Human papillomavirus type 16 variant analysis of E6, E7, and L1 [corrected] genes and long control region in [corrected] cervical carcinomas in patients in northeast China

Human papillomavirus type 16 (HPV 16) plays a cardinal role in the pathogenesis of cervical cancer. HPV 16 has intratypic variants which show different geographical distributions and different oncogenic potentials. To analyze the presence of sequence variations of HPV 16 variants in northeast China, 71 cervical carcinomas were identified by HPV typing. HPV 16-positive specimens were analyzed by PCR-directed sequencing in the E6, E7, and L1 genes and the LCR (long control region). The variation data were compared with those of neighboring districts. In this hospital-based study, HPV 16 was the most common type (73.24%). In HPV 16-positive specimens, 67.31% belonged to the European (E) lineage, while 32.69% were Asian (As) variants. The Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and northern American (NA) lineages were not detected. The most frequently observed variation sites were T178G (32.69%) in E6; A647G (34.62%), G666A (38.46%), and T846C (32.69%) in E7; C6826T (36.17%) and G7060A (61.70%) in L1; and G7521A (98.08%) in the LCR. The most prevalent amino acid variations were D25E in E6 and N29S in E7. In addition, 28 novel variations of HPV 16 were reported. Some covariations between different genes were obtained. In this study, HPV 16 variants belonged to the European lineage and the Asian lineage. Compared with neighboring districts, the distribution of HPV 16 variants in northeast China had a typical pattern. As the first report on HPV 16 variants in northeast China, it should be helpful for designing a HPV vaccine and HPV vaccination program in China.     

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. OBJECTIVES: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. STUDY DESIGN: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. RESULTS: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. CONCLUSIONS: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.     

Human papillomavirus type-16 variants in Quechua aboriginals from Argentina

Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions.     

Human papillomavirus-16 E6 variants in cervical squamous intraepithelial lesions from HIV-negative and HIV-positive women.

We studied 48 human papillomavirus (HPV)-16-positive squamous intraepithelial lesions (SILs) from HIV-negative patients (16 low-grade SILs [LSILs]; 32 high-grade SILs [HSILs]) and 13 HPV-16-positive SILs from HIV-positive patients with AIDS (1 LSIL; 12 HSILs). After HPV typing, the entire HPV-16 E6 coding region was amplified and sequenced in all samples. We detected 12 HPV-16 E6 prototypes and 4 variants among the LSILs in HIV-negative patients, and 15 HPV-16 E6 prototypes and 17 HPV-16 variants in the HSIL group. The most prevalent variant of SIL types was European 350G, present in 3 and 13 cases, respectively. In 3 HSILs and no LSILs we found mixed infection by an HPV-16 E6 prototype and a variant. Two variants (1 each in LSIL and HSIL) were of non-European lineage. The only LSIL in HIV-positive patients had an HPV-16 E6 prototype; in the HSILs, we found 8 HPV-16 E6-prototypes, 4 with mixed infection with HPV-31 and 4 variants, all European 350G. The higher proportion of HPV-16 E6 variants in HSIL than in LSIL in HIV-negative patients suggests a greater risk of progression. However, further studies are needed.     

Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer

Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.     

Human papillomavirus type 16 variants in cervical cancer from an admixtured population in Brazil

Several studies indicate that molecular variants of HPV-16 have different geographic distribution and risk associated with persistent infection and development of high-grade cervical lesions. In the present study, the frequency of HPV-16 variants was determined in 81 biopsies from women with cervical intraepithelial neoplasia grade III or invasive cervical cancer from the city of Belem, Northern Brazil. Host DNAs were also genotyped in order to analyze the ethnicity-related distribution of these variants. Nine different HPV-16 LCR variants belonging to four phylogenetic branches were identified. Among these, two new isolates were characterized. The most prevalent HPV-16 variant detected was the Asian-American B-2, followed by the European B-12 and the European prototype. Infections by multiple variants were observed in both invasive cervical cancer and cervical intraepithelial neoplasia grade III cases. The analysis of a specific polymorphism within the E6 viral gene was performed in a subset of 76 isolates. The E6-350G polymorphism was significantly more frequent in Asian-American variants. The HPV-16 variability detected followed the same pattern of the genetic ancestry observed in Northern Brazil, with European, Amerindian and African roots. Although African ancestry was higher among women infected by the prototype, no correlation between ethnical origin and HPV-16 variants was found. These results corroborate previous data showing a high frequency of Asian-American variants in cervical neoplasia among women with multiethnic origin.     

