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1.
Arch Virol ; 166(3): 853-862, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33486629

RESUMO

The aim of this study was to describe the distribution of human papillomavirus (HPV) genotypes among cervical cancers and pre-cancers in Shaanxi province of western China. A total of 17,341 women who were screened for cervical cancer from January 2014 to December 2016, using HPV genotyping and ThinPrep cytologic test were included. The prevalence and attribution of HPV genotypes were stratified by cervical lesion and age group. Of the subjects, 26.3% were infected with HPV, 28.0% of whom had multiple infections. The crude HPV prevalence increased from atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesions (ASCUS/LSIL, 64.3%) to high-grade squamous intraepithelial lesions (HSIL, 79.8%) and to invasive cervical cancer (ICC, 89.7%, P < 0.001). The three most prevalent genotypes were HPV 16 (8.0%), 58 (4.2%), and 52 (4.0%), and HPV 16, 31 and 33 were positively correlated with increased severity of cervical lesions. Additionally, the divalent vaccine genotypes HPV 16 and 18 accounted for 68.2% of ICC cases. Although 78.5% of ICC and 60.3% of HSIL cases were attributed to 9-valent vaccine genotypes, the other genotypes not covered by any vaccine still resulted in increases in coverage, with 1.5% for ICC, 5.3% for HSIL, and 13.5% for ASCUS/LSIL. HPV prevalence in western China was consistent with other regions of China. Early vaccination with 9-valent HPV vaccine is recommended in this locality for females younger than 26 years with no prior infection, while divalent the vaccine is more appropriate for women between 26 and 45 years, considering the efficacy, safety and cost-effectiveness of vaccines.


Assuntos
Neoplasia Intraepitelial Cervical/epidemiologia , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Papillomavirus Humano 31/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Neoplasias do Colo do Útero/epidemiologia , Adulto , Neoplasia Intraepitelial Cervical/virologia , China/epidemiologia , Feminino , Genótipo , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Papillomavirus Humano 31/genética , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/uso terapêutico , Prevalência , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia , Vacinação
2.
BMC Infect Dis ; 20(1): 482, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32640998

RESUMO

BACKGROUND: Persistent high-risk human papillomavirus (HPV) infection is endorsed by the World Health Organization as an intermediate endpoint for evaluating HPV vaccine effectiveness/efficacy. There are different approaches to estimate the vaccine effectiveness/efficacy against persistent HPV infections. METHODS: We performed a systematic literature search in Pubmed to identify statistical approaches that have been used to estimate the vaccine effectiveness/efficacy against persistent HPV infections. We applied these methods to data of a longitudinal observational study to assess their performance and compare the obtained vaccine effectiveness (VE) estimates. RESULTS: Our literature search identified four approaches: the conditional exact test for comparing two independent Poisson rates using a binomial distribution, Generalized Estimating Equations for Poisson regression, Prentice Williams and Peterson total time (PWP-TT) and Cox proportional hazards regression. These approaches differ regarding underlying assumptions and provide different effect measures. However, they provided similar effectiveness estimates against HPV16/18 and HPV31/33/45 persistent infections in a cohort of young women eligible for routine HPV vaccination (range VE 93.7-95.1% and 60.4-67.7%, respectively) and seemed robust to violations of underlying assumptions. CONCLUSIONS: As the rate of subsequent infections increased in our observational cohort, we recommend PWP-TT as the optimal approach to estimate the vaccine effectiveness against persistent HPV infections in young women. Confirmation of our findings should be undertaken by applying these methods after longer follow-up in our study, as well as in different populations.


Assuntos
Papillomavirus Humano 18/imunologia , Papillomavirus Humano 31/imunologia , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Vacinas contra Papillomavirus/uso terapêutico , Vacinação , Adolescente , Adulto , Feminino , Seguimentos , Humanos , Imunogenicidade da Vacina , Estudos Longitudinais , Infecções por Papillomavirus/virologia , Prevalência , Resultado do Tratamento , Adulto Jovem
3.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32350070

