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1.
Gene ; 782: 145533, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33636291

RESUMO

BACKGROUND: Human papillomavirus is the most common sexually transmitted infection. It is associated with different cancers, mainly cervical cancer, which remains the fourth most frequent cancer among women worldwide; it is also related to anogenital (anus, vulvar, vagina, and penis) and oropharyngeal cancers. Vaccination against HPV infection is the major way of prevention, and it has demonstrated impressive efficacy in reducing cervical cancer incidence. Nowadays, all the licensed HPV recombinant vaccines were designed based on HPV major capsid L1 protein. However, some variations in the HPV L1 gene sequence may induce structural changes within the L1 protein, which may alter the affinity and interaction of monoclonal antibodies (MAbs) with L1 protein epitopes, and influence host immune response and recognition. Hence, the importance of accuracy in delineating epitopes relevant to vaccine design and defining genetic variations within antigenic regions in the L1 gene to predict its impact on prophylactic vaccine efficiency. The present review reports the sequence variations in HR-HPV L1 gene isolates from different countries around the world, which may help to understand the effect of HPV L1 gene variations on vaccine efficiency. METHODS: Research studies were retrieved from PubMed, Google Scholar, Science direct, and the National Center for Biotechnology Information (NCBI) database. A total of 31 articles describing genetic variations within the major capsid L1 gene and conducted in Africa, Europe, America and Asia were found. Only 26 studies conducted on HPV16, 18, 31, 33, 58, 45 and 52 which are the targets of HPV prophylactic vaccines, and which reported genetic variations within the L1 gene, were selected and evaluated in this review. FINDINGS: We found a total of 87, 49, 11, 7, 22, 3, and 17 non-synonymous single nucleotide polymorphisms (SNPs) within HPV16, HPV18, HPV31, HPV58, HPV45, and HPV52 L1 gene, respectively. Four mutations were frequently observed in HPV16 L1 sequences: T353P in the HI loop, H228D in the EF loop, T266A in the FG loop, and T292A in the FG loop. Two mutations in HPV58 L1 sequences: T375N in the HI loop and L150F in the DE loop. Three mutations in HPV33 L1 sequences: T56N in the BC loop, G133S in the DE loop, T266K in the FG loop. Other mutations were found in HPV18, HPV45, and HPV52 L1 sequences. Some were found in different countries, and others were specific to a given population. Furthermore, some variations were located on peptide binding epitopes and lead to a modification of epitopes, which may influence MAbs interactions. Others need further investigations due to the lack of studies. CONCLUSION: This study investigated the major capsid L1 genetic diversity of HPV16, 18, 31, 33, 58, 45, and 52 circulating in different populations around the world. Further investigations should be conducted to confirm their effect on immunogenicity and prophylactic vaccine efficiency.


Assuntos
Proteínas do Capsídeo/genética , Variação Genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Vacinas contra Papillomavirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Proteínas do Capsídeo/imunologia , Saúde Global , Humanos , Imunogenicidade da Vacina/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/fisiologia , Vacinas contra Papillomavirus/genética
2.
Recent Results Cancer Res ; 217: 141-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33200365

RESUMO

Human papillomaviruses (HPVs) are small DNA viruses that infect basal epithelial cells and are the causative agents of cervical, anogenital, as well as oral cancers. High-risk HPVs are responsible for nearly half of all virally induced cancers. Viral replication and amplification are intimately linked to the stratified epithelium differentiation program. The E6 and E7 proteins contribute to the development of cancers in HPV positive individuals by hijacking cellular processes and causing genetic instability. This genetic instability induces a robust DNA damage response and activating both ATM and ATR repair pathways. These pathways are critical for the productive replication of high-risk HPVs, and understanding how they contribute to the viral life cycle can provide important insights into HPV's role in oncogenesis. This review will discuss the role that differentiation and the DNA damage responses play in productive replication of high-risk HPVs as well as in the development of cancer.


