Cervical lesions are associated with human papillomavirus type 16 intratypic variants that have high transcriptional activity and increased usage of common mammalian codons

Jon M. Bible1, Christine Mant1, Jennifer M. Best1,2, Barbara Kell1, William G. Starkeyb,2, K. Shanti Raju3, Paul Seed4, Chandrima Biswas3, Peter Muir2, Jangu E. Banatvala2 and John Cason1

The Richard Dimbleby Laboratory of Cancer Virology1 and the Departments of Infection2, Obstetrics and Gynaecology3 and Public Health Medicine4, Guy’s, King’s College and St Thomas’ Medical and Dental Schools, King’s College London, St Thomas’ Campus, Lambeth Palace Road, London SE1 7EH, UK

Author for correspondence: John Cason. Fax +44 20 7922 8394. e-mail jwcason{at}AOL.com


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human papillomavirus type 16 (HPV-16) is a major cause of cervical neoplasia, but only a minority of HPV-16 infections result in cancer. Whether particular HPV-16 variants are associated with cervical disease has not yet been clearly established. An investigation of whether cervical neoplasia is associated with infection with HPV-16 intratypic variants was undertaken by using RFLP analyses in a study of 100 HPV-16 DNA-positive women with or without neoplasia. RFLP variant 2 was positively associated [odds ratio (OR)=2·57] and variant 5 was negatively associated with disease (OR=0·2). Variant 1, which resembles the reference isolate of HPV-16, was found at a similar prevalence among those with and without neoplasia. Variants 1 and 2 were also more likely to be associated with detectable viral mRNA than variant 5 (respectively P=0·03 and P=0·00). When HPV-16 E5 ORFs in 50 clones from 36 clinical samples were sequenced, 19 variant HPV-16 E5 DNA sequences were identified. Twelve of these DNA sequences encoded variant E5 amino acid sequences, 10 of which were novel. Whilst the associations between HPV-16 E5 RFLP variants and neoplasia could not be attributed to differences in amino acid sequences, correlation was observed in codon usage. DNA sequences of RFLP variant 2 (associated with greatest OR for neoplasia) had a significantly greater usage of common mammalian codons compared with RFLP pattern 1 variants.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Cervical cancer is a major cause of female cancer deaths, with some 450000 incident cases worldwide (Parkin et al., 1988 ). In the UK, human papillomavirus (HPV) types 16 and 18 are the most frequently detected high-risk HPVs in cervical cancers: in our inner-city location, high-risk HPV DNA occurs in about 85% of cervical cancers and 62% are positive for HPV-16 DNA (Cavuslu et al., 1997 ). Since the vast majority of high-risk HPV infections do not result in carcinoma (IARC, 1995 ; Conrad-Stoppler et al., 1996 ), other factors must be involved in malignant progression. Several explanations have been proposed and include exposure to co-carcinogens (Khare et al., 1995 ), genetic predisposition (Magnusson et al., 1999 ), which may consist of the influence of host HLA genotype (Ellis et al., 1995 ) and/or Tp53 polymorphisms (Storey et al., 1998 ), concurrent immunosuppression (Benton et al., 1992 ; Frazer & Tindle, 1992 ), a high virus load in the genital tract (Bavin et al., 1993 ) and virus persistence (Terry et al., 1997 ).

A largely unexplored possibility is that HPV-16 intratypic variants may have differing oncogenic potentials. There has been interest in the phylogeny of HPV-16 variants (Chan et al., 1992 ) and some studies have sought an association between HPV-16 variants and cervical neoplasia (Fujinaga et al., 1994 ; Hecht et al., 1995 ; Londesborough et al., 1996 ; Xi et al., 1997 ; Zehbe et al., 1998 ). However, these reports are largely based on small patient numbers and a limited clinical spectrum of neoplastic lesions and rarely include infected women with normal cytology. Moreover, as yet, no convincing molecular explanation has been proposed for the enhanced pathogenicity of certain HPV-16 variants.