RESUMO

The papillomavirus (PV) E2 protein is a critical regulator of viral transcription and genome replication. We previously reported that tyrosine (Y) 138 of HPV-31 E2 is phosphorylated by the fibroblast growth factor receptor 3 (FGFR3) kinase. In this study, we generated quasiviruses containing G418-selectable HPV-31 genomes with phosphodeficient phenylalanine mutant E2 Y138F and phosphomimetic glutamic acid mutant Y138E. We observed significantly fewer early viral transcripts immediately after infection with these Y138 mutant genomes even though E2 occupancy at the viral origin was equivalent to that of wild-type E2. Keratinocytes infected with Y138F quasiviruses formed stable colonies, and the genomes were maintained as episomes, while those infected with Y138E quasiviruses did not. We previously reported that the HPV-31 E2 Y138 mutation to glutamic acid did not bind to the Brd4 C-terminal motif (CTM). Here, we demonstrate that HPV-16 E2 Y138E bound to full-length Brd4 but not to the Brd4 CTM. We conclude that association of E2 with the Brd4 CTM is necessary for viral genome replication and suggest that this interaction can be regulated by phosphorylation of E2 Y138.IMPORTANCE Papillomavirus (PV) is a double-stranded DNA tumor virus infecting the cutaneous and mucosal epithelium. The PV E2 protein associates with a number of cellular factors to mediate replication of the HPV genome. Fibroblast growth factor receptor 3 (FGFR3) regulates HPV replication through phosphorylation of tyrosine 138 in the HPV E2 protein. Employing a quasivirus infection model and selection for G418 resistant genomes, we demonstrated that Y138 is a critical residue for Brd4 association and that inability to complex with Brd4 does not support episomal replication.


Assuntos
Papillomavirus Humano 31/metabolismo , Queratinócitos/metabolismo , Infecções por Papillomavirus/metabolismo , Plasmídeos/metabolismo , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Humanos , Queratinócitos/patologia , Queratinócitos/virologia , Mutação de Sentido Incorreto , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Fosforilação , Plasmídeos/genética , Tirosina , Proteínas do Envelope Viral/genética
4.
Tumori ; 106(5): 369-377, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32066343

RESUMO

OBJECTIVE: To investigate the prevalence of human papillomavirus (HPV) in archival head and neck cancer (HNC) collected from the Tong-Liao area, which is located in east Inner Mongolia, China. METHODS: The presence of HPV in 54 HNCs and 25 benign biopsies was detected and the sequence variation of the E6 gene in HPV-positive samples was analyzed to determine their lineage/sublineage classification. RESULTS: HPV was detected in only 4 out of 54 HNCs and no benign biopsies were found to be HPV-positive. After further p16INK4a immunostaining, only 3 cases of HNC were positive for both HPV and p16INK4a. Phylogenetic analysis of the isolated E6 gene shows that the HPV 16, HPV 31, and HPV 58 isolated in this study belong to lineage A. CONCLUSIONS: The prevalence of HPV in HNC from this area is very low. The lineage/sublineage classification of the 3 HPV types in HNC in this area is consistent with the previous reported data of HPV lineage distribution in cervical cancer within China.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias de Cabeça e Pescoço/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Adulto , Idoso , China/epidemiologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Neoplasias de Cabeça e Pescoço/epidemiologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 31/isolamento & purificação , Papillomavirus Humano 31/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologia , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Filogenia
5.
Theranostics ; 9(9): 2637-2645, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131058

RESUMO

Rationale: Early and accurate detection of disease is crucial for its prevention, identification, and treatment. However, most of disease diagnostics is still limited in clinical laboratories due to the need of complicated instruments and professional personnel. Herein, we reported a smartphone-based synergistically enhanced colorimetric method for molecular diagnostics in our point of care (POC) smart cup platform. Methods: A disposable microfluidic chip was developed for colorimetric loop-mediated isothermal amplification (LAMP) detection of multiple HPV DNA in our POC smart cup platform. The colorimetric detection takes advantage of synergistic effect of PPi4- and H+ ions, two byproducts of LAMP reaction. Color signal of LAMP assay was recorded and analyzed by our custom Android app (dubbed "Hue Analyzer"). Results: Our method not only significantly improves colorimetric readout, but also provides a 10-fold increase in detection sensitivity. It has been successfully applied for HPV-associated cancer screening with spiked saliva and clinical swab samples. Conclusion: The proposed POC diagnostic platform is completely compatible with other nucleic acid biomarkers and has great potential for personalized health monitoring and disease prevention.