Assuntos
Alphapapillomavirus , Reparo do DNA , Proteínas Oncogênicas Virais , Papillomaviridae , Infecções por Papillomavirus , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Replicação Viral
3.
ACS Appl Mater Interfaces ; 13(1): 298-305, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33382593

RESUMO

Most DNA-based electrochemiluminescence (ECL) biosensors are established through the self-assembly of thiolated single-stranded DNA (ssDNA) probes on the Au electrode surface. Because of this random assembly process, a significant discrepancy exists in the distribution of a modified DNA film on different electrodes, which greatly affects the reproducibility of a biosensor. In this study, a porous bovine serum albumin (BSA) layer was first modified on the electrode surface, which can improve the position distribution and spatial orientation of the self-assembly ssDNA probe. It was then coupled with hyperbranched rolling circle amplification to develop the high-reproducibility-and-sensitivity ECL biosensor for human papillomavirus 16 E6 and E7 oncogene detection. In the presence of the target DNA, the surface of the electrode accumulates abundant amplified products through reaction, which contain double-stranded DNA (dsDNA) fragments of different lengths, followed by plentiful dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen)32+, acting as an ECL indicator) insertion into grooves of dsDNA fragments, and a strong signal can be detected. There is a linear relationship between the signal and the target concentration range from 10 fM to 15 pM, and the detection limit is 7.6 fM (S/N = 3). After the BSA modification step, the relative standard deviation was reduced from 9.20 to 3.96%, thereby achieving good reproducibility. The proposed ECL strategy provides a new method for constructing high-reproducibility-and-sensitivity ECL biosensors.


Assuntos
Técnicas Biossensoriais/métodos , Papillomavirus Humano 16/isolamento & purificação , Proteínas Oncogênicas Virais/análise , Proteínas E7 de Papillomavirus/análise , Proteínas Repressoras/análise , Soroalbumina Bovina/química , Animais , Bovinos , Colo do Útero/virologia , Sondas de DNA/química , Sondas de DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Técnicas Eletroquímicas/métodos , Feminino , Papillomavirus Humano 16/química , Humanos , Limite de Detecção , Substâncias Luminescentes , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Proteínas Oncogênicas Virais/genética , Compostos Organometálicos/química , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/diagnóstico , Fenantrolinas/química , Proteínas Repressoras/genética , Reprodutibilidade dos Testes , Rutênio/química
4.
PLoS One ; 15(12): e0242465, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332365

RESUMO

Peroxiredoxin 2 (PRDX2) is upregulated in various cancers including oral squamous cell carcinoma (OSCC). It is a known tumor promoter in some cancers, but its role in OSCC is unclear. This study aimed to investigate the effect of arecoline, an alkaloid of the betel nut, and human papillomavirus type 16 (HPV16) E6/E7 oncoproteins on induction of PRDX2 expression, and also the effects of PRDX2 overexpression in oral cell lines. Levels of PRDX2 protein were determined using western blot analysis of samples of exfoliated normal oral cells (n = 75) and oral lesion cells from OSCC cases (n = 75). Some OSCC cases were positive for HPV infection and some patients had a history of betel quid chewing. To explore the level of PRDX2 by western blot, the proteins were extracted from oral cell lines that were treated with arecoline or retroviruses containing HPV16 E6 gene and HPV16 E6/E7 expressing vector. For analysis of PRDX2 functions, cell proliferation, cell-cycle progression, apoptosis and migration was compared between oral cells overexpressing PRDX2 and cells with PRDX2-knockdown. PRDX2 expression levels tended to be higher in OSCC samples that were positive for HPV infection and had history of betel quid chewing. Arecoline treatment in vitro at low concentrations and overexpression of HPV16 E6 or E6/E7 in oral cells induced PRDX2 overexpression. Interestingly, in oral cells, PRDX2 promoted cell proliferation, cell-cycle progression (G2/M phase), cell migration and inhibited apoptosis. Upregulation of PRDX2 in oral cells was induced by arecoline and HPV16 oncoproteins and promoted growth of OSCC cells.


Assuntos
Arecolina/farmacologia , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Peroxirredoxinas/genética , Proteínas Repressoras/genética , Idoso , Apoptose/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Papillomavirus Humano 16/química , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/enzimologia , Neoplasias Bucais/patologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Transfecção
5.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333786

