RESUMO
The efficiency of PCR-based diagnostic assays can be impacted by the quality of DNA template, and anal samples can be particularly problematic due to the presence of faecal contaminants. Here, we compared the Quick-DNA Viral Kit (Zymo, Zymo Research, CA) and MagNA Pure 96 DNA and Viral NA Small Volume Kit (MP96, Roche) for use of the Seegene Anyplex II HPV28 assay (Anyplex28, Seegene) with anal samples. A total of 94 anal samples extracted using the MP96 and Zymo kits were tested via the Anyplex28, which detects high-risk human papillomavirus (HR-HPV, Panel A) and low-risk (LR-HPV, Panel B) HPV types. Testing the HR-HPV types (Panel A), 86 (91.5%) MP96 and 84 (89.4%) Zymo samples were deemed assessable. Overall agreement between the two methods was 87/94 (92.6%, 95% CI: 85.3-97.0) with the Kappa value of 0.678 (0.5-0.9). Of the 87 assessable samples, 50 (57.5%) were concordant, 34 (39.1%) partially concordant, and 10 (11.5%)discordant. In conclusion, the Anyplex28 produces comparable HPV genotyping results when using DNA extracts from either of these two methods.
Assuntos
DNA Viral , Papillomaviridae , Infecções por Papillomavirus , Humanos , DNA Viral/genética , DNA Viral/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Papillomaviridae/classificação , Reação em Cadeia da Polimerase/métodos , Canal Anal/virologia , Kit de Reagentes para DiagnósticoRESUMO
OBJECTIVE: The aim of the study was to evaluate clinicopathologic features of cases demonstrating an acanthotic tissue reaction not clearly consistent with psoriasis, lichen simplex chronicus, mycosis, or condyloma. MATERIALS AND METHODS: This is a retrospective pathologic case series of biopsies reported as "benign acanthotic lesion" and "acanthotic tissue reaction" that lacked a clear diagnosis on expert review. Cases with nuclear atypia were excluded. Clinical and histopathologic data were collected, immunohistochemistry for p16 and p53 were obtained, and molecular testing for 28 common anogenital human papillomavirus (HPV) genotypes was undertaken. RESULTS: There were 17 cases with a median age of 47 years. Unilaterality and medial location were clinical reasons for diagnostic difficulty. Histopathologic uncertainty often related to lack of papillary dermal fibrosis to support lichen simplex chronicus or psoriasiform lesions without parakeratosis, subcorneal pustules, and/or mycotic elements. Firm pathologic diagnoses were not possible, but 3 groups emerged: favoring chronic dermatitis, favoring psoriasis, and unusual morphologies. p16 results were negative or nonblock positive while p53 was normal or basal overexpressed. Human papillomavirus testing was negative in 12, low positive for HPV 16 in 1, unassessable in 3, and not requested in 1. CONCLUSIONS: There is a group of acanthotic tissue reactions that cannot be classified with standard histopathologic assessment. Further clinicopathologic research into unilateral acanthotic lesions may provide insight into separation of psoriasis and mycosis when organisms are absent. Once nuclear atypia is excluded, immunohistochemistry for p16 and p53 and HPV molecular testing do not assist in diagnostic identification.
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Alphapapillomavirus , Neurodermatite , Infecções por Papillomavirus , Psoríase , Neoplasias Vulvares , Feminino , Humanos , Pessoa de Meia-Idade , Papillomaviridae , Infecções por Papillomavirus/diagnóstico , Estudos Retrospectivos , Proteína Supressora de Tumor p53 , Neoplasias Vulvares/patologiaAssuntos
Carcinoma de Células Escamosas , Anemia de Fanconi , Neoplasias Bucais , Humanos , Feminino , Neoplasias Bucais/complicações , Neoplasias Bucais/patologia , Neoplasias Bucais/diagnóstico , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/patologia , Anemia de Fanconi/complicações , Anemia de Fanconi/diagnóstico , Neoplasias Vulvares/complicações , Neoplasias Vulvares/patologia , Neoplasias Vulvares/diagnóstico , Neoplasias do Colo do Útero/complicações , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/patologia , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/complicações , Histocitoquímica , Adulto , MicroscopiaRESUMO
CONTEXT.: Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored. OBJECTIVE.: To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples. DESIGN.: We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples. RESULTS.: Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3). CONCLUSIONS.: Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.