Assuntos
Colorimetria/métodos , DNA Viral/genética , Detecção Precoce de Câncer/métodos , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Papillomavirus Humano 31/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Colo do Útero/virologia , Colorimetria/normas , DNA Viral/classificação , DNA Viral/isolamento & purificação , Detecção Precoce de Câncer/instrumentação , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Papillomavirus Humano 31/genética , Humanos , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Teste de Papanicolaou , Infecções por Papillomavirus/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Saliva/virologia , Sensibilidade e Especificidade , Smartphone
6.
Biologicals ; 58: 57-63, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30795963

RESUMO

The main purpose of this paper is to estimate the pre-vaccination prevalence of 12 hrHPV types among 564 women from Vojvodina province (Serbia). The corrected contingency coefficient (Ccorr) was used to estimate the importance of association of examined HPV types and cytological diagnosis. The highest association with the abnormal cytology was observed for HPV 16 (Ccorr = 0.493) in all age groups of participants. The effect of HPV 16 was especially clear within the group of women older than 35 years (Ccorr = 0.691), compared with women younger than 35 (Ccorr = 0.333). The molecular characterization at the level of L1 gene of HPV 16, 18, 31 and 33 variants was for the first time assessed in our region. Nearly all HPV 16 isolates cluster with variant lineage A (96.4%) the remaining isolates clustering with variant lineage D. All of HPV 18 and HPV 33 isolates are clustering within the lineage A while isolates of HPV 31 group with lineages A and C. This contributes to understanding of intrinsic geographical and biological differences of examined HPV types and could be useful for development of cervical cancer screening strategies in Vojvodina (Serbia) and diagnosis of HPV related cervical cancer in general.


Assuntos
Papillomavirus Humano 16 , Papillomavirus Humano 18 , Papillomavirus Humano 31 , Infecções por Papillomavirus/epidemiologia , Neoplasias do Colo do Útero/epidemiologia , Adulto , Idoso , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Prevalência , Sérvia/epidemiologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Adulto Jovem
7.
Gynecol Oncol ; 153(1): 26-33, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30638767

RESUMO

OBJECTIVES: Increasing evidence suggests that extended human papillomavirus (HPV) genotyping (beyond 16/18) is effective for risk stratification in women with normal cytology. This report provides extended genotyping results, using the BD Onclarity HPV Assay, for individual genotypes HPV16, 18, 31, 45, 51, and 52 ̶ and three pooled genotype results for HPV33/58, 35/39/68, and 56/59/66. METHODS: 27,037 women with normal cytology, ≥25 years, were enrolled into the Onclarity HPV trial during routine screening. Women positive for any HPV genotype were referred to colposcopy/biopsy. Hierarchical-ranked prevalence and risk values, associated with cervical intraepithelial neoplasia, grade 2 or worse (≥CIN2) or ≥CIN3, were calculated based on extended genotyping results. RESULTS: HPV 16 and 31 carried the highest risk for ≥CIN2 (11.6% and 12.1%, respectively) and ≥CIN3 (8.1% and 7.5%, respectively); these genotypes were the most prevalent in both ≥CIN2 (29.6% and 19.3%, respectively) and ≥CIN3 (43.7% and 22.5%, respectively). Of the other 12 genotypes, HPV 18, 33/58, and 52 comprised an intermediate risk band (≥CIN2 risk range: 4.9-6.8%; ≥CIN3 risk range: 3.9-5.0%). Genotypes 45, 51, 35/39/68, and 56/59/66 constituted the lowest risk band for both disease grades (≥CIN2 value risk range: 1.7-3.0%; ≥CIN3 value risk range: 1.2-3.6%). CONCLUSIONS: Extended genotyping stratifies risk for ≥CIN2/3 in the ≥25 year-old, normal cytology population. While baseline HPV 16/31 values exceeded the risk threshold for colposcopy referral, the management of women with normal cytology who were positive for the intermediate- or lower-risk genotypes may evolve based on refined risk estimates as well as clinical factors.


Assuntos
Neoplasia Intraepitelial Cervical/virologia , Papillomaviridae/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Adulto , Neoplasia Intraepitelial Cervical/epidemiologia , Neoplasia Intraepitelial Cervical/patologia , Colo do Útero/citologia , Colo do Útero/patologia , Colo do Útero/virologia , Feminino , Genótipo , Papillomavirus Humano 16/genética , Papillomavirus Humano 31/genética , Humanos , Estudos Longitudinais , Gradação de Tumores , Infecções por Papillomavirus/epidemiologia , Prevalência , Risco , Neoplasias do Colo do Útero/epidemiologia
9.
J Infect Dis ; 219(3): 382-390, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30299519