RESUMO

Although the effect of hypoxia on p53 in human papillomavirus (HPV)-positive cancer cells has been studied for decades, the impact of p53 regulation on downstream targets and cellular adaptation processes during different periods under hypoxia remains elusive. Here, we show that, despite continuous repression of HPV16 E6/E7 oncogenes, p53 did not instantly recover but instead showed a biphasic regulation marked by further depletion within 24 h followed by an increase at 72 h. Of note, during E6/E7 oncogene suppression, lysosomal degradation antagonizes p53 reconstitution. Consequently, the transcription of p53 responsive genes associated with senescence (e.g., PML and YPEL3) cannot be upregulated. In contrast, downstream genes involved in autophagy (e.g., DRAM1 and BNIP3) were activated, allowing the evasion of senescence under hypoxic conditions. Hence, dynamic regulation of p53 along with its downstream network of responsive genes favors cellular adaptation and enhances cell survival, although the expression of the viral E6/E7-oncogenes as drivers for proliferation remained inhibited under hypoxia.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Papillomavirus Humano 16/metabolismo , Infecções por Papillomavirus/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Hipóxia Celular/genética , Senescência Celular/genética , Regulação para Baixo , Feminino , Papillomavirus Humano 16/genética , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
6.
Arch Virol ; 165(11): 2589-2597, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32876794

RESUMO

Marek's disease (MD) is a contagious avian viral disease that is responsible for large economic losses to farmers. The disease is caused by Marek's disease virus (species Gallid alphaherpesvirus 2), which causes neurological lesions, immune suppression, and tumor proliferation of lymphoid cells that invade a large number of organs and tissues. Despite widespread vaccination, Marek's disease virus (MDV), has shown a continuous increase in its virulence and has acquired the ability to overcome immune responses induced by vaccines. In the present study, the oncogenic serotype MDV-1 was detected by real-time PCR in DNA samples extracted from organs developing tumor infiltrations. Identification of the pathotype based on a 132-bp tandem repeat and sequencing and phylogenetic analysis of the Meq gene and its encoded protein allowed classification of the isolated viruses as "very virulent", with two new and unique mutations in the Meq gene resulting in amino acid substitutions. Sequencing of pp38, vIl-8, UL1 and UL44 genes did not reveal any new mutations that were characteristic of the Tunisian isolates or correlated with virulence. These results raised concerns about the ability of HVT and CVI988 vaccines, which are currently used in Tunisia and other countries, to protect chickens against highly virulent virus strains.


Assuntos
Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 2/patogenicidade , Proteínas Oncogênicas Virais/genética , Filogenia , Sequência de Aminoácidos , Animais , Galinhas/virologia , DNA Viral/química , Doença de Marek/virologia , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Tunísia , Virulência/genética
7.
PLoS Pathog ; 16(8): e1008792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813746

RESUMO

Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proliferação de Células , Queratinócitos/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/etiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Humanos , Queratinócitos/imunologia , Queratinócitos/virologia , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genética
8.
J Med Microbiol ; 69(7): 960-970, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32510304

RESUMO

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Brasil , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Colo do Útero/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16/patogenicidade , Humanos , Complexo Antígeno L1 Leucocitário/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Carga Viral/métodos , Vírion/imunologia
9.
PLoS Pathog ; 16(6): e1008624, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555725

RESUMO

Human papillomaviruses (HPV) are a major cause of malignancy worldwide. They are the aetiological agents of almost all cervical cancers as well as a sub-set of other anogenital and head and neck cancers. Hijacking of host cellular pathways is essential for virus pathogenesis; however, a major challenge remains to identify key host targets and to define their contribution to HPV-driven malignancy. The Hippo pathway regulates epithelial homeostasis by down-regulating the function of the transcription factor YAP. Increased YAP expression has been observed in cervical cancer but the mechanisms driving this increase remain unclear. We found significant down-regulation of the master Hippo regulatory kinase STK4 (also termed MST1) in cervical disease samples and cervical cancer cell lines compared with healthy controls. Re-introduction of STK4 inhibited the proliferation of HPV positive cervical cells and this corresponded with decreased YAP nuclear localization and decreased YAP-dependent gene expression. The HPV E6 and E7 oncoproteins maintained low STK4 expression in cervical cancer cells by upregulating the oncomiR miR-18a, which directly targeted the STK4 mRNA 3'UTR. Interestingly, miR-18a knockdown increased STK4 expression and activated the Hippo pathway, significantly reducing cervical cancer cell proliferation. Our results identify STK4 as a key cervical cancer tumour suppressor, which is targeted via miR-18a in HPV positive tumours. Our study indicates that activation of the Hippo pathway may offer a therapeutically beneficial option for cervical cancer treatment.


