L1 Open Reading Frame Research Articles

Burning wood in the kitchen increases the risk of cervical neoplasia in HPV-infected women in Honduras.

There is suggestive evidence that the use of wood for cooking increases the risk of invasive cervical cancer. We investigated this association in women with cervical neoplasia in Honduras. Women aged 20-64 years with cervical intraepithelial neoplasia (CIN) grade I (n = 44), CIN II (n = 36) or CIN III (n = 45) were recruited from screening programs in Tegucigalpa City and each was matched by age and clinic to 2 controls (241 total) without cervical abnormalities. The clinics selected women of low socioeconomic status. Cervical scrapes were tested for the presence of human papillomavirus (HPV) DNA using a general primer set directed against the L1 open reading frame, and HPV genotyping was performed. Odds ratios (ORs) were computed through conditional logistic regression; p-values were from tests for linear trend of risk with increasing exposure. HPV DNA was detected in 48% of women with CIN I, 67% with CIN II and 89% with CIN III. The ORs were 1.5, 2.5 and 38.3 respectively. At univariate analysis, age at first intercourse was consistently lower among cases than controls. Risk was reduced by 50% or more in all 3 CIN classes when initiation of sexual activity at age 20 years or older was compared with initiation before age 16 years (p = 0.013 for CIN I). No effect was observed for smoking, oral contraceptives or previous cytologic screening. Effects for number of sexual partners, parity, age at first pregnancy and education were in the expected directions but never persisted after adjustment for HPV. Chronic exposure to wood smoke significantly increased the risk of CIN III (p = 0.022). However, women who said "No" when asked if they ever used wood in the kitchen had a higher risk than those with low or intermediate exposure. This was taken as evidence that the initial screening question had either been misunderstood or that answers were biased. Restricting the analysis to women who reported exposure yielded positive associations in all CIN classes with for CIN III ORs of 2.3 for 25-34 and 9.5 for 35+ years compared with women who had 1-14 years of exposure (p = 0.017). A multivariate analysis of the complete dataset (n = 366) allowed for separate ORs for HPV in each CIN class. Inclusion of age at first intercourse significantly improved this model (p = 0.021). Adding exposure to wood smoke further improved the model only if an interaction between woodsmoke and HPV was allowed for. If, as the data suggest, it was assumed that wood smoke had its effect among HPV-positives only, there was a significant linear dose-response relationship between exposure to woodsmoke and risk of CIN (p = 0.026). This association was independent of other risk factors including education, parity and number of sexual partners. ORs in the final model were 0.37 for age at first intercourse 20 years or higher and 5.69 for more than 35 years of exposure to wood burning in the kitchen. The present study suggests that the use of wood for cooking is a risk factor for cervical neoplasia that deserves further study, given its high prevalence in developing countries.

Association Between Epidermodysplasia Verruciformis-Associated Human Papillomavirus DNA in Plucked Eyebrow Hair and Solar Keratoses

Association Between Epidermodysplasia Verruciformis-Associated Human Papillomavirus DNA in Plucked Eyebrow Hair and Solar Keratoses

Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5'-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.

Characterization of a novel genital human papillomavirus by overlapping PCR: candHPV86 identified in cervicovaginal cells of a woman with cervical neoplasia.

A novel human papillomavirus (HPV), candHPV86, was cloned and characterized from cervicovaginal cells obtained from a 37-year-old Hispanic woman with cervical intraepithelial neoplasia grade 1 (CIN1) using an overlapping PCR technique. Primers were designed by phylogenetic alignment of closely related HPV genomes using the L1 fragment sequence amplified by GP5+/6+. The 7983 bp complete nucleotide sequence of the HPV genome was determined by sequence walking. A basic local alignment sequence tool (BLAST) homology search using the L1 open reading frame demonstrated that this HPV was most closely related to HPVHAN2294 (GenBank, AJ400628; 86% homology) and HPV84 (84% homology). candHPV86 was placed in the HPV genome homology group A3 by phylogenetic analyses. The overlapping PCR technique is applicable for characterizing the complete spectrum and variation of HPVs in a population.