In this study, we have combined a PCR assay (Cavuslu et al., 1996a ; Mant et al., 1997 ) and RFLP analyses to identify HPV-16 variants amongst HPV-16-infected women with or without cervical neoplasia. Our PCR amplifies the wild-type HPV-16 E5 gene between nt 3866 and 4077. For the reference HPV-16 sequence, this region contains more than 45 restriction endonuclease (RE) cleavage sites, at least three of which [XcmI (3872CCANNNNN{downarrow}NNNNTGG3886), SspI (3978AAT{downarrow}ATT3983) and NspI (4077ACATG{downarrow}C4082)] are disrupted in different reported HPV-16 variants (Chan et al., 1992 ). These RE were thus used in an RFLP assay to identify eight HPV-16 variants and determine their associations with neoplasia. As early region viral mRNA is more commonly detected amongst women with more severe lesions (Biswas et al., 1997 ), we also sought to determine whether particular HPV-16 variants were associated with evidence of virus transcription, which might result in high virus copy number, a factor associated with lesion severity (Bavin et al., 1993 ).

Our RFLP analyses indicated that certain variants were associated with neoplasia, raising the possibility that variation of the E5 protein might explain these associations directly. Indeed, HPV-16 E5 plays an integral role in the early stages of neoplastic transformation, with E5 mRNA and protein detected in low-grade cervical intraepithelial neoplasias (CIN) (Stoler et al., 1992 ; Kell et al., 1994 ). E5-stimulated mitogenesis might increase the number of HPV-16-infected keratinocytes (Leechanachai et al., 1992 ; Leptak et al., 1991 ; Pim et al., 1992 ) as well as inducing up-regulation of virus transcription via an E5-induced increase of AP-1 and its effect on the long control region of HPV-16. To date, the functional domains of HPV-16 E5 have not been delineated, although molecular modelling suggests that the protein consists of three anchor-like {alpha}-helices (residues 8–30, 37–52 and 58–76). The first helix contains two Cys-X-Cys motifs at amino acids 18–20 and 24–26 and a Phe15-X-X-Cys18-Phe19 sequence that is conserved in mucosal HPV types 6b, 16, 18, 31, 33, 35 and 58. The third {alpha}-helix contains nine amino acids that are homologous to connexins in the motif 55Ser-X2-Arg-X5-Iso-Iso-Phe-Val-X2-Pro-X2-Leu-X3-His77 (Ullman et al., 1994 ). Therefore, we sequenced HPV-16 isolates and interpreted nucleotide changes with respect to the associations between RFLP patterns, neoplasia and E5 protein structures and motifs. As it has been suggested recently that codon usage may be important in the regulation of papillomavirus early gene expression (Zhou et al., 1999 ), we also investigated this possibility.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Reference samples.
The HPV-16 reference isolate, pAt-16 (Seedorf et al., 1985 ; Halbert & Galloway, 1988 ), was kindly provided by E. M. De Villiers (DKFZ, Heidelberg, Germany). Two HPV-16 DNA-positive cell lines (CaSki and SiHa) and an HPV-16 DNA-negative cell line (A431) were purchased from the ATCC (Manassas, VA, USA).

{blacksquare} Clinical samples.
For the investigation of variants with the RFLP assay, cervical brush smears were collected with an Axibrush (Colgate Medical) from 89 HPV-16 E5 DNA-positive women attending local well-woman centres and gynaecological outpatient clinics at St Thomas’ Hospital (London, UK). These included 25 women with normal cytology to act as controls, six women with borderline lesions (cytologically determined HPV-associated change), 18 women with biopsy-proven CIN-I, 17 women with CIN-II, 19 women with CIN-III and four women with cervical carcinoma. Mean ages and the age range for these women are shown in Table 1. In addition, HPV-16 E5-positive samples from 10 µm sections of formalin-fixed, paraffin-embedded cervical cancers from four women (obtained from J. Goodlad, Department of Histopathology, St Thomas’ Hospital) and DNA from seven Swedish women (three CIN-III and four cervical carcinomas) from I. Zehbe (DKFZ, Heidelberg, Germany) were investigated. All samples were positive for human {beta}-globin DNA.


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Table 1. Prevalence of different HPV-16 RFLP variants

 
For the analyses of HPV-16 DNA sequences, samples included buccal swabs from 11 children aged between 6 and 11 years and cervical brush smears from three women with normal cytology, one woman with CIN-I, six women with CIN-II, nine women with CIN-III and six women with cervical carcinoma. Of these 36 clinical samples, eight had RFLP patterns that resembled the reference isolate (RFLP pattern 1; Fig. 1), 27 were RFLP pattern 2 and one was RFLP pattern 5. Fifty clones were sequenced from these 36 clinical samples. Permission for the collection of clinical specimens was provided by the Research Ethics Committee of St Thomas’ Hospital.



