomaviruses (de Villiers et al., 2004a). We report here the isolation and characterization of seven novel HPV types, all of which were initially identified as partial ORF L1 sequences.

The complete genomes of seven novel HPV types were amplified through rolling-circle amplification of cellular DNA and subsequent long PCR amplification (Leppik et al., 2007), using primers that had been designed on the respective partial L1 sequence on which the respective putative HPV types had initially been identified (de Villiers et al., 1997; Astori et al., 1998). The seven types and the respective partial L1 sequences on which they were identified are as follows: HPV 94 (DL40), HPV 98 (GA1-3), HPV 99 (GA3-1), HPV 100 (DL267), HPV 104 (DL253), HPV 105 (DL294) and HPV 113 (DL250). An additional partial sequence, DL285 (de Villiers et al., 1997), forms part of the recently described HPV 107 (Vasiljevic et al., 2008). Patient samples that had, over the course of 20 years, been identified as harbouring the respective partial putative novel HPV type were used for rolling-circle amplification. These patient samples originated from a multitude of different clinics worldwide and, although the present data include unpublished data, the majority of samples formed part of previously published studies performed by our laboratory (Shamanin et al., 1994, 1996; de Villiers et al., 1997, 1999, 2004b; Astori et al., 1998; Lavergne & de Villiers, 1999; Koppikar et al., 2005; Forslund et al., 2007; Asgari et al., 2008). The number of included samples that harboured the respective HPV type varied. Samples in which these novel HPV types had been identified as single infections were used to isolate complete genomes.

Cellular DNA was amplified by using either GenomiPhi or TempliPhi kits (catalogue no. 25-6600-01 and 25-6400-10, respectively; Amersham Biosciences). Products were purified by using MicroSpin G-25 columns (catalogue no. 27-5325-01; Amersham Biosciences). Long PCR amplification (Expand Long Template PCR system; catalogue no. 1681-834; Roche) using these respective primer pairs was performed by an initial amplification for 10 cycles of 30 s at the respective annealing temperatures (55 °C for DL267 primers, 65 °C for all other primers), followed by 6 min elongation at 68 °C. The following 20 cycles were performed under the same conditions except that the length of every subsequent elongation step was increased by 20 s. Amplicons of putative full-length genomes (approx. 8 kb) were eluted and cloned into vector pMOSBlue (pMOSBlue Blunt End kit; catalogue no. RPN5110; Amersham Biosciences). Sequencing of the respective clones was performed in an ABI Prism 3100 Genetic Analyzer with BigDye Terminator chemistry (Perkin-Elmer Applied Biosystems). Alignment of full-length ORF L1 sequences was performed by using the clustal w program (Thompson et al., 1994) by using a gap-creation penalty of 10 and a gap-extension penalty of 5. Phylogenetic reconstruction was performed by using the maximum-parsimony and neighbour-joining methods; trees were calculated by heuristic search implementing tree bisection–reconnection [paupsearch (Maddison et al., 1997); husar (Senger et al., 1998)] and data were bootstrap-resampled 1000 times. The phylogenetic tree was displayed by using the TreeView program (University of Glasgow; ).

Verification as bona fide papillomavirus genomes was done by analyses of the genome organizations and comparisons with all previously characterized HPV types (de Villiers et al., 2004a). A summary of the genome lengths (nt) and the positions of the ORFs is presented in Table 1(a)⇓. The separation between early and late regions of HPV 98 (70 bp), HPV 99 (90 bp), HPV 100 (68 bp), HPV 104 (66 bp), HPV 105 (87 bp) and HPV 113 (47 bp) did not allow E5 genes (Hirsch-Behnam et al., 1999), whereas the HPV 94 genome has a putative E5 ORF (nt 4121–4261). Alignments of individual ORFs with those of closely related HPV types suggest that the second ATGs present in the E6 ORFs of HPV 94, HPV 98 and HPV 113 are probably used in vivo. Similarly, HPV 100 will use the third ATG of the E6 ORF. All HPV types described here are predicted to use the second ATG in the L1 ORF, except for HPV 98 and HPV 113, which will use the first ATG. The putative E4 ORFs of HPV 99 and HPV 105 do not have start codons, but ORFs that could potentially encode these genes are present in each genome. Transcripts for E4 genes of other HPV types have been shown to start in the E1 ORF and are then spliced into the E4 gene (Nasseri et al., 1987; Brown et al., 1999).

Nucleotide and amino acid sequence similarities to the closest known HPV types are given in Table 1(b)⇑. HPV 94 is related most closely to HPV 10 of the genus Alphapapillomavirus, species 2. Nucleotide sequence identity between their L1 ORFs is 86 % and, although they share 90 % amino acid sequence similarity in L1, an L1 nucleotide sequence similarity value <90 % defines this isolate as a novel HPV type (de Villiers et al., 2004a).