Human papillomavirus 16 E6, L1, L2 and E2 gene variants in cervical lesion progression

The human papillomavirus (HPV) 16 E6 genome variant 350G has been found to be more prevalent in women with persistent infection and cervical disease progression than the HPV16 E6 prototype 350T. In this study, we examined whether women who progressed to a high-grade lesion, yet were infected with the prototype 350T, showed variants in other HPV genes such as L1, L2 and E2. Although we detected variants within these genes, they could not explain this phenomenon. Indeed they correlated similarly with variant 350G and prototype 350T. These data indicate that polymorphisms in HPV16 E6 rather than in the other analyzed genes play a role in determining the risk for cervical lesion progression and that additional factors are likely to be required as well.     

Association of human papillomavirus type 16 and its genetic variants with cervical lesion in Korea

Persistent human papillomavirus type 16 (HPV16) is the major risk factor for cervical cancer. HPV16 intratypic variants differ in their geographical distribution and oncogenic potential. This study aimed to analyze the distribution of HPV16 variants and their association with cervical lesion histopathology in Korean women. In total, 133 HPV16‐positive cervical samples from women admitted to Seoul National University Boramae Hospital were analyzed by sequencing E6, E7, and L1 genes and the long control region (LCR), and the variant distribution according to cervical lesion grade was determined. Isolates were grouped into a phylogenetic lineage, and A1‐3, A4, C, and D sublineages were detected in 54.1, 37.8, 0.7, and 7.4% of samples, respectively. The most commonly observed LCR variations were 7521G>A (91.5%), 7730A>C (59.6%), and 7842G>A (59.6%). Furthermore, A4 or D sublineage‐positive women had a higher risk for cervical cancer than women who were positive for A1–3. Among HPV phylogenetic clusters, A1–3 was the predominant sublineage, and within A1–3, the 350G polymorphism was highly frequent. These results differed from those of previous studies in Korea and other Asian countries. The findings suggest that cervical neoplasia incidence in HPV16‐infected patients could be affected by the distribution of HPV16 variants in the population.     

Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients.

Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age-matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA-negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18-positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Müller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa.     

Variation of human papillomavirus 16 in cervical and lung cancers in Sichuan, China

Although the crucial role of human papillomaviruses (HPVs), especially HPV-16 in various cancers has been confirmed, the variation of HPV-16 among different cancers have not been investigated in a specific geographic location. In order to elucidate whether similar HPV-16 variants are involved in different kinds of cancers in the same geographic location, the analysis of sequence variants of E6 and E7 oncogenes and L1 gene of HPV-16 in cervical and lung cancers in Sichuan, China, was carried out. Tissue samples from 122 cervical cancers, 104 lung cancers, and 138 controls were subjected to RT-PCR or PCR, sequencing, and sequence analysis. The infection rates of HPV-16 in cervical, lung cancers, and non-malignant controls were 68.9%, 17.3%, and 37.0%, respectively. Asian prototype variants prevailed in cervical and lung cancers, while European prototype variants in non-malignant controls. In comparison to the lung cancer, cervical cancer showed a much higher diversity of HPV-16 oncogenes. These results indicate that in Sichuan, China, Asian prototype variants of HPV-16 are more pathogenic than their European counterparts.     

Antibodies to HPV-16 E6 and E7 proteins as markers for HPV-16-associated invasive cervical cancer.

