RESUMO
Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.
Assuntos
Anticorpos Antivirais , Papillomavirus Humano 11 , Papillomavirus Humano 6 , Neoplasias Laríngeas/imunologia , Papiloma/imunologia , DNA Viral , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 6/fisiologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Laríngeas/patologia , Neoplasias Laríngeas/virologia , Papiloma/patologia , Papiloma/virologia , Infecções por Papillomavirus/imunologia , RNA Mensageiro/metabolismo , Carga ViralRESUMO
BACKGROUND: Recurrent Respiratory Papillomatosis (RRP) is a rare disease characterized by the growth of papillomas in the airway and especially the larynx. The clinical course is highly variable among individuals and there is poor understanding of the factors that drive an aggressive vs an indolent course. METHODS: A convenience cohort of 339 affected subjects with papillomas positive for only HPV6 or HPV11 and clinical course data available for 1 year or more, from a large multicenter international study were included. Exploratory data analysis was conducted followed by inferential analyses with frequentist and Bayesian statistics. RESULTS: We examined 339 subjects: 82% were diagnosed prior to the age of 18 years, 65% were infected with HPV6, and 69% had an aggressive clinical course. When comparing age at diagnosis with clinical course, the probability of aggressiveness is high for children under five years of age then drops rapidly. For patients diagnosed after the age of 10 years, an indolent course is more common. After accounting for confounding between HPV11 and young age, HPV type was minimally associated with aggressiveness. Fast and Frugal Trees (FFTs) were utilized to determine which algorithms yield the highest accuracy to classify patients as having an indolent or aggressive clinical course and consistently created a branch for diagnostic age at ~5 years old. There was no reliable strong association between clinical course and socioeconomic or parental factors. CONCLUSION: In the largest cohort of its type, we have identified a critical age at diagnosis which demarcates a more aggressive from less aggressive clinical course.
Assuntos
Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 6/fisiologia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Adulto , Fatores Etários , Pré-Escolar , Condiloma Acuminado/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mães , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/cirurgia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/cirurgiaRESUMO
Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 11/fisiologia , Proteínas Nucleares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Linhagem Celular , Humanos , Ligação ProteicaRESUMO
The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication.
Assuntos
DNA Polimerase II/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 11/fisiologia , Ativação Transcricional , Proteínas Virais/metabolismo , Humanos , Mapeamento de Interação de ProteínasRESUMO
CONTEXT: - Human papillomavirus (HPV) has a well-known role in the pathogenesis of squamous cell carcinoma and precursor lesions of the cervix, anogenital region, and head and neck, but its role in the development of squamous neoplasms of the eye, particularly the conjunctiva, remains unclear. OBJECTIVE: - To review recent evidence implicating HPV in the pathophysiology of ocular lesions. DATA SOURCES: - Published articles obtained from a PubMed search of the English literature were the primary sources for this review. CONCLUSIONS: - The low-risk HPV types 6 and 11 appear to play a role in the development of at least a subset of conjunctival squamous papillomas. The role of HPV in the pathogenesis of pterygium and ocular surface squamous neoplasia is less well defined. There is evidence to suggest that HPV may be a cofactor in the development of these lesions, acting in concert with ultraviolet radiation and/or human immunodeficiency virus infection in a subgroup of cases.
Assuntos
Carcinoma de Células Escamosas/virologia , Neoplasias Oculares/virologia , Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Pterígio/virologia , Carcinoma de Células Escamosas/fisiopatologia , Neoplasias da Túnica Conjuntiva/fisiopatologia , Neoplasias da Túnica Conjuntiva/virologia , Neoplasias Oculares/fisiopatologia , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 16/fisiologia , Humanos , Papiloma/fisiopatologia , Papiloma/virologia , Infecções por Papillomavirus/fisiopatologia , Pterígio/fisiopatologia , Raios UltravioletaRESUMO
HPV-11 and HPV-6 are the etiological agents of about 90â% of genital warts (GWs). The intra-typic variability of HPV-11 and its association with infection persistence and GW development remains undetermined. Here, HPV infection in men (HIM) participants who had an HPV-11 genital swab and/or GW, preceded or not by a normal skin genital swab were analysed. Genomic variants were characterized by PCR-sequencing and classified within lineages (A, B) and sublineages (A1, A2, A3, A4). HPV-11 A2 variants were the most frequently detected in the genital swab samples from controls and in both genital swabs and GW samples from cases. The same HPV-11 variant was detected in the GW sample and its preceding genital swab. There was a lack of association between any particular HPV-11 variant and the increased risk for GW development.