RESUMO

Background: Proactive recommendations for human papillomavirus (HPV) vaccines in Japan have been suspended for 5 years because of safety concerns. While no scientific evidence exists to substantiate these concerns, one reason given for not reinstating recommendations is the lack of reliable vaccine effectiveness (VE) data in a Japanese population. This study reports the VE of the bivalent HPV vaccine in Japanese women aged 20-22 years. Methods: During cervical screening between 2014 and 2016, women had Papanicolaou smears and HPV tests performed and provided data about their sexual history. Estimates of VE for vaccine-targeted HPV type 16 (HPV16) and 18 and cross-protection against other types were calculated. Results: Overall, 2197 women were tested, and 1814 were included in the analysis. Of these, 1355 (74.6%) were vaccinated, and 1295 (95.5%) completed the 3-dose schedule. In women sexually naive at vaccination, the pooled VEs against HPV16 and 18 and for HPV31, 45, and 52 were 95.5% (P < .01) and 71.9% (P < .01), respectively. When adjusted for number of sex partners and birth year, pooled VEs were 93.9% (P = .01) and 67.7% (P = .01) for HPV16 and 18 and HPV31, 45, and 52, respectively. Conclusions: The bivalent HPV vaccine is highly effective against HPV16 and 18. Furthermore, significant cross-protection against HPV31, 45, and 52 was demonstrated and sustained up to 6 years after vaccination. These findings should reassure politicians about the VE of bivalent HPV vaccine in a Japanese population.


Assuntos
Proteção Cruzada/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Adulto , Colo do Útero/imunologia , Detecção Precoce de Câncer , Feminino , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Papillomavirus Humano 31/imunologia , Humanos , Imunização , Programas de Imunização , Japão , Infecções por Papillomavirus/diagnóstico , Vacinas contra Papillomavirus/administração & dosagem , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia , Vacinação , Adulto Jovem
10.
PLoS Pathog ; 14(10): e1007367, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30312361

RESUMO

The life cycle of HPV is tied to the differentiation status of its host cell, with productive replication, late gene expression and virion production restricted to the uppermost layers of the stratified epithelium. HPV DNA is histone-associated, exhibiting a chromatin structure similar to that of the host chromosome. Although HPV chromatin is subject to histone post-translational modifications, how the viral life cycle is epigenetically regulated is not well understood. SETD2 is a histone methyltransferase that places the trimethyl mark on H3K36 (H3K36me3), a mark of active transcription. Here, we define a role for SETD2 and H3K36me3 in the viral life cycle. We have found that HPV positive cells exhibit increased levels of SETD2, with SETD2 depletion leading to defects in productive viral replication and splicing of late viral RNAs. Reducing H3K36me3 by overexpression of KDM4A, an H3K36me3 demethylase, or an H3.3K36M transgene also blocks productive viral replication, indicating a significant role for this histone modification in facilitating viral processes. H3K36me3 is enriched on the 3' end of the early region of the high-risk HPV31 genome in a SETD2-dependent manner, suggesting that SETD2 may regulate the viral life cycle through the recruitment of H3K36me3 readers to viral DNA. Intriguingly, we have found that activation of the ATM DNA damage kinase, which is required for productive viral replication, is necessary for the maintenance of H3K36me3 on viral chromatin and for processing of late viral RNAs. Additionally, we have found that the HPV31 E7 protein maintains the increased SETD2 levels in infected cells through an extension of protein half-life. Collectively, our findings highlight the importance of epigenetic modifications in driving the viral life cycle and identify a novel role for E7 as well as the DNA damage response in the regulation of viral processes through epigenetic modifications.


Assuntos
Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Papillomavirus Humano 31/fisiologia , Queratinócitos/virologia , Infecções por Papillomavirus/virologia , Replicação Viral , Células Cultivadas , Cromatina , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Queratinócitos/metabolismo , Metilação , Infecções por Papillomavirus/genética , Ligação Proteica , RNA Viral/genética
11.
PLoS One ; 13(10): e0205933, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30356257