Assuntos
Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Papillomavirus Humano , Infecções por Papillomavirus/diagnóstico , Inclusão em Parafina/métodos , Papillomaviridae/genética , Genótipo , DNA Viral/genética , DNA Viral/análise , FormaldeídoRESUMO
OBJECTIVE: WHO recommends human papillomavirus (HPV) testing for cervical screening, with triage of high-risk HPV (hrHPV) positive women. However, there are limitations to effective triage for low-resource, high-burden settings, such as Papua New Guinea. In this exploratory study, we assessed the performance of host methylation as triage tools for predicting high-grade squamous intraepithelial lesions (HSIL) in self-collected and clinician-collected samples. DESIGN: Exploratory observational study. SETTING: Provincial hospital, same-day cervical screen-and-treat trial, Papua New Guinea. PARTICIPANTS: 44 hrHPV+women, with paired self/clinician-collected samples (4 squamous cell carcinomas (SCC), 19 HSIL, 4 low-grade squamous intraepithelial lesions, 17 normal). PRIMARY AND SECONDARY OUTCOME MEASURES: Methylation levels of CADM1, MAL and miR124-2 analysed by methylation-specific PCRs against the clinical endpoint of HSIL or SCC (HSIL+) measured using liquid-based-cytology/p16-Ki67 stain. RESULTS: In clinician-collected samples, MAL and miR124-2 methylation levels were significantly higher with increasing grade of disease (p=0.0046 and p<0.0015, respectively). miR124-2 was the best predictor of HSIL (area under the curve, AUC 0.819) while MAL of SCC (AUC 0.856). In self-collected samples, MAL best predicted HSIL (AUC 0.595) while miR124-2 SCC (AUC 0.812). Combined miR124-2/MAL methylation yielded sensitivity and specificity for HSIL+ of 90.5% (95% CI 69.6% to 98.8%) and 70% (95% CI 45.7% to 88.1%), respectively, in clinician-collected samples, and 81.8% (95% CI 59.7% to 94.8%) and 47.6% (95% CI 25.7% to 70.2%), respectively, in self-collected samples. miR124-2/MAL plus HPV16/HPV18 improved sensitivity for HSIL+ (95.2%, 95% CI 76.2% to 99.9%) but decreased specificity (55.0%, 95% CI 31.5% to 76.9%). CONCLUSION: miR124-2/MAL methylation is a potential triage strategy for the detection of HSIL/SCC in low-income and middle-income country.
Assuntos
Molécula 1 de Adesão Celular , Metilação de DNA , Detecção Precoce de Câncer , MicroRNAs , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Infecções por Papillomavirus , Triagem , Neoplasias do Colo do Útero , Humanos , Feminino , MicroRNAs/genética , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Papua Nova Guiné , Detecção Precoce de Câncer/métodos , Molécula 1 de Adesão Celular/genética , Adulto , Triagem/métodos , Pessoa de Meia-Idade , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Infecções por Papillomavirus/diagnóstico , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/diagnóstico , Manejo de Espécimes/métodos , Adulto Jovem , Sensibilidade e Especificidade , Esfregaço VaginalRESUMO
The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.
Assuntos
Hepacivirus/genética , Hepacivirus/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Catálise , Linhagem Celular , Genoma Viral , Genótipo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Transporte Proteico , RNA Viral/metabolismo , Proteínas não Estruturais Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus/genética , Replicação Viral/genéticaRESUMO
IMPORTANCE: The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.
Assuntos
Papillomavirus Humano 16 , Infecções por Papillomavirus , Humanos , Papillomavirus Humano 16/genética , Infecções por Papillomavirus/diagnóstico , Canal Anal , Reação em Cadeia da Polimerase , Papillomaviridae/genéticaRESUMO
GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-gamma ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.
Assuntos
Vírus GB B/isolamento & purificação , Hepatite Viral Animal/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Callithrix , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Vírus GB B/imunologia , Hepatite Viral Animal/virologia , Antígenos de Histocompatibilidade Classe I/imunologia , Dados de Sequência MolecularRESUMO
Intracellular pathogens use complex and tightly regulated processes to enter host cells. Upon initial interactions with signaling proteins at the surface of target cells, intracellular microbes activate and co-opt specific host signaling pathways that mediate cell surface-cytosol communications to facilitate pathogen internalization. Here, we discuss the roles of host receptor tyrosine kinases (RTKs) in the establishment of productive infections by major intracellular pathogens. We evaluate the gaps in the current understanding of this process and propose a comprehensive approach for assessing the role of host cell signaling in the biology of intracellular microorganisms and viruses. We also discuss RTK-targeting strategies for the treatment of various infections.