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Fig. 1. HPV-16 E5 RFLP variants. Negative images of UV light-transilluminated agarose gels containing ethidium bromide. M, Molecular size ladder (with sizes in bp). Each panel shows (from left to right) the undigested amplicon and the results of SspI, XcmI and NspI digestion of a clinical sample. Variant 1 (which resembles the reference isolate pAt-16) is digested by all three RE; variant 2 does not have the SspI site; variant 3 has no XcmI site; variant 4 has no NspI site; variant 5 has no SspI or XcmI sites; variant 6 has no SspI or NspI sites; variant 7 no XcmI and NspI sites; and variant 8 has lost all three sites.

 
{blacksquare} PCR.
Cells from the cervical brush smears were resuspended in four 1 ml aliquots in PBS for PCR and in a further 1 ml aliquot in RNAzol for RT–PCR (see below) and stored at -70 °C. A 1 ml sample in PBS was then centrifuged at 10000 g and the cell pellet was resuspended in 200 µl proteinase K (PK) solution and digested overnight at 55 °C. A 20 µl aliquot of the PK solution was then used in a 200 µl non-nested PCR to amplify nt 3866–4077 of the HPV-16 genome, as described previously (Cavuslu et al., 1996a ; Mant et al., 1997 ). For each batch of 20 clinical samples, two negative controls (water and A431 cells) and two positive controls (either pAt-16 and CaSki or pAt-16 and SiHa DNA) were included. Amplicons were analysed by electrophoresis into 2% (w/v) agarose gels containing ethidium bromide. Samples were tested in a randomized, blind fashion.

{blacksquare} RFLP analyses of E5 PCR products.
Amplicons from PCRs were purified by ethanol precipitation and DNA pellets were resuspended in 50 µl molecular biology-grade distilled water. Four 5 µl aliquots were prepared, three of which were subjected to an overnight digestion at 37 °C with 5 U of one RE (SspI, XcmI or NspI; New England Biolabs and Boehringer-Mannheim) in appropriate buffer before being analysed in agarose gels. Spiking experiments indicated that mixed infections could only be identified in the RFLP assay when the least-frequent variant exceeded 10% (w/w) of the total HPV-16 E5 DNA present (data not shown).

{blacksquare} RT–PCR assay for HPV-16 early-region mRNA (EmRNA).
These were performed by using the method of Biswas et al. (1997) to analyse 50 of the clinical samples, all of which contained sufficient RNA for analysis, as indicated by positivity by RT–PCR for human keratin mRNA (Bosma et al., 1995 ; data not shown). Briefly, RT was primed by oligo(dT)25–30 with Moloney murine leukaemia virus reverse transcriptase and then samples were subjected to a nested PCR with primers located within the E5 ORF. This assay detects the majority of HPV-16 early region transcripts.

{blacksquare} Precautions against contamination.
Strict precautions against PCR contamination were taken and included the use of four geographically remote rooms and good laboratory practice (Mant et al., 1997 ).

{blacksquare} DNA sequencing.
E5 PCR amplicons were purified by using Qiagen columns and cloned into pGEM-T (Promega). Plasmids were transformed into Escherichia coli JM109 cells and grown overnight at 37 °C on agar plates. White colonies were selected and grown in midi-cultures. Plasmid DNA was purified by using Qiagen columns and inserts were sequenced in both orientations (with T7 and SP6 primers that recognize sequences in pGEM-T that flank the inserted E5 DNA) by using a commercial kit (Sequenase, Pharmacia). The resulting E5 products representing wild-type HPV-16 E5 DNA sequence between nucleotides 3866 and 4077 were analysed on a semi-automated DNA sequencer (ALF, Pharmacia). To permit comparisons with previous studies, HPV-16 DNA sequences were classified as being of European or African origin (see Table 2 for details) and arranged as subclasses according to their RFLP pattern (e.g. 1, 2 or 5) or variation within an RFLP pattern (1.1, 1.2 etc.).