Transforming proteins E6 and E7 of human papillomaviruses (HPVs) are consistently expressed in HPV-associated cervical cancers. In ELISA with four HPV-16 E6-E7 peptides, patients with HPV-16-associated invasive cervical cancer (group 1) had a greater seroreactivity than all other groups, which included patients with HPV-16-associated cervical intraepithelial neoplasia, invasive cervical cancer patients without HPVs, and unaffected controls. A larger proportion of group 1 sera, as compared to sera of all other groups, was reactive with at least one peptide (49% vs 17-27%), and with two or more peptides (22% vs 0-6%). A clear difference between group 1 and all other groups was also found for high ELISA absorbance values to at least one peptide (22% vs 0-8%). This high seroreactivity of group 1 sera was confirmed by a radioimmunoprecipitation assay with in vitro transcribed and translated HPV-16 E7 protein. Sera from 50% of group 1 but only 3% of controls were reactive in this test. Antibodies to HPV-16 E6 and E7 proteins appear to be virus-specific and disease state-specific markers of HPV-associated cervical cancer.     

Genomic diversity of human papillomavirus genotype 53 in an ethnogeographically closed cohort of white European women

Human papillomavirus (HPV) genotype 53 is classified taxonomically in alpha HPV genus-species 6, together with HPV-30, HPV-56, and HPV-66 and is considered to be one of three "probable high-risk" HPV genotypes. Recent worldwide comparison of 44 isolates of HPV-53 showed the existence of nine long control region (LCR) genomic variants, which formed a phylogenetic tree with two deep dichotomic branches. In order to investigate further the genomic diversity of HPV-53, a total of 94 isolates of HPV-53 obtained from an ethnogeographically closed cohort of 70 white European women was analyzed. The identification and characterization of HPV-53 genomic variants was based on analysis of three different HPV genomic regions: LCR, E6 and E7. A higher genomic diversity of HPV-53 was identified in the ethnogeographically closed cohort of white European women than has been reported previously on isolates collected worldwide. Altogether, 19 HPV-53 genomic variants, composed of 13 LCR, 13 E6, and 5 E7 genomic variants, were identified. Eleven out of 13 LCR, all E6, and four out of five E7 genomic variants were described for the first time. The present study confirmed dichotomic phylogeny of HPV-53 described previously and, in addition, showed for the first time that after a dichotomic split, both groups of HPV-53 genomic variants formed star-like phylogenetic clusters. In women with persistent HPV-53 infection, HPV-53 genomic variants remained unchanged for up to 51 months. In rare cases, infection with multiple HPV-53 genomic variants is possible. Taking into account the results of this and previous studies, at least 26 different HPV-53 genomic variants exist today.     

Analysis of relative binding affinity of E7-pRB of human papillomavirus 16 clinical variants using the yeast two-hybrid system

A number of genotypes of the human papillomaviruses (HPV) are associated with malignancies of the uterine cervix. Sequencing work has revealed the existence of intratype HPV variants with minor differences in the nucleotide sequence. More recent data suggest the possibility that some of the variants may have different modes of clinical manifestation. In this study, sequences of the E6 and E7 oncogenes of 17 HPV16 isolates derived from PAP smear samples of Taiwanese patients were analyzed. A number of E6 and E7 novel variants were found. Particularly, a prevalent (64.7%) E6 polymorphic site A442C with an E113D amino acid substitution seems specific to Taiwanese patients. In E7, two novel but silent polymorphic sites G663A (41.2%) and T846C (88.2%) were also prevalent in the samples analyzed. The yeast two-hybrid system was adopted for rapid assessment of relative E7-pRb binding affinity in the variants. The relative binding affinities of the E7 proteins of different HPV types to pRB were in close agreement with previous biochemical data. A T663G/C24W polymorphic change in E7 correlated with a decrease in E7-pRb relative binding affinity the significance of which remains to be clarified. This semi-quantitative biochemical and genetic approach may be useful as a first step in the development of clinical protocols for the screening and identification of important HPV variants for clinical interpretation and for further functional analysis by transfection or other bioassays.     