Assuntos
Transformação Celular Viral , MicroRNAs/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteínas Serina-Treonina Quinases/genética , RNA Neoplásico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
10.
Int J Exp Pathol ; 101(1-2): 45-54, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32436348

RESUMO

Oral cancer causes significant global mortality and has a five-year survival rate of around 64%. Poor prognosis results from late-stage diagnosis, highlighting an important need to develop better approaches to detect oral premalignant lesions (OPLs) and identify which OPLs are at highest risk of progression to oral squamous cell carcinoma (OSCC). An appropriate animal model that reflects the genetic, histologic, immunologic, molecular and gross visual features of human OSCC would aid in the development and evaluation of early detection and risk assessment strategies. Here, we present an experimental PIK3CA + 4NQO transgenic mouse model of oral carcinogenesis that combines the PIK3CA oncogene mutation with oral exposure to the chemical carcinogen 4NQO, an alternate experimental transgenic mouse model with PIK3CA as well as E6 and E7 mutations, and an existing wild-type mouse model based on oral exposure to 4NQO alone. We compare changes in dorsal and ventral tongue gross visual appearance, histologic features and molecular biomarker expression over a time course of carcinogenesis. Both transgenic models exhibit cytological and architectural features of dysplasia that mimic human disease and exhibit slightly increased staining for Ki-67, a cell proliferation marker. The PIK3CA + 4NQO model additionally exhibits consistent lymphocytic infiltration, presents with prominent dorsal and ventral tongue tumours, and develops cancer quickly relative to the other models. Thus, the PIK3CA + 4NQO model recapitulates the multistep genetic model of human oral carcinogenesis and host immune response in carcinogen-induced tongue cancer, making it a useful resource for future OSCC studies.


Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação , Quinolonas , Carcinoma de Células Escamosas de Cabeça e Pescoço/induzido quimicamente , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Neoplasias da Língua/induzido quimicamente , Neoplasias da Língua/genética , 4-Nitroquinolina-1-Óxido , Animais , Proliferação de Células , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Progressão da Doença , Linfócitos/patologia , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Tempo , Neoplasias da Língua/patologia
11.
Gene ; 747: 144682, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304786

RESUMO

Human Papillomavirus 16 (HPV16) is the most oncogenic HPV and the most associated genotype with cervical cancer development and progression. Currently, all developed vaccines are targeting HPV16 and were designed based on the major L1 capsid protein. Thus, evaluation of the diversity of HPV16 L1 sequence, mainly in the antigenic regions, will be of a great interest to assess the efficacy of the prophylactic vaccines and to predict the impact of genetic variations in these regions on the vaccination-induced immunity. A total of 377 HPV16 L1 sequences, published in public domain GenBank database, from the Americas, Africa, Asia, and Europe were collected and assembled. A total of 626 mutation events affecting 83 distinct nucleotides into the five antigenic regions of L1 gene of HPV16 were reported, and most SNPs were located in DE (27.38%, 23/83) and FG (31%, 26/83) loops. Overall, 4 mutations were frequently found in HPV16 sequences: T176N and N181T in EF loop; A266T in the FG loop and T353P/I/N HI loop. Of particular interest, some SNPs are ubiquitous and were found in all populations whereas others were population specific and their presence was limited to one or 2 at the maximum. Association between mutations in the antigenic regions and ethnicity was also investigated and showed that mutations in BC and DE loops were present with no significant difference in sequences from Europe, Asia, America and Africa. However, most mutations in FG loop are reported in sequences from European cases and are less pronounced in cases from America and Asia, whereas mutations EF and HI loops prevail in Asian cases. These data highlight a high number of variant amino acid residues that could affect the vaccination-induced immunity and impact the effectiveness of the prophylactic vaccination to fight against HPV, warranting the need of further investigation for vaccines and natural history studies of HPV16.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Variação Genética , Papillomavirus Humano 16/genética , Imunidade , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Vacinação , Aminoácidos/genética , Antígenos Virais/imunologia , Sequência de Bases , Grupos Étnicos/genética , Humanos , Modelos Moleculares , Mutação/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética
12.
PLoS One ; 15(4): e0232105, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320451