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Table 2. Nucleotide changes in the HPV-16 E5 ORF in 50 clones from clinical isolates

 
{blacksquare} Codon usage.
Changes of codon usage compared with the reference sequence of HPV-16 were determined for each altered ‘in-frame’ codon from tables documenting codon usage in mammalian cells and calculated as the change in frequency of usage per 1000 codons (Ellington & Cherry, 1994 ). For each DNA sequence, the sum of the values of individual codon changes was calculated; thus, any value greater than or less than zero (the value for the reference isolate of HPV-16) represents a net increased or decreased use of more-common mammalian codons.

{blacksquare} Statistical analyses.
It was assumed that each RFLP pattern represented a unique, or a cluster of related, HPV-16 nucleotide variant(s). Odds ratios (OR) were estimated by logistic regression to determine the association between RFLP variants and neoplasia and detectable EmRNA; ordinal logistic regression was used for the association of RFLP variants and grade of lesion (including no neoplasia, asymptomatic patients, as grade 0) by using Stata 5.0 software (StataCorp). Student’s unpaired, two-tailed t-test and Fisher’s exact test were used to assist in the analyses of codon usage data.

{blacksquare} HPV-16 E5 protein structure prediction.
The secondary structure of the reference HPV-16 amino acid sequence was estimated by using the SOSUI programme (http://azusa.proteome.bio.tuat.ac.jp/sosui/).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Prevalence of HPV-16 variants among women with and without neoplasia
The RFLP assay can detect eight HPV-16 variants in clinical samples (Fig. 1, Table 1). Overall, variant 2 was most common, present in 55 of 100 (55%) cases investigated, while variant 1, which resembles the reference isolate, was detected in 23 (23%) samples and variant 5 in 14 (14%) samples. Other variants were infrequent. Variant 1 was detected at a similar prevalence amongst those with (16 of 75, 21 %) and without (7 of 25, 28%) neoplasia. Variant 2 was twice as common among those with neoplasia (47 of 75, 63%) than amongst asymptomatic women (8 of 25, 32%: P=0·007) and was more likely to be associated with neoplasia than was variant 1, although this difference was not statistically significant [OR=2·57; P=0·111; 95% confidence interval (CI) 0·80–8·21]. Variant 5 was six times more prevalent amongst asymptomatic women (9 of 25, 36%) than those with neoplasia (5 of 75, 7%; P=0·0009) and, compared with variant 1, was significantly less likely to be associated with neoplasia (OR=0·24; P=0·049; 95% CI, 0·06–0·99). Odds of neoplasia were tenfold higher for those infected with variant 2 than with variant 5 (OR=10·57; P=0·000; 95% CI, 2·8–39·8). Variant 5 was significantly negatively associated with lesion severity (OR=0·197; P=0·015; 95% CI, 0·05–0·71). Mixed infections were not observed with this assay (which is relatively insensitive for this purpose; see Methods).

Correlation of HPV-16 E5 variants with evidence of virus transcription
EmRNA was more commonly detected among those infected with variant 1 (P=0·03) and those with variant 2 (P=0·00) than in patients infected with variant 5 (Fig. 2). Amongst those with neoplasia, EmRNA was detected in 73% (8 of 11 samples) of those with variant 1, 85% (17 of 20) of those with variant 2 and 40% (2 of 5) of those with variant 5, although this difference was not significant (P>0·05).



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Fig. 2. Association between RFLP variants and detection of HPV-16 EmRNA. Numbers above each bar indicate the actual number of subjects positive for viral EmRNA of the total number tested.

 
HPV-16 E5 DNA sequence variation
In order to investigate these observations further, we sequenced 50 clones from 36 clinical samples and three from HPV-16-positive controls (pAt-16, SiHa and CaSki DNA). The latter had DNA sequences (respectively 1.1, 2.15 and 5.1) identical to those reported previously (Table 2). Forty-three clones differed from the reference sequence of HPV-16 (pAt-16) and consisted of 19 variant E5 DNA sequences. Only one example (of ten attempted) of the pattern 5 variant (5.2) was cloned successfully.

RFLP variant pattern 1 consisted of two DNA sequences, 1.1 and 1.2 (Table 2), with the former being most prevalent (7 of 8 clones). For RFLP pattern 2, there were 17 different sequences (2.1–2.14, 2.16–2.18) amongst 42 clones; 20 clones had sequence 2.1. RFLP pattern 5 consisted of one unique sequence. Evidence for homogeneous and mixed HPV-16 variant infections was obtained when DNA sequencing was used. All clones from samples 721 (a–d), 1709 (a–c), HMNO1 (a, b) and TMN6 (a, b) had the same sequence. In contrast, just two (a, c) of four clones from sample TSS2 and two (a, b) of three from HAP1 had the same sequence. All three clones from HEP1 and both from samples TRL and 161 were different.