Nucleotide polymorphisms of the human papillomavirus 16 E1 gene

The E1 ORF is one of the most conserved regions in the human papillomavirus (HPV) genome. The complete E1 gene of the HPV16 genome was amplified with four overlapping primer sets in 16 high-grade (CIN II, III) and 13 low-grade cervical (CIN I) intraepithelial neoplasias as well as in one cervical cancer case. Sequence analysis of the E6 and E7 genes was also carried out in the same cervical samples in order to confirm the association between nucleotide sequence variations in the HPV16 E1 ORF and HPV16 variant lineages. Analysis of the E1 ORF revealed 27 nucleotide changes, and these changes were correlated with those found in HPV16 Asian American and African type II variants. Of these nucleotide variations, A1668G, G2073A, T2169C, T2189C, A2453T, C2454T, A2587T and G2650A were identified only in high-grade dysplasia cases. A phylogenetic tree of the E1 ORF and nucleotide sequence analysis of the E1, E6 and E7 genes revealed that intratypic nucleotide sequence polymorphisms located in the E1 ORF can be used to identify the major phylogenetic branch to which a HPV16 genome belongs. Moreover, amplification of the E1 ORF revealed a disruption between nucleotides 878 and 1523 in five high- and two low-grade cervical cases, indicating that integration of HPV DNA occurs at an early stage of viral infection.     

Human papillomavirus type 45 E7 is a transforming protein inducing retinoblastoma protein degradation and anchorage-independent cell cycle progression

High-risk human papillomaviruses (HPV) cause cervical cancer. The biological properties of HPV-45, the third most prevalent high-risk HPV-genotype, are unknown. We demonstrate here that the HPV-45 E7 protein transforms immortalized NIH3T3 fibroblasts, while mutations in either the conserved LXCXE sequence (C28G) or the carboxyl-terminus (Δ87LQQLF91) significantly abolish this activity. To address the mechanisms underlying cell transformation by HPV-45 E7, we investigated its impact on the cell cycle. We show that HPV-45 E7 associates with the hypophosphorylated form of the retinoblastoma protein (pRb) and induces a significant reduction in the pRb half-life which can be blocked by epoxomicin. Moreover, HPV-45 E7 induces anchorage-independent cell cycle progression of NIH3T3 cells and extends the lifespan of primary human keratinocytes. HPV-45 E7C28G did not bind pRb and could neither induce pRb-proteolysis nor promote cell cycle progression. HPV-45 E7Δ87LQQLF91 had intermediate pRb-binding affinity and retained a residual activity to induce the degradation of pRb but lost the capability to promote cell cycle progression in suspension. Another carboxyl-terminal mutant, HPV-45 E7Δ81AEDL84, showed a trend to reduced transforming activity, had reduced pRb-binding activity and lost the capability to induce pRb-degradation; however, this mutant could induce anchorage-independent cell cycle progression with the same efficiency as HPV-45 E7 wild type. In summary, these data suggest that HPV-45 E7 is a transforming protein and that abrogation of cell cycle control contributes to its oncogenic potential.     

Genetic diversity of human papillomavirus type 16 E6, E7, and L1 genes in Italian women with different grades of cervical lesions

High‐risk human papillomaviruses (HPVs) are risk factors for the development of cervical cancer. HPV 16 is the most common type, being present in about 60% of cervical cancers worldwide. Previous studies have reported upon the association between HPV 16 E6 variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In this study, the presence of HPV 16 polymorphisms in the E6, E7, and L1 genes was investigated in relation to the presence of high‐grade lesions. Sequencing of the E6 gene revealed the presence of nucleotide mutations resulting in 15 amino acid changes. Of these, the G134D and C136R fall within the CXXC zinger finger domain important for p53 binding. In the E7 gene, four nucleotide variations were identified with two leading to the amino acid substitutions L15V and S31R. The L1 gene showed 13 nucleotide changes leading to 11 amino acid substitutions. Among these, the R364C and N367D are located at the base of the HI‐loop of the L1 protein, considered to be the immunodominant epitope of HPV 16. No significant relationship between HPV 16 variants and high‐grade lesions was found. Phylogenetic analysis showed that all the HPV 16 variants identified belonged to the European lineage, except one which was of the Asian‐American lineage. The European‐350G variant was detected most frequently (22 of 34, 64.7%). The study provides some new data on the genetic diversity of HPV 16 which may help to understand the oncogenic potential of the virus and to improve management of patients. J. Med. Virol. 81:1627–1634, 2009. © 2009 Wiley‐Liss, Inc.