RESUMO

Human papillomaviruses (HPVs) are a group of circular double-stranded DNA viruses, showing severe tropism to mucosal tissues. A subset of HPVs, especially HPV16 and 18, are the primary etiological cause for several epithelial cell malignancies, causing about 5.2% of all cancers worldwide. Due to the high prevalence and mortality, HPV-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. Achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. Previous information has shown that E5, E6, and E7 early proteins are responsible for the induction and maintenance of HPV-associated cancers. Therefore, the prediction of major histocompatibility complex (MHC) class I T cell epitopes of HPV16, 18, 31 and 45 oncoproteins was targeted in this study. For this purpose, a two-step plan was designed to identify the most probable CD8+ T cell epitopes. In the first step, MHC-I and II binding, MHC-I processing, MHC-I population coverage and MHC-I immunogenicity prediction analyses, and in the second step, MHC-I and II protein-peptide docking, epitope conservation, and cross-reactivity with host antigens' analyses were carried out successively by different tools. Finally, we introduced five probable CD8+ T cell epitopes for each oncoprotein of the HPV genotypes (60 epitopes in total), which obtained better scores by an integrated approach. These predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the HPV-associated cancers. Additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for DNA- or peptide-based vaccine development.


Assuntos
Epitopos/análise , Proteínas Oncogênicas Virais/química , Papillomaviridae/metabolismo , Alelos , Sequência de Aminoácidos , Simulação por Computador , Epitopos/química , Antígenos de Histocompatibilidade Classe I/química , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 31/metabolismo , Humanos , Peptídeos/química , Prevalência
12.
Hum Vaccin Immunother ; 14(8): 2025-2033, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29683766

RESUMO

Current available human papillomavirus (HPV) vaccines are based on the major capsid protein L1 virus-like particles (VLPs), which mainly induce type-specific neutralizing antibodies against vaccine types. Continuing to add more types of VLPs in a vaccine raises the complexity and cost of production which remains the principal impediment to achieve broad implementation of HPV vaccines, particularly in developing regions. In this study, we constructed 16L1-31L2 chimeric VLP (cVLP) by displaying HPV31 L2 aa.17-38 on the h4 coil surface region of HPV16 L1, and assessed its immunogenicity in mouse model. We found that the cVLP adjuvanted with alum plus monophosphoryl lipid A could induce cross-neutralizing antibody responses against 16 out of 17 tested HPV pseudoviruses, and the titer against HPV16 was as high as that was induced by HPV16 L1VLP (titer > 105), more importantly, titers over 103 were observed against two HR-HPVs including HPV31 (titer, 2,200) and -59 (titer, 1,013), among which HPV59 was not covered by Gardasil-9, and medium or low titers of cross-neutralizing antibodies against other 13 tested HPV pseudoviruses were also observed. Our data demonstrate that 16L1-31L2 cVLP is a promising candidate for the formulation of broader spectrum HPV vaccines.


Assuntos
Papillomavirus Humano 16/imunologia , Papillomavirus Humano 31/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteção Cruzada/genética , Proteção Cruzada/imunologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 31/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/genética , Peptídeos , Engenharia de Proteínas , Vacinas de Partículas Semelhantes a Vírus/genética
13.
Mol Med Rep ; 17(4): 5498-5507, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29393441

RESUMO

Human papillomavirus (HPV) type 31 is an important pathogenic subtype associated with cervical cancer. The aims of the present study were to analyze E5, E6, E7 and L1 gene mutations of HPV­31 among females, and to elucidate the evolutionary associations between them. In total, 87 positive samples were collected. The E5, E6, E7 and L1 genes were amplified by polymerase chain reaction and sequenced. Subsequently, two phylogenetic trees were constructed from the nucleotide sequences of the E5, E6 and E7 and the L1 variants of HPV­31. In total, 31 mutation sites of E5, E6 and E7 genes were identified, of which 16 were non­synonymous. T4053A (F80I), C285T (H60Y), C520T (A138V) and A743G (K62E) were the most common non­synonymous mutations. A total of 30 mutation sites of L1 genes were identified, of which four were non­synonymous. The most common non­synonymous mutations of L1 genes were A6350G (T29A) and C6372A (T36N). By phylogenetic analysis, A and C variants were most frequently detected, while B variants were less frequently detected in this population. The sequence variation data obtained in the present study provides a foundation for future research regarding HPV­induced oncogenesis, and may prove valuable for developing diagnostic probes and in the design of HPV vaccines for targeted populations.