Deduced E5 amino acid sequences
The 19 variant E5 DNA sequences (excluding CaSki and SiHa DNA) encoded amino acid changes or silent codon changes: no termination codons, insertions or deletions were observed. Twelve variant E5 amino acid sequences differed from the HPV-16 reference sequence; of these, ten (DNA sequences 1.2, 2.4, 2.6, 2.7, 2.8, 2.10, 2.12, 2.14, 2.18 and 5.2) have not been described previously (Chan et al., 1992 ; Eriksson et al., 1999 ). These ten protein sequences differed from the reference isolate by between one and four amino acids at twelve positions (Table 3).


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Table 3. Deduced amino acid changes in the E5 protein sequence for isolates with RFLP patterns 1, 2 and 5

 
Amongst our clinical samples, there were two E5 amino acid sequences amongst clones with RFLP pattern 1 (1.1 and 1.2), 11 amongst RFLP pattern 2 and one RFLP pattern 5 isolate. The RFLP pattern 5 sequence 5.2 had an unique change at amino acid 12 (Ser->Pro). When all variant E5 amino acid sequences in Table 3 were considered, in the first {alpha}-helix, 4 of 23 (17·4%) positions were subject to variation, the Cys-X-Cys motifs were conserved in all cases and the Phe15-X-X-Cys18-Phe19 signature was unchanged in all sequences except SiHa (2.15). The second {alpha}-helix was most variable, with 10 of 16 (62·5%) residues subject to change and the third {alpha}-helix had five of 19 (26·3%) variable residues. Amongst our samples, the connexin motif was retained in all pattern 1.1 amino acid sequences, but pattern 1.2 had an Iso->Thr change at amino acid 64, and an Iso->Val/Leu change at residue 65 was common to all except two pattern 2 sequences and sequence 5.2. However, this last change has previously been reported in a pattern 1 sequence (IS.244); hence, there were no unique amino acid differences between the pattern 1 and pattern 2 RFLP variants.

Codon usage
Codon changes for 43 different HPV-16 E5 DNA sequences (i.e. 19 variant DNA sequences described in this study and 24 other unique E5 sequences from the studies mentioned in Table 3) were examined for changes from the reference isolate (Table 4). Twenty-six of 38 (68·4%) RFLP pattern 2 DNA variants and none of RFLP pattern 1 sequences utilized codons that enhanced the usage of mammalian codons by a frequency of more than 2 per 1000 codons (Fisher’s exact test, P= 0·006; Fig. 3). We also analysed codon usage in DNA sequences reported in previous studies that demonstrated associations between E6 and/or E7 DNA sequence variants and disease (Xi et al., 1997 ; Londesborough et al., 1996 ; Zehbe et al., 1998 ). Variants identified by these authors as being most pathogenic had more-commonly used mammalian codons than the less-pathogenic, reference-like (RL) variants and, in two studies, these differences were significant (unpaired t-test; P=0·0 and P=0·04) (Fig. 4).


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Table 4. Codon usage changes amongst isolates with RFLP patterns 1, 2 and 5

 


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Fig. 3. Change in codon frequency usage for the E5 DNA sequences corresponding to RFLP patterns 1 and 2. Data are shown as individual points for each unique DNA sequence encoding the amino acid sequences shown in Table 3 and thus take no account of the relative frequency of each variant. {circ}, Data from the current study; {bullet}, data from previous studies. Means±SEM are indicated by bars. Data are expressed as the net change in frequency of codon usage per 1000 mammalian codons compared with the sequence of the reference isolate. Compared with the RFLP pattern 1 variants, significantly more RFLP pattern 2 variants had an increase in the usage of more common mammalian codons; >2 per 1000 codons (P=0·006). The RFLP pattern 5 variants 5.1 and 5.2 had codon changes of 0·3 and -35·1, respectively (data not shown). These data could not be analysed according to clinical status as many sequences from DNA databases did not have clinical details.