Assuntos
Condiloma Acuminado/virologia , Papillomavirus Humano 11/isolamento & purificação , Adolescente , Adulto , Condiloma Acuminado/patologia , Progressão da Doença , Genótipo , Papillomavirus Humano 11/classificação , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/fisiologia , Humanos , Masculino , Filogenia , Adulto JovemAssuntos
Micropartículas Derivadas de Células/metabolismo , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 6/fisiologia , Macrófagos/virologia , Infecções por Papillomavirus/virologia , Micropartículas Derivadas de Células/ultraestrutura , Papillomavirus Humano 11/ultraestrutura , Papillomavirus Humano 6/ultraestrutura , Humanos , Macrófagos/patologia , Macrófagos/ultraestrutura , Infecções por Papillomavirus/patologiaAssuntos
Cesárea/estatística & dados numéricos , Condiloma Acuminado/terapia , Papillomavirus Humano 11/patogenicidade , Papillomavirus Humano 6/patogenicidade , Infecções por Papillomavirus/terapia , Infecções Respiratórias/terapia , Adolescente , Adulto , Fatores Etários , Aminoquinolinas/uso terapêutico , Condiloma Acuminado/complicações , Condiloma Acuminado/epidemiologia , Condiloma Acuminado/cirurgia , Gerenciamento Clínico , Feminino , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 6/fisiologia , Humanos , Imiquimode , Indutores de Interferon/uso terapêutico , Japão/epidemiologia , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/etiologia , Infecções por Papillomavirus/cirurgia , Guias de Prática Clínica como Assunto , Gravidez , Prevalência , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/etiologia , Infecções Respiratórias/cirurgiaRESUMO
UNLABELLED: The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE: While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 11/fisiologia , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Papillomavirus Humano 11/genética , Humanos , Estrutura Terciária de Proteína , Deleção de Sequência , Proteínas Virais/genéticaRESUMO
BACKGROUND: Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is the clear unmet medical need in order to justify the development of drugs targeting human papillomavirus replication. The native host cells of HPVs are human primary keratinocytes which can be cultivated in raft cultures. However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains. METHODS: U2OS cells were transfected with HPV11 wt or E8- minicircle genomes and their gene expression was studied via 3' RACE, 5' RACE or via real time PCR methods. The DNA replication of these genomes was detected by Southern blot methods. RESULTS: The analysis of HPV11 transcripts in U2OS cells showed that the patterns of promoter use, splice sites and polyadenylation cleavage sites are identical to those previously characterized in human HPV-related lesions, human squamous carcinoma cell lines (e.g., SSC-4) and laryngeal papillomas. Transcriptional initiation from the three previously described HPV11 promoters in the E6 and E7 ORFs (P90, P264, and P674-714) were functional, and these promoters were used together with two promoter regions in the E1 ORF (P1092 and P1372). Mutating the E8 ORF ATG start codon to ACG eliminated the translation of fusion proteins from the E8 ORF coupled to E1 and E2 proteins C-terminal sequences, leading to the de-repression of gene expression (particularly from the P1092 promoter) and to the activation of genome replication. These data suggested that the expression of the functional E8^E2 protein is used to control viral gene expression and copy number of the HPV11 genome. The analysis of HPV11 E1 expression plasmids showed that the E6/E7 region, together with the E1 coding region, is crucial for the production of functionally active E1 protein. CONCLUSIONS: The data presented in this paper suggest that in human osteosarcoma cell line U2OS the gene expression pattern of the HPV11 truly reflect the expression profile of the replicating HPV genome and therefore this cellular system is suitable for drug development program targeting HPV replication.