Assuntos
Variação Genética , Papillomavirus Humano 31/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Virais/genética , DNA Viral , Evolução Molecular , Genótipo , Humanos , Filogenia , Seleção Genética , Análise de Sequência de DNA
14.
J Virol ; 92(4)2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29167339

RESUMO

The papillomavirus E2 protein regulates transcription, replication, and nuclear retention of viral genomes. Phosphorylation of E2 in the hinge region has been suggested to modulate protein stability, DNA-binding activity, and chromosomal attachment. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. Mass spectrometry analyses of human papillomavirus 31 (HPV31) E8^E2 and E2 proteins identify phosphorylated S78, S81, and S100 in E8^E2 and S266 and S269 in E2 in their hinge regions. Phos-tag analyses of wild-type and mutant proteins indicate that S78 is a major phosphorylation site in E8^E2, but the corresponding S266 in E2 is not. Phosphorylation at S78 regulates E8^E2's repression activity of reporter constructs, whereas the corresponding E2 mutants do not display a phenotype. Phosphorylation at S78 does not alter E8^E2's protein stability, nuclear localization, or binding to DNA or to cellular NCoR/SMRT complexes. Surprisingly, in the context of HPV31 genomes, mutation of E8^E2 S78 does not modulate viral replication or transcription in undifferentiated or differentiated cells. However, comparative transcriptome analyses of differentiated HPV31 E8^E2 S78A and S78E cell lines reveal that the expression of a small number of cellular genes is changed. Validation experiments suggest that the transcription of the cellular LYPD2 gene is altered in a phospho-S78 E8^E2-dependent manner. In summary, our data suggest that phosphorylation of S78 in E8^E2 regulates its repression activity by a novel mechanism, and this seems to be important for the modulation of host cell gene expression but not viral replication.IMPORTANCE Posttranslational modification of viral proteins is a common feature to modulate their activities. Phosphorylation of serine residues S298 and S301 in the hinge region of the bovine papillomavirus type 1 E2 protein has been shown to restrict viral replication. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. A large fraction of HPV31 E8^E2 is phosphorylated at S78 in the hinge region, and this is important for E8^E2's repression activity. Surprisingly, phosphorylation at S78 in E8^E2 has no impact on viral replication in tissue culture but rather seems to modulate the expression of a small number of cellular genes. This may indicate that phosphorylation of viral transcription factors serves to broaden their target gene specificity.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/química , Papillomavirus Humano 31 , Fosforilação , Proteínas Virais/química , Regulação Viral da Expressão Gênica , Genoma Viral , Células HeLa , Humanos , Queratinócitos/virologia , Mutação , Transcrição Genética , Replicação Viral
15.
Theranostics ; 7(16): 3814-3823, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29109779

RESUMO

BACKGROUND: The most recent (2012) worldwide estimates from International Agency for Research on Cancer indicate that approximately 528,000 new cases and 270,000 deaths per year are attributed to cervical cancer worldwide. The disease is preventable with HPV vaccination and with early detection and treatment of pre-invasive cervical intraepithelial neoplasia, CIN. Antibodies (Abs) to HPV proteins are under investigation as potential biomarkers for early detection. METHODS: To detect circulating HPV-specific IgG Abs, we developed programmable protein arrays (NAPPA) that display the proteomes of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). Arrays were probed with sera from women with CIN 0/I (n=78), CIN II/III (n=84), or invasive cervical cancer (ICC, n=83). RESULTS: Abs to any early (E) HPV protein were detected less frequently in women with CIN 0/I (23.7%) than women with CIN II/III (39.0%) and ICC (46.1%, p<0.04). Of the E Abs, anti-E7 Abs were the most frequently detected (6.6%, 19.5%, and 30.3%, respectively). The least frequently detected Abs were E1 and E2-Abs in CIN 0/I (1.3%) and E1-Abs in CIN II/III (1.2%) and ICC (7.9%). HPV16-specific Abs correlated with HPV16 DNA detected in the cervix in 0% of CIN 0/I, 21.2% of CIN II/III, and 45.5% of ICC. A significant number (29 - 73%) of E4, E7, L1, and L2 Abs had cross-reactivity between HPV types. CONCLUSION: HPV protein arrays provide a valuable high-throughput tool for measuring the breadth, specificity, and heterogeneity of the serologic response to HPV in cervical disease.