 


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Fig. 4. Codon usage in previous studies of HPV-16 variants. Studies are indicated as A (Xi et al., 1997 ), B (Londesborough et al., 1996 ) and C (Zehbe et al., 1998 ). RL, Reference-like HPV-16 DNA sequences; n, number of samples in each group. Data are expressed as the mean change of codon usage for each group ±SEM and, unlike Fig. 3, include the differences in incidence of HPV-16 variants in each population. Differences between RL and variant sequences were statistically significant in studies B (P=0·00) and C (P=0·04).

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
We have demonstrated that infection with HPV-16 RFLP variant 2 is more likely, and infection with variant 5 less likely, to be associated with cervical lesions than infection with variant 1, which resembles the reference isolate. Moreover, HPV-16 RFLP variant 2 was most prevalent in every grade of cervical lesion and, thus, did not just reflect virus persistence. We have also shown that HPV-16 EmRNA is more commonly detected in women infected with variants 1 and 2 than amongst those with variant 5, suggesting that high-transcription variants of HPV-16 are more oncogenic.

We are confident that these results are not artefactual, as (i) stringent precautions were taken against PCR contamination (Mant et al., 1997 ); (ii) DNA sequencing indicated the presence of many unique sequence variants, suggesting that cross-contamination from a single source was unlikely; (iii) samples were collected, processed and analysed in a random, blind fashion; and (iv) RFLP results were reproducible, in that the positive controls (pAt-16, SiHa and CaSki) produced the correct RFLP patterns (1, 2 and 5, respectively) consistently in repeat analyses. Similarly, repeat analysis of 20 original clinical samples produced concordant RFLP patterns on both occasions (data not shown). Subjects were recruited for study on the basis of HPV-16 E5 DNA positivity, which might possibly introduce a bias against those with integrated viral DNA (where the E5 ORF can be discontinuous). However, this is unlikely to influence our findings, as an intact E5 HPV-16 ORF is found in the majority of cervical carcinomas (Cavuslu et al., 1996b ; Eriksson et al., 1999 ). The distribution of HPV-16 variants can differ geographically (Chan et al., 1992 ) and it is conceivable that the inclusion of the seven Swedish patients may potentially have confounded our analysis. However, re-analysis of the data after exclusion of these patients did not alter our findings.

Nucleotide variation in the HPV-16 E6 ORF has been investigated by others and revealed variants that are associated positively with persistent infections (Londesborough et al., 1996 ; Xi et al., 1995 ), high grades of CIN (Xi et al., 1995 ; Ellis et al., 1997 ) or cervical cancer (Zehbe et al., 1998 ; Ellis et al., 1995 ). The enhanced pathogenicity of such HPV-16 E6 variants may be due to a decreased ability to degrade p53 and alter keratinocyte differentiation (Conrad-Stoppler et al., 1996 ), an inability to elicit neutralizing antibodies (Ellis et al., 1997 ) and/or alteration of dominant cytotoxic T-cell epitopes (Ellis et al., 1995 ). We investigated whether our E5 RFLP variants co-segregated with the E6 variant described by Ellis et al. (1995) . However, only one of 66 subjects analysed had this E6 variant (data not shown) and we therefore conclude that this HPV-16 variant is rare in our locality (Luxton et al., 2000 ). Whilst the reason for the lack of co-segregation of our E5 variants with this E6 variant is not known, it may reflect demographic and lifestyle differences between the populations studied.

In the present study, viral EmRNA was commonly detected in subjects infected with RFLP variants 1 or 2, but only rarely in those with variant 5. It is possible that these findings were confounded by the differing clinical status of subjects infected with these HPV-16 variants, although when only those with neoplasia were considered, a similar trend was observed. Alternatively, it is conceivable that nucleotide variation in the primer sites adversely effected the detectability of mRNA from different variants. This is unlikely, as pattern 1 variants would therefore be expected to have most mRNA transcripts: this was not the case. Whilst in vitro studies are required to resolve this issue, these data imply that HPV-16 variants 1 and 2 have high transcriptional activity. HPV-16 transcription is regulated by the P97 promoter and the E2 protein; hence, our E5 variants may co-segregate with E2 variants (Terry et al., 1997 ; Hecht et al., 1995 ) that explain the apparent differences in transcriptional activity.