Assuntos
Genoma Viral , Papillomavirus Humano 11/genética , Infecções por Papillomavirus/virologia , Transcrição Gênica , Replicação Viral , Linhagem Celular Tumoral , Replicação do DNA , Regulação Viral da Expressão Gênica , Papillomavirus Humano 11/fisiologia , Humanos , Regiões Promotoras Genéticas , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Condyloma acuminatum is one of the most commonly occurring sexually transmitted diseases. HNP1 is a small antimicrobial peptide that has been reported to have antiviral activities. AIM: Using the condyloma acuminatum tissue culture to resemble the situation more closely in vivo, we investigate the therapeutic effect of a recombinant plasmid encoding HNP1 gene in condyloma acuminatum tissue. METHODS: Recombinant plasmid DNA carrying HNP1 cDNA was constructed and identified. Then the recombinant plasmid was transfected into a condyloma acuminatum tissue fragment, and the HNP1 expression was determined on these tissue fragments by immunohistochemistry. TUNEL staining and flow cytometry techniques were used to examine cell apoptosis of condyloma acuminatum tissue. Relative real-time polymerase chain reaction was used to validate antihuman papillomavirus therapeutics of the treatment groups. RESULTS: Transfected HNP1 gene was expressed mainly in the cytoplasmic granules of the condyloma acuminatum tissues. Positive apoptotic cells were observed in condyloma acuminatum tissues transfected with the HNP1 gene. In addition, the HPV expression was lower in the HNP1 treatment tissues as compared to their corresponding control tissues. CONCLUSION: The results indicate that HNP1 can directly promote condyloma acuminatum cell apoptosis and play an antivirus role in the condyloma acuminatum tissue by limiting viral replication. These observations suggest a possible application for human HNP1 on condyloma acuminatum therapy.
Assuntos
Condiloma Acuminado/genética , Dermatopatias/genética , alfa-Defensinas/genética , Apoptose/genética , Condiloma Acuminado/metabolismo , Condiloma Acuminado/terapia , Grânulos Citoplasmáticos/química , Feminino , Terapia Genética , Papillomavirus Humano 11/fisiologia , Humanos , Plasmídeos , Dermatopatias/metabolismo , Dermatopatias/terapia , Técnicas de Cultura de Tecidos , Transfecção , Replicação Viral , alfa-Defensinas/análiseRESUMO
BACKGROUND: We present a long-term safety, immunogenicity, and effectiveness study of a quadrivalent human papillomavirus (HPV4) vaccine. METHODS: Sexually naive boys and girls aged 9 to 15 years (N = 1781) were assigned (2:1) to receive HPV4 vaccine or saline placebo at day 1 and months 2 and 6. At month 30, the placebo group (n = 482) received HPV4 vaccine following the same regimen and both cohorts were followed through month 96. Subjects ≥ 16 years were eligible for effectiveness evaluations. The primary objective was to evaluate the long-term anti-HPV6/11/16/18 serological levels. The secondary objective was to estimate vaccine effectiveness against HPV6/11/16/18-related persistent infection or disease. RESULTS: For each of the HPV4 vaccine types, vaccination-induced anti-HPV response persisted through month 96. Among 429 subjects who received HPV4 vaccine at a mean age of 12, none developed HPV6/11/16/18-related disease or persistent infection of ≥ 12 months' duration. Acquisition of new sexual partners (among those ≥ 16 years) was â¼1 per year. Subjects receiving HPV4 vaccine at month 30 (mean age 15 years) had a similar baseline rate of seropositivity to ≥ 1 of the 4 HPV types to those vaccinated at day 1 (mean age 12 years; 1.9% [9 of 474] vs 1.7% [20 of 1157]); however, 4 of the 9 subjects vaccinated at the later age were seropositive to 3 vaccine types, indicating previous HPV exposure. No new significant serious adverse events were observed for 8 years postvaccination in both genders. CONCLUSIONS: When administered to adolescents, the HPV4 vaccine demonstrated durability in clinically effective protection and sustained antibody titers over 8 years.
Assuntos
Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Vacinação/tendências , Adolescente , Criança , Método Duplo-Cego , Feminino , Seguimentos , Papillomavirus Humano 11/efeitos dos fármacos , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/fisiologia , Papillomavirus Humano 18/efeitos dos fármacos , Papillomavirus Humano 18/fisiologia , Papillomavirus Humano 6/efeitos dos fármacos , Papillomavirus Humano 6/fisiologia , Humanos , Masculino , Infecções por Papillomavirus/sangue , Infecções por Papillomavirus/epidemiologia , Vacinas contra Papillomavirus/sangue , Fatores de Tempo , Resultado do TratamentoRESUMO
We previously discovered that nuclear import of low risk HPV11 E7 is mediated by its zinc-binding domain via a pathway that is independent of karyopherins/importins (Piccioli et al., 2010. Virology 407, 100-109). In this study we mapped and characterized a leucine-rich nuclear export signal (NES), 76IRQLQDLLL84, within the zinc-binding domain that mediates the nuclear export of HPV11 E7 in a CRM1-dependent manner. We also identified a mostly hydrophobic patch 65VRLVV69 within the zinc-binding domain that mediates nuclear import of HPV11 E7 via hydrophobic interactions with the FG-repeats domain of Nup62. Substitutions of hydrophobic residues to alanine within the 65VRLVV69 sequence disrupt the nuclear localization of 11E7, whereas the R66A mutation has no effect. Overall the data support a model of nuclear entry of HPV11 E7 protein via hydrophobic interactions with FG nucleoporins at the nuclear pore complex.
