Programme in Infectious Diseases, Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore 117597, Republic of Singapore1
Author for correspondence: Vincent Chow.Fax +65 776 6872. e-mail micctk{at}nus.edu.sg
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The source of the viral genome was a cervical scraping taken from a female sex worker undergoing colposcopy in the Department of STD Control Clinic, Singapore, on 23 April 1996. While no malignant cells were observed by cervical cytology, histopathological examination of a concurrent punch biopsy of the cervix demonstrated features of a wart virus infection of the squamous epithelium. Screening of DNA extracted from both cervical specimens (scraping and biopsy) by PCR with consensus primers PVCOU [5' KKIKKRACCGAAAICGGT 3', from the long control region (LCR)] and PVCOD (5' YIICIRMAWACTTTCGTTTTA 3', from the E1 gene), designed to amplify DNA of several genital HPVs (including HPV types 6, 11, 16, 18, 31 and 33), produced target fragments of ~1·1 kb. Further, nested PCR with type- specific primer pairs for common genital HPV types 11, 16 and 18, which have been described previously (Chow et al., 1990a , b
; Tham et al., 1991
), proved negative. Preliminary DNA sequencing of the ~1·1 kb consensus PCR product by cycle sequencing (Chow et al., 1998
) by using 33P-labelled primers and the AmpliCycle Sequencing kit (Perkin Elmer) revealed nucleotide similarities of its 5' portion to HPV- 18 (78% identity) and of its 3' portion to HPV-59 (77% identity). We then proceeded to obtain the full-length viral genome via a strategy of overlapping PCR fragments amplified from template DNA of the cervical scrape. Thus, by using consensus L1 gene primers MY11 and MY09 (Bauer et al., 1991
), purchased from Maxim Biotech, a fragment of ~450 bp was next derived and sequenced. Based on nucleotide sequence information of the two consensus PCR fragments, two pairs of specific primers were synthesized to amplify two overlapping fragments of 1267 and 5796 bp, flanked by primer pairs FC1F/FC1R (spanning nucleotides 68396859 and 293272) and FD1F/FD1R (spanning nucleotides 925944 and 67206701), by classical PCR with AmpliTaq DNA polymerase (Perkin Elmer) and by long-distance PCR with Advantage Tth polymerase (Clontech), respectively. All four sub-genomic fragments were subjected to cycle sequencing in both directions by using the ABI PRISM BigDye Terminator cycle sequencing ready reaction kit and an ABI PRISM 377 DNA sequencer (Perkin Elmer). To amplify the full-length viral genome from the cervical scrape DNA template by long-distance PCR, a pair of back-to- back L1 gene-specific primers, FE74F (5' CGGAATTCCTAATCCAGTTCCATCTATA 3') and FE74R (5' CGGAATTCTAGTTGCAGTAGATAAGGTA 3'), were designed to flank a ~7·8 kb target fragment spanning nucleotides 66556654. This amplified full-length viral genome was used as template in nested PCR screening experiments using seven pairs of specific primers that targeted overlapping segments, and which also served as sequencing primers. The almost full-length viral DNA was generated by semi-nested PCR with primers NFE74F (nt 67016720) and FE74R and the amplified complete viral genomic DNA as template, and then subjected to RFLP analysis with restriction endonucleases Ase I, PvuI, SpeI, StuI and TaqI. Computer software for processing sequence data included DNASIS and PROSIS for DNA translation, prediction of open reading frames (ORFs), DNA motif search and hydrophilicity profiles; BCM for mapping of restriction sites; BLAST and GCG for sequence comparison and protein motifs search; CLUSTALW for multiple alignments and PHYLIP for constructing phylogenetic trees.
Fig. 1(a)(lanes 14) displays the four overlapping amplified fragments that were sequenced to generate the complete nucleotide sequence of HLT7474-S shown in Fig. 1( b)
. Although cloning of overlapping PCR products is a feasible way of acquiring full-length HPV sequence data (Forslund & Hansson, 1996
), there is a possibility of co- amplifying subgenomic parts of other HPV types present simultaneously in a given cervical specimen. In order to exclude this possibility, the whole viral genome was amplified and confirmed by screening by nested PCR with seven specific primer pairs, which yielded products of expected sizes (data not shown). For further authentication, nearly complete viral genomic DNA of 7766 bp was digested with five informative restriction enzymes and produced RFLP profiles consistent with those predicted from the restriction site mapping derived from the nucleotide sequence (Fig. 1a
, lanes 610). The viral genome contains 7812 bp with a G+C content of ~38 mol%, well within the expected range for a typical HPV. Table 1
summarizes the locations of the ORFs and their putative encoded proteins, as well as motifs within the LCR, numbered in accordance with the related prototype HPV-18 (Cole & Danos, 1987
).