We sequenced HPV-16 E5 DNA from clinical isolates to determine why HPV-16 E5 RFLP variants are associated differentially with neoplasia, having conjectured that variants may encode variant E5 proteins that explain these associations directly. We experienced problems in cloning sufficient examples of pattern 5 variants, perhaps as a result of the low copy number that is implicit in our observation of the rare detection of mRNA amongst those infected with these variants. Overall, RFLP pattern 2 variants were more heterogeneous (17 of 41, 41·4%, differed from one another) than pattern 1 variants (two sequences of eight; 25% variation). Our sequencing data are unlikely to represent PCR artefacts, since (i) the proof-reading enzyme rTth was used and (ii) multiple analyses of the HPV-16 reference and CaSki DNA isolates did not reveal any deviation from published sequences and repeat sequencing of 12 wild-type isolates produced concordant sequences on both occasions. Whilst no evidence of mixed HPV-16 variant infections was observed when the RFLP assay was used, sequencing revealed the presence of such mixed variant infections: this probably reflects the sensitivity limits of the former method for detecting mixed variant infections, as described in the Methods.

Whilst there were no consistent amino acid differences in E5 proteins encoded by pattern 1 and pattern 2 RFLP variants, we did find marked changes in codon usage. The HPV-16 RFLP pattern 2 family had nucleotide substitutions that resulted in the increased usage of more-frequently used mammalian codons in 68·4% of instances compared with the RFLP pattern 1 group. Whilst this increased usage of more-common mammalian codons was relatively small overall, it is conceivable that, if similar increases of mammalian codon usage are present in other ORFs of these isolates, they may cumulatively confer a selective growth advantage on such variants. Interestingly, increased use of common mammalian codons was not restricted to this study or to the E5 ORF, as HPV-16 E6 and/or E7 variants have exhibited similar characteristics. Londesborough et al. (1996) found that HPV-16 E6 350G variants were associated with more persistent infections than reference-like (RL) variants: 350G variants had a higher use of mammalian common codons than the latter (P=0·00). Similarly, Zehbe et al. (1998) noted that E6 variants were more prevalent than RL sequences in invasive carcinomas compared with CIN-III lesions: again the former had a greater use of common codons (P=0·04). Xi et al. (1997) conducted a longitudinal study that showed that women infected with non-RL variants were more likely to develop CIN-II/III as opposed to those infected with RL variants. Here too, disease-associated variants had a higher, though not statistically significant, use of common codons. This is probably due to inclusion of five 350G variants amongst their RL group. Other studies are more difficult to interpret. The paper by Terry et al. (1997) described coding changes but not synonymous codon changes, and thus could not be analysed. Fujinaga et al. (1994) included only a small number of subjects; nevertheless, nine carcinoma patients exhibited a moderate increase in codon usage (mean increase of 1·74 per 1000 codons) compared with the reference isolate.

In conclusion, our results suggest that RFLP variant 2 (characterized by loss of the SspI site only) is associated predominantly with cervical neoplasia and with high transcriptional activity. Longitudinal studies of patients are now required to determine whether infection with particular variants is prognostic for disease.


   Acknowledgments
 
C.M. and J.M.B. contributed equally to this paper. We are grateful for financial support from the Richard Dimbleby Cancer Fund, Smith’s Charities, the British Council and the Special Trustees of St Thomas’ Hospital. We thank Dr P. Rice for collecting some samples included in this study and for assistance with the cloning. We would also like to acknowledge our respect for the late Dr Jian Zhou, who kindly provided us with a preprint of his last paper (Zhou et al., 1999 ).


   Footnotes
 
The EMBL accession numbers of the HPV-16 DNA sequences reported in this study are AJ244833–AJ244883.

b Present address: Institute of Aquaculture, University of Stirling, Perthshire, UK.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Biswas, C., Kell, B., Mant, C., Jewers, R. J., Cason, J., Muir, P., Raju, K. S. & Best, J. M. (1997). Detection of human papillomavirus type 16 early-gene transcription by reverse transcription-PCR is associated with abnormal cervical cytology. Journal of Clinical Microbiology 35, 1560-1564.[Abstract]

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Cavuslu, S., Starkey, W. G., Kaye, J. N., Biswas, C., Mant, C., Kell, B., Rice, P., Best, J. M. & Cason, J. (1996a). Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system.Journal of Virological Methods 58, 59-69.[Medline]

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Received 5 October 1999; accepted 24 January 2000.