Assuntos
Transporte Ativo do Núcleo Celular , Papillomavirus Humano 11/fisiologia , Proteínas E7 de Papillomavirus/metabolismo , Sinais Direcionadores de Proteínas , Papillomavirus Humano 11/genética , Humanos , Carioferinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas E7 de Papillomavirus/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Exportina 1RESUMO
BACKGROUND: The etiology and the molecular mechanisms related to breast carcinogenesis remain poorly understood. Some recent reports have examined the role of Human Papillomavirus (HPV) in this disease. The purpose of this study was to determine the prevalence of HPV in breast cancer. METHODS: Sixty one fresh frozen breast cancers samples were analyzed. Samples were tested for HPV by PCR, and products were automatically sequenced. Findings were correlated with clinical and pathological characteristics. RESULTS: The HPV DNA prevalence in the breast cancer samples was 26% (16/61). Clinical parameters were not statistically associated with HPV presence (p>0.05 χ(2) test). Sequence analysis in a subgroup of cases indicates the prevalence of low risk HPV11, followed by high risk HPV16. We found no HPV transcriptional activity. CONCLUSION: The present study demonstrated for the first time in Argentina the presence of HPV in a proportion of the malignant breast tissues. This finding suggests that HPV may have a biological significance in breast carcinogenesis.
Assuntos
Neoplasias da Mama/virologia , Papillomavirus Humano 11/isolamento & purificação , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Argentina , Neoplasias da Mama/patologia , Feminino , Papillomavirus Humano 11/genética , Papillomavirus Humano 16/genética , Humanos , Pessoa de Meia-Idade , RiscoRESUMO
Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6) or HPV-11. Specific HLA-DR haplotypes DRB1*01:02 and DRB1*03:01 are associated with the development of RRP, disease severity, and Th2-like responses to HPV early proteins. Th1-like responses to HPV proteins have been shown to be protective in animal models. Therefore, we investigated the hypothesis that RRP patients have dysfunctional Th1-like, HPV-specific T cell responses. Using MHC class II tetramers, we identified immunogenic peptides within HPV-11 early proteins. Two distinct peptides (E6(113-132) and E2(1-20)) contained DRB1*01:02- or DRB1*03:01-restricted epitopes, respectively. An additional peptide (E2(281-300)) contained an epitope presented by both alleles. Peptide binding, tetramer, and proliferation assays identified minimal epitopes within these peptides. These epitopes elicited E2/E6-specific CD4(+) T cell responses in RRP patients and healthy control subjects, allowing the isolation of HPV-specific T cell lines using tetramers. The cytokine profiles and STAT signaling of these tetramer-positive T cells were measured to compare the polarization and responsiveness of HPV-specific T cells from patients with RRP and healthy subjects. HPV-specific IFN-γ secretion was substantially lower in T cells from RRP patients. HPV-specific IL-13 secretion was seen at modest levels in T cells from RRP patients and was absent in T cells from healthy control subjects. HPV-specific T cells from RRP patients exhibited reduced STAT-5 phosphorylation and reduced IL-2 secretion, suggesting anergy. Levels of STAT-5 phosphorylation and IFN-γ secretion could be improved through addition of IL-2 to HPV-specific T cell lines from RRP patients. Therapeutic vaccination or interventions aimed at restoring Th1-like cytokine responses to HPV proteins and reversing anergy could improve clinical outcomes for RRP patients.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Papillomavirus Humano 11/imunologia , Fator de Transcrição STAT5/imunologia , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Antígenos HLA-DR/imunologia , Cadeias alfa de HLA-DR , Cadeias HLA-DRB1 , Interações Hospedeiro-Patógeno/imunologia , Papillomavirus Humano 11/fisiologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Peptídeos/imunologia , Fosforilação/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/virologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Virais/química , Proteínas Virais/imunologiaRESUMO
The goal was to determine recurrent or second primary status for late stomal malignancies, 16 and 17 years post-total laryngectomy in two laryngeal squamous cell carcinoma (LSCC) patients, based on DNA methylation signatures and HPV typing. Adopting a literature review based definition of late stomal recurrences as new primaries at the site of the stoma or neopharynx occurring >5 years after total laryngectomy, we employed a multi-gene candidate approach to examine promoter methylation in 24 tumor suppressor genes and PCR-based assays for HPV status offered additional insights into whether the late stomal tumors post-total laryngectomy were related or not. The primary tumor for Patient 1 was negative for HPV but had aberrant hypermethylation of APC, MLH1 and BRCA1. The stomal biopsy 17-years later showed presence of HPV-16 without any methylated genes. In Patient 2, HPV-11 and promoter methylation of APC identified in the primary tumor was also observed in the stomal malignancy 16 years post-total laryngectomy. Additional information provided by molecular typing for HPV and methylation markers underscored Patient 1's and 2's late stomal presentation as most likely a second primary and recurrence, respectively. DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable marker. Molecular marks to discern genetic heterogeneity or relatedness of stomal malignancies several years post-total laryngectomy can provide clues to their status as either second primaries or likely recurrences. Our results support the hypothesis that a subset of stomal recurrences after total laryngectomy represents second primary tumors.