Detection of Human Papillomaviruses in Cervical Neoplasias Using Multiple Sets of Generic Polymerase Chain Reaction Primers

Objective.The aim of this study was to evaluate precisely the differences in the spectra of human papillomavirus (HPV) types detected by different generic primer pairs commonly used for detection of this extraordinarily heterogeneous virus.Methods. Three sets of polymerase chain reaction (PCR) primers for the L1 open reading frame (ORF) and two sets for E6/E7 ORFs were used to detect HPVs in DNAs from 107 cervical tissues, including 77 cervical neoplasias. HPV types were determined by analysis of restriction fragment length polymorphisms (RFLPs) and nucleotide sequencing.Results. A high overall detection rate of HPV in cervical neoplasias (76/77, 98.7%) was achieved by polymerase chain reaction (PCR) amplification with multiple sets of generic primers, while the detection rate for each individual primer pair varied from 48/77 (62%) to 70/77 (91%). Only in 34 of 77 cases (44%) were HPV DNAs positive for all sets of primer pairs. Further determination of HPV types by RFLPs and nucleotide sequencing showed inconsistencies between the PCR primer pairs used.Conclusion. Our study revealed that the HPV detection rate is critically affected by the choice of PCR primers, and that appropriate use of combinations of generic PCR primer sets followed by RFLP analyses is both necessary and sufficient for typing most HPVs in cervical lesions. More precise methods such as sequencing would be necessary in only a few cases.

Complete Nucleotide Sequence and Analysis of a Novel Human Papillomavirus (HPV 84) Genome Cloned by an Overlapping PCR Method

Molecular diagnosis of human papillomaviruses (HPVs) in cervicovaginal samples reveals a plethora of known and novel HPV genomes. We describe the use of an overlapping PCR method to clone and analyze the complete genome of HPV 84 from cervicovaginal cells obtained from a 21-year-old Caucasian female with a normal Pap smear. The 7948-bp complete nucleotide sequence of HPV 84 was determined from five overlapping PCR products by sequence walking. A BLAST homology search demonstrated that HPV 84 was most closely related to HPV 61 (89%), HPV 72 (86%), and HPV 83 (85%) by nucleotide sequence analysis of the L1 open reading frame, placing it in the HPV genome homology group A3. Previously, this virus had been identified as Pap155. Based on extensive epidemiological data, HPV 84 is a highly prevalent genital papillomavirus primarily detected in normal and HIV-infected women.

Improved amplification of genital human papillomaviruses.

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

The integration of HPV-18 DNA in cervical carcinoma.

AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV-18) DNA, which is associated with up...

A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin.

A pair of degenerate PCR primers (FAP59/64) was designed from two relatively conserved regions of the L1 open reading frame of most human papillomaviruses (HPV). The size of the generated amplicon was about 480 bp. PCR using these primers was found capable of amplifying DNA from 87% (65/75) of the HPV types tested, its sensitivity being 1-10 copies for HPV-5, -20 and -30 clones. HPV was found in 63% (5/8) of tumour samples and in 63% (5/8) of normal skin biopsies from patients with various cutaneous tumours. HPV-5, HPV-8, HPV-12, HPVvs20-4 and six putatively novel HPV types were identified. No correlation was found to exist between specific HPV and tumour types. Skin surface swab samples from one or more sites on three of four healthy volunteers were found to contain HPV, types 12 and 49 being identified, as well as eight novel HPV types, two of which were also found among the patients. In all, HPV was detected in 75% (9/12) of those tested, five HPV types and 12 novel candidate types being identified, and 37% (7/19) of HPV-positive samples were found to manifest more than one HPV type. All the HPV detected manifested high degrees of nucleotide sequence similarity with HPV types associated with skin lesions and epidermodysplasia verruciformis. The overall HPV finding in the skin samples was 50% (20/40) using the FAP primers as compared to 18% (7/40) using another PCR test designed for skin types. The results thus suggest the new method to be sensitive and generally applicable for detecting cutaneous HPV.