All other novel HPV types are related most closely to members of the genus Betapapillomavirus. The HPV 98 L1 ORF shares 83 % nucleotide sequence identity with the L1 ORF of HPV 24. HPV 99 and HPV 105 are both related most closely (82 %) to HPV 8, whereas their L1 ORFs share 80 % nucleotide sequence identity with each other, thus defining each as an individual, novel HPV type (de Villiers et al., 2004a). Phylogenetic analyses based on L1 ORF nucleotide sequences indicate that these HPV types all group into species 1 of the genus Betapapillomavirus. HPV 100, HPV 104 and HPV 113 group into species 2 of the same genus (Fig. 1⇓). The sequence of HPV 100 L1 is 79 % identical to the L1 ORFs of both HPV 22 and HPV 23. The HPV 104 ORF L1 nucleotide sequence is 78 % identical to that of HPV 107, and the HPV 113 ORF L1 nucleotide sequence is 83 % identical to that of HPV 111 (Vasiljevic et al., 2008) and 78 % identical to that of HPV 9.

The E6 genes of the novel HPV types all contained the two characteristic zinc-binding domains CxxC(x)_29–30CxxC, separated by 36 aa. The putative E7 genes contained one zinc-binding domain [CxxC(x)₂₉CxxC], which was modified slightly in HPV 98 as CxxxC(x)₂₉CxxC and in HPV 113 as CxC(x)₂₉CxxC. HPV 94 E7 does not have the LxCxE motif necessary for binding to the pRB protein, whereas all other novel types do. Potential casein kinase II phosphorylation sites were observed in HPV 98 (LHCEEELPEQDTEVEPERTSYK) and HPV 99 (LFCEEELPTEQETEEE), but not in the other types. ATP-binding sites were present in all putative E1 ORFs: in HPV 94 at aa 511–518 (GPADTGKS), in HPV 98 at aa 438–445 (GPPNTGKS), in HPV 99 at aa 432–439 (GPPNSGKS), in HPV 100 at aa 434–441 (GPPDTGKS), in HPV 104 at aa 438–445 (GPPDSGKS), in HPV 105 at aa 431–438 (GPPNSGKS) and in HPV 113 at aa 434–441 (GPPDTGKS). Only HPV 94 has a leucine zipper [L(x)₆L(x)₆L(x)₆L, aa 303–324] in its E2 ORF. Potential polyadenylation signals were present in all HPV types described here, for both early and late mRNA (HPV 94, nt 69 and 4366; HPV 98, nt 224 and 4296; HPV 99, nt 118 and 4382; HPV 100, nt 191 and 4245; HPV 104, nt 170 and 4185; HPV 105, nt 109 and 4280; HPV 113, nt 105 and 4044). The upstream regulatory region (URR) consisted of 690 bp in the HPV 94 genome, which is in the size range of URRs of other HPV types in the genus Alphapapillomavirus (Chen et al., 2007). The URRs in the other novel HPV types were much smaller (HPV 98, 349 bp; HPV 99, 468 bp; HPV 100, 369 bp; HPV 104, 419 bp; HPV 105, 451 bp; HPV 113, 373 bp), corresponding to the sizes of URRs in other HPV types of the genus Betapapillomavirus (Vasiljevic et al., 2007). Putative E2-binding sites were present in all novel HPV types, although the majority of these sites were related to, rather than bona fide [ACC(N)₆GGT], E2-binding sites. In vitro analyses are required for each of these putative E2-binding sites to determine whether they will be functional.

The number of biopsies harbouring the novel HPV types was generally too low to define prevalences (Table 2⇓). In total, 2117 biopsies from skin, mucosa of the head and neck region and oesophagus were analysed during the course of several years, using the same primers and amplification conditions for each specimen as were used in the original identification of these novel HPV types. The specimens analysed originated from a large number of clinics in different countries. They can be grouped, based on the histopathological information provided by these clinics, as normal (n=374), perilesional (n=623), benign lesions (n=202), squamous cell carcinomas (n=631) and in addition, actinic keratoses (n=125) and basal cell carcinomas (n=162) of the skin. HPV 94 DNA was present in nine samples, HPV 98 in 25, HPV 99 in five, HPV 100 in 44, HPV 104 in 14, HPV 105 in four and HPV 113 in nine biopsies. HPV 98, HPV 100, HPV 104 and HPV 113 were also demonstrated in malignant mucosal lesions. These HPV types appeared either as single infections (sample numbers varied) or in combination with other HPV types in the same sample. Functional analyses in organotypic raft cultures of the intact genomes of the respective HPV types are required to enable insight into the pathogenicity of these papillomaviruses.