Assuntos
Anticorpos/sangue , Neoplasia Intraepitelial Cervical/sangue , Neoplasia Intraepitelial Cervical/diagnóstico , Detecção Precoce de Câncer/métodos , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/diagnóstico , Adulto , Neoplasia Intraepitelial Cervical/imunologia , Feminino , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Papillomavirus Humano 31/imunologia , Humanos , Análise Serial de Proteínas/métodos , Neoplasias do Colo do Útero/imunologia
16.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28768864

RESUMO

The papillomavirus (PV) E2 protein is a DNA binding, protein interaction platform that recruits viral and host factors necessary for transcription and replication. We recently discovered phosphorylation of a tyrosine (Y102) in bovine PV (BPV) E2. To identify the responsible factor, we tested several candidate tyrosine kinases that are highly expressed in keratinocytes for binding to BPV-1 E2. Fibroblast growth factor receptor 3 (FGFR3) coimmunoprecipitated with the BPV-1 E2 protein, as did human papillomavirus 31 (HPV-31) E2, which also colocalized with FGFR3 within the nucleus. A constitutively active mutant form of FGFR3 decreased BPV-1 and HPV-31 transient replication although this result also occurred in a BPV-1 E2 mutant lacking a previously identified phosphorylation site of interest (Y102). Furthermore, FGFR3 depletion in cell lines that maintain HPV-31 episomes increased viral copy number. These results suggest that FGFR3 kinase activity may regulate the PV reproductive program through phosphorylation of the E2 protein although this is unlikely to occur through the Y102 residue of HPV E2.IMPORTANCE The papillomavirus (PV) is a double-stranded DNA tumor virus infecting cervix, mouth, and throat tissues. The viral protein E2 is responsible for the replication of the virus. Understanding the mechanisms of the replicative life cycle of the virus may bring to light direct targets and treatments against viral infection. We recently found that the fibroblast growth factor receptor 3 (FGFR3) interacts with and mediates PV E2 function through phosphorylation of the E2 protein. Our study suggests that the function of the E2 protein may be regulated through a direct FGFR3 target during the maintenance stage of the PV life cycle.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 31/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Fosfotransferases/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Animais , Bovinos , Replicação do DNA , Papillomavirus Humano 31/enzimologia , Humanos , Fosforilação , Plasmídeos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Tirosina/química
17.
J Virol ; 91(22)2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28835500

RESUMO

The human papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase E6AP/UBE3A to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP autoubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a protein kinase A phosphoacceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study, we have analyzed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced autodegradation of E6AP but does affect HPV-18 E6-induced autodegradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and nonphosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas nonphosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phosphoregulation of E6AP, both in terms of its enzymatic activity and its subcellular distribution.IMPORTANCE Recent reports demonstrate the importance of phosphoregulation of E6AP for its normal enzymatic activity. Here, we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to autodegradation and suggests that this is not a simple stimulation of an already-existing activity but rather a redirection of E6AP activity toward itself. Furthermore, E6-mediated regulation of the subcellular distribution of phospho-E6AP appears to be dependent, in part, upon the 14-3-3 family of proteins.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 31/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/genética , Núcleo Celular/virologia , Citoplasma/genética , Citoplasma/virologia , Proteínas de Ligação a DNA/genética , Proteína 1 Homóloga a Discs-Large , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Papillomavirus Humano 31/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/genética , Fosforilação , Transporte Proteico , Proteólise , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
Protein Expr Purif ; 133: 110-120, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28267627

RESUMO

Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be prevented by vaccination with HPV vaccines derived from eukaryotic expression systems. Here, we report the soluble expression of the major capsid protein L1 of HPV31, a dominant carcinogenic HPV genotype, in Escherichia coli. HPV31 L1 protein and its elongated form (L1+) were observed in SDS-PAGE and CE-SDS analysis, generated by the native HPV31 L1 gene with a TAA stop codon. Replacing the TAA with TAG but not TGA could completely terminate protein translation. Mass spectrometry sequencing showed that L1+ comprised L1 with a C-terminal extension of 38 amino acids (aa). RNA folding analysis revealed that the unfaithful L1+ expression may result from translational read-through, as TAG is more stable and accessible than the other stop codons. The 38-aa elongated fragment perturbs self-assembly of HPV31 L1+, as shown in size and morphology analyses. By 3D cryo-electron microscopy structure determination, we show self-assembly of purified HPV31 L1 (TAG) VLPs into T = 7 icosahedral symmetry particles, resembling the native HPV virion. Finally, through additional characterization and antigenicity/immunogenicity assays, we verified that the E.coli-derived HPV31 VLPs are an ideal immunogen for HPV vaccine development. Our findings outline a codon optimization stratagem for protein expression and provide a method for the in-depth investigation of prokaryotic translation regulation.