Detection of human papillomavirus (HPV) in laryngeal carcinoma cell lines provides evidence for a heterogeneic cell population

The role of human papillomavirus (HPV) has been studied in laryngeal carcinomas with contradictory results. To evaluate the causal relationship between HPV infection and epithelial malignancies of the larynx, 27 laryngeal carcinoma cell lines from 22 patients were studied. Also, paraffin-embedded biopsy samples of the original tumours were available from 12 patients. First, Southern blot hybridisation (SBH) was used for the analysis of 18 cell lines and 12 original tumour sections were studied by in situ hybridisation (ISH) to detect HPV. Further, cell lines and tumour biopsy samples were investigated with polymerase chain reaction (PCR) using three sets of consensus primers directed to L1 and E1 ORFs (open reading frames) and type-specific primers to HPV 16 E6 region. The adjacent apparently normal epithelium of one original biopsy sample showed positive signals for HPV by ISH. All other samples were HPV negative with these methods. The study was then extended to 27 laryngeal carcinoma cell lines, including the 18 cell lines studied earlier. A new nested PCR method was used with MY as external and general primers (GP) as internal primers for the cell lines and original tumour samples to achieve a maximal sensitivity. Subsequent SBH was performed to confirm the specificity of PCR products with both low- and high-risk HPV oligonucleotide probe mixtures and also with the HPV 16 oligoprobe. With this method, seven of 27 (26%) cell lines and seven of 12 (58%) tumour samples were found to harbour high-risk HPV. In two cases both the original tumour sample and the derived cell line showed HPV positivity. These results indicate that HPV copy numbers are low and only a minority of tumour cells harbour HPV DNA, explaining partly the controversial results reported earlier.

Consensus polymerase chain reaction and enzyme-linked immunosorbent assay for human papillomavirus detection and typing in cervical specimens.

Human papillomavirus (HPV) infection is common in cervical intraepithelial neoplasia (CIN). This study investigates HPV detection and typing assay based on polymerase chain reaction amplification of L1 open reading frame with general primers GP5/GP6, followed by enzyme-linked immunosorbent assay detection with type-specific DNA probes. To determine the sensitivity of this assay, formalin-fixed CaSki cells were used as reference cell lines. Fifty copies of viral DNA diluted in DNA from 100,000 noninfected cells could be detected. This assay was also investigated for HPV detection and typing of 67 cervical specimens diagnosed with with CIN III or carcinoma in situ (CIS) and their adjacent squamous epithelium. The CIN III lesions were infected in approximately 80% of the samples, 81% in the neighboring CIN II, and 68% in CIN I. The HPV infection was even detectable in 54% of nondysplastic epithelium located near a CIN III lesion.

Evaluation of human papillomavirus-consensus primers for HPV detection by the polymerase chain reaction

Cervical cancer is one of the most frequently found cancers in women and appears to have a viral aetiology. Substantial evidence points to the human papillomaviruses (HPV) as the infectious agents and there is considerable interest in identifying and accurately typing the viruses. Since HPVs now comprise more than 100 different HPV types, the polymerase chain reaction (PCR) has been the preferred methodology for virus identification and typing on isolated DNA. In that context, five commonly employed PCR consensus primers have been evaluated for the detection and typing of HPV. The five consensus primer pairs were derived from the consensus sequences of either the L1 and E1 open reading frames. All primers exhibited approximately equal sensitivity, as defined by the ability to detect HPV DNA, on a series of standard HPV DNA-containing preparations. However, the five primer pairs performed differently on 24 HPV-positive and 34 HPV-negative samples obtained from cervical scrapes which had been typed by type-specific PCR for HPV 6/11, 16, 18 and 33. The values for agreement between identification of samples by a HPV type-specific PCR and the consensus primer PCR were 78, 84, 91, 93 and 98%. Three samples, which were positive with only one of the five consensus primer pairs and were negative with the PCR for HPV types 6/11, 16, 18 and 33, contained other HPV sequences or HPV-related sequences as determined by DNA sequence analysis. To our knowledge, this report represents the first extensive comparison of five different consensus primers in a polymerase chain reaction for the detection of HPV. Our results suggest that PCR typing for human papillomaviruses requires more than one consensus primer pair to identify all HPV-infected samples.

Prevalence of antibodies to human papillomaviruses in the general population of the Czech Republic.