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Phylogenetic trees (Ho et al., 1991 ; Chan et al., 1992
) based on the individual ORFs, putative proteins and LCRs were constructed to determine the relationships between HLT7474-S and the known HPVs from group A7. The E1, E2, E5 and L2 proteins of the novel HPV were more closely associated with those of HPV-70 and HPV-39. However, greater similarities were demonstrated for the E4, E6 and L1 proteins of the novel type to those of HPV-18 and HPV- 45, and for the novel HPV E7 protein and LCR to its HPV-59 counterparts (Fig. 2
).
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The E7 protein of HLT7474-S harboured three important conserved regions. At aa 42108 there was a metal-binding domain that has been shown to be important for the transforming ability, dimerization and stability of the E7 protein. The metal-binding domain contained two CXXC zinc finger motifs, the C-terminal motif of HPV-16 E7 having been shown to be essential for its transactivating function as well as for the immortalization of human keratinocytes. The pRb-binding and CKII phosphorylation motifs were detected within the CR2 domain, which is involved in the efficient dissociation of E2F from pRb as well as in the induction of DNA synthesis. The occurrence of aspartic acid as the first residue of the pRb-binding motif (DLYCYEE) is notable, given that this residue occurs in high-risk HPVs and is critical for the transforming activity of E7. The serine residue in the CKII phosphorylation site (NSEEEIDE) has also been implicated in transformation by E7. The CR1 domain (MHGPKPTVHDIVLDL) of the novel E7 protein shared several conserved residues found in genital HPVs, especially HPV-18, the sequence of which differed only by four of 15 residues (Munger & Halpern, 1997 ).
The motif MXYXH, which is conserved in genital HPVs (Chan et al., 1995 ), was detected in the L1 protein of the novel HPV. Similarly, the L2 protein possessed the motif TTPA(I/V)L(D/N)(I/V), which is highly conserved in mucosal HPVs and is thought to play a role in tissue tropism (Rho et al., 1994
). Comparison of the hydrophilicity plots of the L1 proteins of HLT7474-S and other HPVs within group A7 showed strikingly similar profiles despite some differences in amino acid sequence. The corresponding L2 hydrophilicity plots depicted generally similar patterns, albeit with distinct variations, especially in the C-terminal third of L2. These observations are congruent with the notions that the L1 major capsid proteins constitute group-specific antigens of papillomaviruses while the L2 proteins may act as type-specific antigens.
Spanning nt 7125104, the HLT7474-S LCR contains a diverse array of signature motifs, including the TATA box representing the E6/E7 promoter, the polyadenylation signal and putative binding sites for E1, E2, transcription factors AP1, NF1, SP1, TEF1 and YY1, as listed in Table 1. The putative origin of replication, to which the E1 protein binds to initiate DNA replication, was identified within the 3' segment of the LCR upstream of the TATA box. There were four binding sites for the E2 protein, which regulates viral DNA replication and gene expression. Of additional interest is a sequence spanning nt 78002, conforming to the consensus motif TACANNNTGTTCT of the glucocorticoid-responsive element (GRE), which may serve to upregulate expression from the E6/E7 promoter (O'Connor et al., 1995
). Also of note within the LCR is the existence of a 20-mer motif TGCTTTTAGGCACATATTTT (nt 76477666), which as yet has no characterized function but which is extremely well-conserved in intermediate- and high-risk anogenital HPV types and is proposed to contribute to their oncogenicity (Marich et al., 1992
).
In conclusion, we have isolated a novel genital HPV type, HLT7474-S, and characterized its complete genomic sequence and organization and its putative proteins. The features of this isolate warrant its designation as a co-evolved member of the A7 group of genital HPVs. Taken together, the phylogenetic relationships and the occurrence of conserved motifs within the individual viral genes, their encoded proteins and the LCR suggest strongly that this novel HPV type is potentially a highly oncogenic virus with a predilection for genital mucosa. Of conspicuous prominence is the remarkable resemblance of the sequences and conserved motifs of its transforming E6 and E7 proteins with those of HPV-18, which has a well-established causative link with highly malignant genital cancers associated with poor prognosis (Burger et al., 1996 ; Comerford et al., 1995
). Finally, the availability of sequence information for this novel HPV may facilitate future experiments in vitro as well as clinical studies to ascertain its prevalence of infection and carcinogenic potential (Meyer et al., 1998
).
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References |
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Received 2 June 1999;
accepted 3 August 1999.