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/cirurgia , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/cirurgia , Recidiva Local de Neoplasia/genética , Estomas Cirúrgicos/patologia , Idoso , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/patologia , Metilação de DNA , Epigenômica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Neoplasias Laríngeas/complicações , Neoplasias Laríngeas/patologia , Laringectomia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/complicações , Recidiva Local de Neoplasia/patologia , Segunda Neoplasia Primária/complicações , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/patologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologiaRESUMO
The papillomavirus life cycle is regulated by a family of proteins encoded by the E2 open reading frame; E2 proteins regulate viral gene expression, DNA replication and genome maintenance. We have previously shown that the bovine papillomavirus (BPV1) full-length E2 protein forms heterodimers with repressor forms of E2, and these E2 heterodimers serve as activators of transcription and replication during the viral life cycle. In the present study, using the single-chain E2 heterodimer as a model, we show that human papillomavirus (HPV) 11 and 18 E2 heterodimers with single activation domain are able to initiate replication of URR-containing plasmid in transient assay. Single-chain E2 heterodimer in the context of HPV18 genome initiates genome replication, but is not sufficient for long-term replication of HPV18 genome. We also show that HPV18 genome has a capacity to encode truncated E2 repressor E8/E2 which acts as a negative regulator of HPV18 genome replication.
Assuntos
Genoma Viral , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/fisiologia , Células COS , Bovinos , Linhagem Celular , Chlorocebus aethiops , DNA Viral/biossíntese , DNA Viral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dimerização , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/fisiologia , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Plasmídeos/biossíntese , Plasmídeos/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/fisiologiaRESUMO
Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer.
Assuntos
Dano ao DNA , Replicação do DNA/genética , Replicação do DNA/fisiologia , Papillomaviridae/genética , Papillomaviridae/fisiologia , Proteínas Virais/genética , Proteínas Virais/fisiologia , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/fisiologia , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Viral/biossíntese , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Etoposídeo/farmacologia , Feminino , Instabilidade Genômica , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/patogenicidade , Papillomavirus Humano 11/fisiologia , Humanos , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Local hyperthermia has been successfully used in the treatment of viral warts by mechanisms that have largely remained unclear. Using an organotypic culture system, we found that hyperthermia at 42 °C and 45 °C could induce a significant increase in the transcriptional expression of interferon (IFN)-α, IFN-ß and IFN-γ, in a temperature-dependent manner in condyloma acuminata (CA), but not in normal skin. Accordingly, local hyperthermia could enhance the expression of 2'-5' oligoadenylate synthase and double-stranded RNA (dsRNA)-dependent protein kinase, two antiviral enzymes downstream of the IFN-dependant pathway. Hyperthermia led to an increase in IFN-α/ß receptor transcripts, and an increase in the levels in phospho-Stat1 and phospho-Stat2 in CA, though it had no influence on the levels of Jak1, Tyk2, Stat1 and Stat2 transcriptional expression. Local hyperthermia was proved effective in treating human papillomavirus-infected skin. These results suggested that hyperthermia took effect partly by inducing the expression of local endogenous IFN and partly by subsequent IFN-induced antiviral activity via Jak-STATs signalling pathway in CA.
Assuntos
Antivirais/metabolismo , Condiloma Acuminado/terapia , Hipertermia Induzida , Interferons/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Condiloma Acuminado/patologia , Condiloma Acuminado/virologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Temperatura Alta , Papillomavirus Humano 11/fisiologia , Papillomavirus Humano 6/fisiologia , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interferons/genética , Masculino , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Replicação Viral , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7(39-98) localized mostly to the nucleus. The GST-11E7 and GST-11cE7(39-98) were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.