Sera from 450 individuals between the age of 1 and 80 years, representing the general population of the Czech Republic, were tested for the presence of antibodies to human-papillomavirus(HPV)-derived antigens. The following antigens were used: (i) HPV1 virions; (ii) HPV16, -18 and -33-virus-like particles (VLP); (iii) peptides derived from L2 open reading frames (ORFs) of HPV16 and HPV6/11; (iv) peptides derived from HPV16 E2, E4 and E7 ORFs of HPV16. The prevalence of antibodies reactive with the capsid-derived antigens was age-dependent, while no clear age dependence was observed in the distribution of antibodies to peptides derived from HPV16 early proteins. In individual sera, high correlations between the presence of antibodies reactive with the 2 L2 peptides, also between the antibodies reactive with different VLPs, were found. While the simultaneous presence of the 2 L2 antibodies was frequently detected in individual sera in all age groups, the simultaneous occurrence of VLP antibodies was detected mostly in subjects older than 20 years. There were no significant differences in HPV-antibody distribution between men and women.

Detection of Human Papillomavirus DNA in PUVA-Associated Non-Melanoma Skin Cancers

Psoralen and UVA (PUVA) photochemotherapy is associated with a dose-dependent increased risk of nonmelanoma skin cancer in patients treated for psoriasis. Like ultraviolet B radiation, PUVA is both mutagenic and immunosuppressive and may thus act as a complete carcinogen; however, the reversed squamous to basal cell carcinoma ratio (SCC:BCC) in PUVA-treated patients, also seen in immunosuppressed renal transplant recipients, suggests a possible cofactor role for human papillomavirus (HPV) infection. In this study we examine a large series of benign and malignant cutaneous lesions for the presence of HPV DNA from patients treated with high dose (> or =500 J per cm2) ultraviolet A. A panel of degenerate primers based on the L1 (major capsid protein) open reading frame was employed, designed to detect mucosal, cutaneous, and epidermodysplasia verruciformis HPV types with high sensitivity and specificity. HPV DNA was detected in 15 of 20 (75%) non-melanoma skin cancer, seven of 17 (41.2%) dysplastic PUVA keratoses, four of five (80%) skin warts, and four of 12 (33%) PUVA-exposed normal skin samples. The majority of HPV positive lesions contained epidermodysplasia verruciformis-related HPV including HPV-5, -20, -21, -23, -24, and -38. Possible novel epidermodysplasia verruciformis types were identified in further lesions. Mixed infection with epidermodysplasia verruciformis, cutaneous, and/or mucosal types was present in six of 30 (20%) of all HPV positive lesions, including in normal skin, warts, dysplastic PUVA keratoses, and squamous cell carcinomas. The prevalence and type of HPV infection in cutaneous lesions from PUVA-treated patients is similar to that previously reported in renal transplant-associated skin lesions, and suggests that the role of HPV in PUVA-associated carcinogenesis merits further study.

Temporal usage of multiple promoters during the life cycle of human papillomavirus type 31b.

The life cycles of human papillomaviruses (HPVs) are dependent upon the differentiation of the epithelial cells they infect. HPV type 31b (HPV31b) virions can be purified following the growth of a latently HPV-infected cell line (CIN-612 9E) in the organotypic or raft system. Treatment of the CIN-612 9E raft tissues with protein kinase C (PKC) activators is required for upregulation of late gene expression and efficient production of virions. We employed the raft culture system to study the temporal usage of HPV31b promoters during the viral life cycle. We compared monolayer cultures of CIN-612 9E cells, untreated CIN-612 9E raft tissues, and PKC-induced CIN-612 9E raft tissues harvested at various time points during epithelial differentiation. We found that the HPV31b major early promoter precisely maps to nucleotide (nt) 99 (P99). A transcriptional start site for both early and late gene transcripts mapped upstream of P99 at nt 77 (P77). The P77 and P99 promoters were used constitutively throughout the HPV31b life cycle; however, initiation from P99 was much stronger than from P77. Mapping of the differentiation-induced P742 promoter revealed multiple start sites. These start sites were difficult to detect in monolayer cultures, were induced in untreated rafts, and were greatest in PKC-induced raft tissues at 8 to 12 days. A constitutively active promoter, P3320, was also defined and is responsible for the transcription of unspliced and spliced RNAs containing E5a, E5b, L2, and L1 open reading frames.