RESUMO

Cervical cancer is a significant public health problem, especially in low- and middle-income countries, where women have little access to cervical cancer screening; consequently 80% of cervical cancer related mortality occurs in these regions. The development of screening methods that need less infrastructure thus represents an urgent medical need. The study aims to compare the detection rates of high-risk human papillomavirus 16 and 18 E6 oncoprotein in urine, vaginal self-collected, and cervical scrapes of women using the OncoE6™ Cervical Test and compare the HPV16 and/or HPV18 E6 detection rates with the HPV DNA testing. Paired urine, vaginal self-collected and cervical specimens were collected from 124 women who participated in cervical cancer screening or treatment in this proof-of-concept study and underwent to HPV16/18-E6 testing and high-risk HPV DNA testing prior to treatment of cervical neoplasia or cancer. Concordance between urinary, vaginal and cervical HPV16/18-E6 and HPV-DNA testing was evaluated for patients classified as negative group (

Assuntos
Proteínas de Ligação a DNA/urina , Imunoensaio/métodos , Proteínas Oncogênicas Virais/urina , Proteínas Repressoras/urina , Adulto , Proteínas de Ligação a DNA/genética , Feminino , Testes de DNA para Papilomavírus Humano , Papillomavirus Humano 16/genética , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/virologia , Vagina/virologia
13.
PLoS One ; 15(4): e0227900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320400

RESUMO

OBJECTIVE: Identify the prevalence of HPV infections in the uterine cervix and oral cavity and HPV16 variants in HIV+ women. METHODS: A total of 174 HIV+ women attended an HIV+ specialized clinic in Mexico City. Cells were obtained from the oral cavity and cervix to extract DNA. Polymerase chain reaction (PCR) was used to amplify the HPV sequence with generic primers. We detected specific HPV types using the INNO-LiPA HPV Genotyping Extra II Kit (INNOGENETICS). The identification of variants was studied by sequencing the E6 gene with a Big Dye Terminator Kit and an Applied Biosystems 3500/3500xL genetic analyzer. RESULTS: HPV infection was very high in the uterine cervix (168/174, 96.6%) and oral cavity (161/174, 92.5%). The prevalence of HPV concurrent infections in the cervix and oral cavity was 155/174 (89.1%). We found hrHPVs to be more prevalent than low-risk HPVs (lrHPVs) in the oral cavity (90.2% versus 45.4%) and that infections simultaneously affected the cervix (94.3% versus 36.2%) and oral cavity (85.1% versus 20.1%). Surprisingly, only European variants of HPV type 16 were found in the uterine cervix of women and the oral cavity of all tested samples (52 oral cavity samples and 52 uterine cervix samples). CONCLUSIONS: The high prevalence of HPV, multiple infections and presence of the EP350G intravariant in both anatomical regions are strongly related to the persistence of the virus, which is fundamental for the development of cancer. Therefore, it is very important to control and monitor this high-risk population as well as implement programs for the early detection of HPV and vaccination.


Assuntos
Infecções por HIV/imunologia , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/epidemiologia , Neoplasias do Colo do Útero/prevenção & controle , Adulto , Fatores Etários , Colo do Útero/virologia , DNA Viral/isolamento & purificação , Feminino , Infecções por HIV/tratamento farmacológico , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , México/epidemiologia , Pessoa de Meia-Idade , Boca/virologia , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/transmissão , Infecções por Papillomavirus/virologia , Prevalência , Proteínas Repressoras/genética , Fatores de Risco , Comportamento Sexual/estatística & dados numéricos , Neoplasias do Colo do Útero/virologia
14.
Nat Commun ; 11(1): 1985, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332752

RESUMO

The unsatisfactory response rate of immune checkpoint blockade (ICB) immunotherapy severely limits its clinical application as a tumor therapy. Here, we generate a vaccine-based nanosystem by integrating siRNA for Cd274 into the commercial human papillomavirus (HPV) L1 (HPV16 L1) protein. This nanosystem has good biosafety and enhances the therapeutic response rate of anti-tumor immunotherapy. The HPV16 L1 protein activates innate immunity through the type I interferon pathway and exhibits an efficient anti-cancer effect when cooperating with ICB therapy. For both resectable and unresectable breast tumors, the nanosystem decreases 71% tumor recurrence and extends progression-free survival by 67%. Most importantly, the nanosystem successfully induces high response rates in various genetically modified breast cancer models with different antigen loads. The strong immune stimulation elicited by this vaccine-based nanosystem might constitute an approach to significantly improve current ICB immunotherapy.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/terapia , Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Nanopartículas/administração & dosagem , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Humanos , Imunidade Inata/genética , Camundongos , Recidiva Local de Neoplasia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Intervalo Livre de Progressão , RNA Interferente Pequeno/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
15.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238586