Assuntos
Proteínas do Capsídeo , Códon de Terminação , Expressão Gênica , Papillomavirus Humano 31/genética , Mutagênese , Proteínas Oncogênicas Virais , Vacinas contra Papillomavirus , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Papillomavirus Humano 31/metabolismo , Humanos , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Vacinas contra Papillomavirus/biossíntese , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
19.
Eur J Obstet Gynecol Reprod Biol ; 210: 157-165, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28039759

RESUMO

OBJECTIVE: High-grade vaginal intraepithelial neoplasia (vaginal HSIL) represents an uncommon entity. Here, we sought to identify predictors for recurrence and risk factor for developing genital cancers after primary treatment for vaginal HSIL. METHODS: Data of consecutive 5104 women who had human papillomavirus (HPV) DNA test were searched for identify women with histological confirmed vaginal HSIL. Disease-free interval and the risk of developing HPV-related gynecological cancers were assessed using Kaplan-Meier and Cox proportional hazard models. RESULTS: Overall, 77 patients were included. After a mean (SD) follow-up of 69.3 (33.0) months, 11 (14%) and 4 (5%) patients experienced vaginal HSIL recurrence and the occurrence of HPV-related gynecological cancers, respectively. Via multivariate analysis factors predicting for vaginal HSIL recurrence were infection from HPV31 at diagnosis (HR: 5.0 (95%CI:1.17, 21.3); p=0.03) and persistence of HPV infection after treatment (HR: 7.0 (95%CI:1.54, 31.6); p=0.01). Additionally, patients who had LASER ablation experienced a trend toward a lower risk of recurrence in comparison to medical treatment (HR: 0.20 (95%CI:0.03, 1.09); p=0.06). Considering the occurrence of HPV-related gynecological cancers, we observed that no factors independently correlated with this risk; while, a trend towards higher risk was observed for women with HIV infection (HR:16.4 (95%CI:0.90, 300.1); p=0.06) and persistence of HPV infection (HR: 13.3 (95%CI:0.76, 230.2); p=0.07). CONCLUSIONS: Patients affected by vaginal HSIL experienced a relatively high risk of recurrence. Persistence of HPV after treatment and pretreatment HPV-31 infection predicts for high-grade vaginal intraepithelial neoplasia recurrence. Further investigations are warranted in order to corroborate our data.


Assuntos
Carcinoma in Situ/virologia , Papillomavirus Humano 31/isolamento & purificação , Recidiva Local de Neoplasia/virologia , Neoplasias Vaginais/virologia , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos
20.
Int J Cancer ; 140(8): 1747-1756, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28052328

RESUMO

Studies of the clinical relevance of human papillomavirus (HPV) DNA load have focused mainly on HPV16 and HPV18. Data on other oncogenic types are rare. Study subjects were women enrolled in the atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL) triage study who had ≥1 of 11 non-HPV16/18 oncogenic types detected during a 2-year follow-up at 6-month intervals. Viral load measurements were performed on the first type-specific HPV-positive specimens. The association of cervical intraepithelial neoplasia grades 2-3 (CIN2/3) with type-specific HPV DNA load was assessed with discrete-time Cox regression. Overall, the increase in the cumulative risk of CIN2/3 per 1 unit increase in log10 -transformed viral load was statistically significant for four types within species 9 including HPV31 (adjusted hazard ratio [HR adjusted ] = 1.32: 95% confidence interval [CI], 1.14-1.52), HPV35 (HR adjusted = 1.47; 95% CI, 1.23-1.76), HPV52 (HR adjusted = 1.14; 95% CI, 1.01-1.30) and HPV58 (HR adjusted = 1.49; 95% CI, 1.23-1.82). The association was marginally significant for HPV33 (species 9) and HPV45 (species 7) and was not appreciable for other types. The per 1 log10 -unit increase in viral load of a group of species 9 non-HPV16 oncogenic types was statistically significantly associated with risk of CIN2/3 for women with a cytologic diagnosis of within normal limits, ASC-US, or LSIL at the first HPV-positive visit but not for those with high-grade SIL. Findings suggest that the viral load-associated risk of CIN2/3 is type-dependent, and mainly restricted to the species of HPV types related to HPV16, which shares this association.


Assuntos
Neoplasia Intraepitelial Cervical/virologia , DNA Viral/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/virologia , Neoplasia Intraepitelial Cervical/epidemiologia , DNA Viral/isolamento & purificação , Detecção Precoce de Câncer , Feminino , Genótipo , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/patogenicidade , Papillomavirus Humano 31/genética , Papillomavirus Humano 31/patogenicidade , Humanos , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/epidemiologia , Esfregaço Vaginal , Carga Viral
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