Evaluation of immunoassays for the detection and typing of PCR amplified human papillomavirus DNA.

AIMS: To evaluate different hybridisation techniques to detect and type human papillomavirus (HPV) DNAs amplified by consensus primer polymerase chain reaction (PCR) in biopsy and cytological specimens. METHODS: A hybrid...

Molecular analysis of episomal human papillomavirus type 16 DNA in a cervical carcinoma cell line

Integration of human papillomavirus type 16 DNA sequences into host DNA is a frequent event in cervical carcinogenesis. However, recent studies showing that HPV16 is present exclusively in an episomal form in many primary cervical cancers suggest that HPV16 can transform target cells by mechanisms that do not require viral integration. We have established a cervical carcinoma cell line that harbors episomal copies of HPV16 DNA of approximately 10 kb. Restriction enzyme and two-dimensional gel analysis confirmed that HPV16 DNA was extrachromosomal with both monomeric and multimeric forms present. HPV16 was maintained as episomes with passage both in culture and after subcutaneous growth in nude mice. The 10 kb viral genome, consisting of a full-length copy of HPV16 and a partial duplication of the long control region and the L1 open reading frame, exhibited transforming activity comparable to prototype HPV16. This cell line should provide a useful model system for studying the biological significance of the physical state of the HPV16 genome in cervical carcinoma cells.

Nucleotide and amino acid sequence variations in the L1 open reading frame of human papillomavirus type 6

Nucleotide and amino acid sequence variations in the L1 open reading frame of human papillomavirus type 6

Nucleotide and amino acid sequence variations in the L1 open reading frame of human papillomavirus type 6

Human papillomavirus (HPV) type 6 induces benign tumors such as condyloma acuminata and laryngeal papilloma. The HPV-6 DNA has been thought to be a heterogeneous group of subtypes and variant-types. To examine sequence variations of the HPV-6 L1 ORF, we analyzed by single-strand conformation polymorphism (SSCP) and by DNA sequencing 21 specimens from condyloma acuminata and laryngeal papilloma that harbor HPV-6 DNA. PCR products of HPV-6 DNA were digested with 6 restriction enzymes yielding 8 fragments, which were then analyzed by SSCP. The resolution patterns showed that the L1 coding sequences were separated into three SSCP groups, I, II, and III, and two minor groups, (I) and (III). By sequencing the five representatives of each SSCP group and by comparing these sequences with those of HPV-6a [Hofmann et al., 1995] and HPV-6b [Schwarz et al., 1983], we identified base substitutions at 20 positions in the L1 coding region and an amino acid substitution in one case.

Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b.

Human papillomaviruses (HPVs) are etiologic agents of anogenital cancers. The lack of an efficient in vitro system with which to study the differentiation-dependent viral life cycle has impeded most investigations of viral transcription and gene expression. The CIN-612 clone 9E cell line latently maintains episomal copies of HPV type 31b (HPV31b). The complete replicative life cycle of HPV31b can be studied by using the organotypic (raft) culture system. A number of spliced HPV31b early gene transcripts and two late gene transcripts have been described in studies using the raft system. An HPV31b early promoter, P97, and a differentiation-induced promoter, P742, have been characterized by using this system. In this study, we used the raft system to analyze the temporal expression patterns of HPV31b late gene transcripts during the viral life cycle. The expression of late RNAs peaked at day 12 after lifting to the air-liquid interface; the levels then declined dramatically by day 16. The peak of late RNA expression was coincident with the appearance of virus particles in the raft tissues. We characterized transcripts with the potential to encode late gene products, including 19 RNAs containing the L1 region and 4 RNAs containing the E5b and L2 open reading frames. We also found evidence for two novel promoters. Transcription of both L1- and L2-containing RNAs initiated at a region upstream of the early promoter. In addition, late gene RNAs were also transcribed by using a promoter in the E4 reading frame.