RESUMO

Beta genus human papillomaviruses (ß-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, ß-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, ß-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how ß-HPV E6 causes this change in cell signaling. An in silico screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including proproliferative genes associated with HP inactivation. ß-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B [H2CB]) to induce failed cytokinesis. This system allowed us to show that ß-HPV 8E6 reduced activation of large tumor suppressor kinase (LATS), an HP kinase. LATS is required for p53 accumulation following failed cytokinesis. These phenotypes were dependent on ß-HPV 8E6 destabilizing p300 and did not completely attenuate the HP. It did not alter H2CB-induced nuclear exclusion of the transcription factor YAP. ß-HPV 8E6 also did not decrease HP activation in cells grown to a high density. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge, this marks the first time that a ß-HPV E6 protein has been shown to hinder HP signaling.IMPORTANCE ß-HPVs contribute to cSCC development in immunocompromised populations. However, it is unclear if these common cutaneous viruses are tumorigenic in the general population. Thus, a more thorough investigation of ß-HPV biology is warranted. If ß-HPV infections do promote cSCCs, they are hypothesized to destabilize the cellular genome. In vitro data support this idea by demonstrating the ability of the ß-HPV E6 protein to disrupt DNA repair signaling events following UV exposure. We show that ß-HPV E6 more broadly impairs cellular signaling, indicating that the viral protein dysregulates the HP. The HP protects genome fidelity by regulating cell growth and apoptosis in response to a myriad of deleterious stimuli, including failed cytokinesis. After failed cytokinesis, ß-HPV 8E6 attenuates phosphorylation of the HP kinase (LATS). This decreases some, but not all, HP signaling events. Notably, ß-HPV 8E6 does not limit senescence associated with failed cytokinesis.


Assuntos
Citocinese/genética , Interações Hospedeiro-Patógeno/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocalasina B/análogos & derivados , Citocalasina B/farmacologia , Citocinese/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteína p300 Associada a E1A/deficiência , Proteína p300 Associada a E1A/genética , Regulação da Expressão Gênica , Células HCT116 , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/virologia , Proteínas Oncogênicas Virais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/virologia , Papillomaviridae/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
16.
Comp Immunol Microbiol Infect Dis ; 70: 101463, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32146261

RESUMO

This study aimed to provide mechanistic insights into mitophagy pathway associated with papillomavirus infection in urothelial cells of cattle. The elimination of mitochondria via autophagy, termed mitophagy, is an evolutionarily conserved mechanism for mitochondrial quality control and homeostasis. PINK1/parkin-mediated mitophagy, a ubiquitin-dependent selective autophagy of dysfunctional mitochondria, has been described here, for the first time, in urothelial cells from 25 bladder cancers in cattle infected by bovine papillomavirus (BPV). The expression of BPV-2 and BPV-13 E5 oncoprotein was detected by RT-PCR. Abnormal mitochondria delimited by expanding phagophores, were peculiar ultrastructural features of neoplastic urothelial cells. High levels of mitochondrial phosphorylated PINK1/parkin were observed in neoplastic urothelial cells infected by BPVs. Phosphoparkin interacted with mitofusin 2 (Mfn2) and ubiquitin (Ub), which confirmed that Mfn2 is a parkin receptor at the mitochondrial level, where parkin interacted also with Ub. Furthermore, parkin established a complex that was comprised of optineurin, p62, LC3, laforin, and embryonic stem cell-expressed Ras (ERAS), that interacted with BPV E5 oncoprotein, and Bag3, which, in turn, regulated the formation of a complex composed of Hpc70/Hsp70, CHIP, an HSC70-interacting E3 ubiquitin ligase. It is conceivable that ERAS is involved in mitophagosome maturation via phosphatidylinositol 3-kinase (PI3K) pathway. Bag3, in association with Hsc70/Hsp70, may contribute to the transport and degradation of CHIP-ubiquitinated cargo as this complex recognises ubiquitinated cargos and transports them to aggresomes to be degraded. Furthermore, Bag3 may be involved in mitophagosome formation as it interacted with synaptopodin 2, which is known to play a role in mitophagosome biogenesis.


Assuntos
Carcinoma Papilar/veterinária , Mitofagia , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/veterinária , Ubiquitina-Proteína Ligases/genética , Neoplasias da Bexiga Urinária/veterinária , Animais , Carcinoma Papilar/virologia , Bovinos , Doenças dos Bovinos/virologia , Feminino , Mitocôndrias/patologia , Infecções por Papillomavirus/virologia , Regulação para Cima , Neoplasias da Bexiga Urinária/virologia , Urotélio/patologia , Urotélio/virologia
17.
Artif Cells Nanomed Biotechnol ; 48(1): 515-524, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32048523

RESUMO

Vulvar intraepithelial neoplasia (VIN) is associated with human papillomavirus (HPV) infection. Curcumin is a natural bioactive compound with antineoplastic properties. The use of nanoparticles containing curcumin could allow a better performance of this compound in therapies. So, VIN biopsies were collected and HPV DNA detection was performed by PCR, positive samples were genotyped by Restriction Fragment Length Polymorphism (RFLP) and HPV-16 variants were determined by sequencing. HPV-16 positive vulva carcinoma cells (A431) were transduced with E-P and E-350G HPV-16 E6 variants. The viability of the transduced cells treated with nanoemulsions was determined by MTT assay. Besides, apoptosis was evaluated by enzymatic activity of Caspase-3/7. The cell viability assay showed that both the empty nanoemulsion (NE-V) and the nanoemulsion of curcumin (NE-CUR) had little effect on cell viability as compared to control cells. Additionally, we observed that cells irradiated in the presence of NE-CUR presented 90% of cell death. The apoptosis assay further revealed a significant increase in the activity of caspases 3 and 7 in A431 cells expressing both HPV-16 E6 variants after treatment with NE-CUR. Finally, we submitted the HPV transduced A431 cells to organotypic cultures and observed that the combination of treatments affected tissue architecture with evident signals of tissue damage. We concluded that nanoemulsions attain good biocompatibility, since no cytotoxicity was observed and NE-CUR associated with photoactivation showed promising results, leading to death only in cells subjected to irradiation. This drug delivery system associated with photodynamic therapy may become promising in the treatment of vulva lesions.


Assuntos
Antivirais/farmacologia , Curcumina/farmacologia , Papillomavirus Humano 16/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Adulto , Carcinoma in Situ/virologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Curcumina/química , Emulsões , Feminino , Genótipo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , Luz , Nanopartículas/química , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Neoplasias Vulvares/virologia
18.
Int J Oral Sci ; 12(1): 3, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911577

RESUMO

High-risk human papillomaviruses (HPVs) are involved in the development of several human cancers, including oropharyngeal squamous cell carcinomas. However, many studies have demonstrated that HPV alone is not sufficient for the oncogenic transformation of normal human epithelial cells, indicating that additional cofactors are required for the oncogenic conversion of HPV-infected cells. Inasmuch as chronic inflammation is also closely associated with carcinogenesis, we investigated the effect of chronic exposure to tumor necrosis factor α (TNFα), the major proinflammatory cytokine, on oncogenesis in two immortalized oral keratinocyte cell lines, namely, HPV16-immortalized and human telomerase reverse transcriptase (hTERT)-immortalized cells. TNFα treatment led to the acquisition of malignant growth properties in HPV16-immortalized cells, such as (1) calcium resistance, (2) anchorage independence, and (3) increased cell proliferation in vivo. Moreover, TNFα increased the cancer stem cell-like population and stemness phenotype in HPV16-immortalized cells. However, such transforming effects were not observed in hTERT-immortalized cells, suggesting an HPV-specific role in TNFα-promoted oncogenesis. We also generated hTERT-immortalized cells that express HPV16 E6 and E7. Chronic TNFα exposure successfully induced the malignant growth and stemness phenotype in the E6-expressing cells but not in the control and E7-expressing cells. We further demonstrated that HPV16 E6 played a key role in TNFα-induced cancer stemness via suppression of the stemness-inhibiting microRNAs miR-203 and miR-200c. Overexpression of miR-203 and miR-200c suppressed cancer stemness in TNFα-treated HPV16-immortalized cells. Overall, our study suggests that chronic inflammation promotes cancer stemness in HPV-infected cells, thereby promoting HPV-associated oral carcinogenesis.


Assuntos
Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/metabolismo , MicroRNAs/metabolismo , Neoplasias Bucais/genética , Boca/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Telomerase/genética , Fator de Necrose Tumoral alfa/metabolismo , Carcinogênese/genética , Carcinogênese/imunologia , Carcinoma de Células Escamosas/patologia , Transformação Celular Viral/genética , Regulação da Expressão Gênica , Genes Virais , Papillomavirus Humano 16/genética , Humanos , MicroRNAs/genética , Boca/virologia , Neoplasias Bucais/patologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Telomerase/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
19.
Sci Rep ; 10(1): 1097, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974410

RESUMO

p53 and aldehyde dehydrogenase (ALDH) have been implicated in key tumorigenesis processes including cancer initiating cell (CIC) maintenance; however, the relationship between these two mediators remains poorly defined. In this study, ALDH isoform expression diversity was revealed in CICs with disparate p53 functional states: gain of function, high risk p53 mutation (p53HRmut) and wildtype p53 (p53WT) inactivated by the human papillomavirus 16 (HPV16) E6 oncogene. Interrogation of head and neck squamous cell carcinoma (HNSCC) cell lines and patient tumors showed that HPV16+/p53WT cases have higher ALDH variance score (AVS), a measure of tumor ALDH isoform expression diversity, compared to HPV-/p53HRmut cases (p = 0.03). AVS and several individual ALDH isoforms were associated with prognosis in HPV16+/p53WT HNSCC but not in HPV-/p53HRmut HNSCC. Knockdown of the dominant ALDH isoform in high AVS HNSCC depleted the CIC pool in vitro and in vivo. Our results demonstrate that p53 functional states are associated with distinct ALDH isoform transcriptomic signatures. Moreover, tumor ALDH profiling may provide insight on which ALDH isoform to target in high AVS HNSCC tumors to deplete the CIC population.


Assuntos
Aldeído Desidrogenase/genética , Infecções por Papillomavirus/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/enzimologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcriptoma , Proteína Supressora de Tumor p53/genética
20.
J Virol ; 94(8)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31996427

RESUMO

Human papillomavirus (HPV) type 58 is the third most commonly detected HPV type in cervical cancer among Eastern Asians. Our previous international epidemiological studies revealed that HPV58 carrying an E7 natural variant, T20I/G63S (designated V1), was associated with a higher risk of cervical cancer. We recently showed that V1 possesses a greater ability to immortalize and transform primary cells, as well as degrading pRB more effectively, than the prototype and other common variants. In this study, we performed a series of phenotypic and molecular assays using physiologically relevant in vitro and in vivo models to compare the oncogenicity of V1 with that of the prototype and other common natural variants. Through activation of the AKT and K-Ras/extracellular signal-regulated kinase (ERK) signaling pathways, V1 consistently showed greater oncogenicity than the prototype and other variants, as demonstrated by increased cell proliferation, migration, and invasion, as well as induction of larger tumors in athymic nude mice. This study complements our previous epidemiological and molecular observations pinpointing the higher oncogenicity of V1 than that of the prototype and all other common variants. Since V1 is more commonly found in eastern Asia, our report provides insight into the design of HPV screening assays and selection of components for HPV vaccines in this region.IMPORTANCE Epidemiological studies have revealed that a wild-type variant of HPV58 carrying an E7 variation, T20I/G63S (V1), is associated with a higher risk of cervical cancer. We previously reported that this increased oncogenicity could be the result of the virus's greater ability to degrade pRB, thereby leading to an increased ability to grow in an anchorage-independent manner. In addition to this, this report further showed that this HPV variant induced activation of the AKT and K-Ras/ERK signaling pathways, thereby explaining its genuine oncogenicity in promoting cell proliferation, migration, invasion, and formation of tumors, all to a greater extent than the prototype HPV58 and other common variants.


Assuntos
Papillomaviridae/classificação , Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Animais , Grupo com Ancestrais do Continente Asiático , Proliferação de Células , Modelos Animais de Doenças , Feminino , Variação Genética , Humanos , Camundongos , Camundongos Nus , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Vacinas contra Papillomavirus , Ratos , Neoplasias do Colo do Útero/virologia
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