Assuntos
Infecções Bacterianas/microbiologia , Espaço Intracelular/microbiologia , Espaço Intracelular/virologia , Receptores Proteína Tirosina Quinases/metabolismo , Viroses/virologia , Animais , Infecções Bacterianas/prevenção & controle , Membrana Celular/efeitos dos fármacos , Membrana Celular/microbiologia , Membrana Celular/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Viroses/prevenção & controleRESUMO
Wolbachia-infected mosquitoes are refractory to super-infection with arthropod-borne pathogens, but the role of host cell signaling proteins in pathogen-blocking mechanisms remains to be elucidated. Here, we use an antibody microarray approach to provide a comprehensive picture of the signaling response of Aedes aegypti-derived cells to Wolbachia. This approach identifies the host cell insulin receptor as being downregulated by the bacterium. Furthermore, siRNA-mediated knockdown and treatment with a small-molecule inhibitor of the insulin receptor kinase concur to assign a crucial role for this enzyme in the replication of dengue and Zika viruses in cultured mosquito cells. Finally, we show that the production of Zika virus in Wolbachia-free live mosquitoes is impaired by treatment with the selective inhibitor mimicking Wolbachia infection. This study identifies Wolbachia-mediated downregulation of insulin receptor kinase activity as a mechanism contributing to the blocking of super-infection by arboviruses.
Assuntos
Vírus da Dengue/patogenicidade , Receptor de Insulina/uso terapêutico , Wolbachia/química , Zika virus/patogenicidade , Animais , Culicidae , Receptor de Insulina/farmacologiaRESUMO
Host cell signalling during infection with intracellular pathogens remains poorly understood. Here we report on the use of antibody microarray technology to detect variations in the expression levels and phosphorylation status of host cell signalling proteins during hepatitis C virus (HCV) replication. Following transfection with HCV RNA, the JNK and NF-κB pathways are suppressed, while the JAK/STAT5 pathway is activated; furthermore, components of the apoptosis and cell cycle control machineries are affected in the expression and/or phosphorylation status. RNAi-based hit validation identifies components of the JAK/STAT, NF-κB, MAPK and calcium-induced pathways as modulators of HCV replication. Selective chemical inhibition of one of the identified targets, the JNK activator kinase MAP4K2, does impair HCV replication. Thus this study provides a comprehensive picture of host cell pathway mobilization by HCV and uncovers potential therapeutic targets. The strategy of identifying targets for anti-infective intervention within the host cell signalome can be applied to any intracellular pathogen.
Assuntos
Hepacivirus/fisiologia , Hepatite C/metabolismo , Transdução de Sinais , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Replicação ViralRESUMO
Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.
RESUMO
Finding novel and effective antibiotics for treatment of Legionella disease is a challenging field. Treatment with antibiotics usually cures Legionella infection; however, if the resultant disease is not timely recognized and treated properly, it leads to poor prognosis and high case fatality rate. Legionella pneumophila DrrA protein (Defects in Rab1 recruitment protein A)/also known as SidM affects host cell vesicular trafficking through modification of the activity of cellular small guanosine triphosphatase )GTPase( Rab (Ras-related in brain) function which facilitates intracellular bacterial replication within a supporter vacuole. Also, Legionella pneumophila LepA and LepB (Legionella effector protein A and B) proteins suppress host-cell Rab1 protein's function resulting in the cell lysis and release of bacteria that subsequently infect neighbour cells. Legionella readily develops resistant to antibiotics and, therefore, new drugs with different modes of action and therapeutic strategic approaches are urgently required among antimicrobial drug therapies;gene therapy is a novel approach for Legionnaires disease treatment. On the contrary to the conventional treatment approaches that target bacterial proteins, new treatment interventions target DNA (Deoxyribonucleic acid), RNA (Ribonucleic acid) species, and different protein families or macromolecular complexes of these components. The above approaches can overcome the problems in therapy of Legionella infections caused by antibiotics resistance pathogens. Targeting Legionella genes involved in manipulating cellular vesicular trafficking using a dendrimer-mediated antisense therapy is a promising approach to inhibit bacterial replication within the target cells.
RESUMO
A chemically synthesized peptide representing the C-terminal subunit (p13-C) of the p13 protein of GB virus B (GBV-B), the most closely related virus to hepatitis C virus (HCV) showed ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to potassium ions than to chloride ions. Amantadine but not hexamethylene amiloride (HMA) inhibited the ion channel function of p13-C in the lipid membranes. However, neither agent was able to inhibit the replication and secretion of GBV-B from virus-infected cultured marmoset hepatocytes, which were harvested from a marmoset that was infected in vivo or inhibit replication after in vitro infection of naive hepatocytes. These data suggest that the GBV-B ion channel, contrary to the data derived from the lipid membranes, is either resistant to amantadine or that virus replication and secretion are independent of ion channel function. As the p7 protein of HCV also has ion channel activity that is apparently resistant to amantadine in vivo, the former possibility is most likely. Ion channels are likely to have an important role in the life cycle of many viruses and compounds that block these channels may prove to be useful antiviral agents.
Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Vírus GB B/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Callithrix , Células Cultivadas , Infecções por Flaviviridae/virologia , Vírus GB B/genética , Vírus GB B/metabolismo , Vírus GB B/fisiologia , Hepatite Viral Animal/virologia , Hepatócitos/virologia , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Bicamadas Lipídicas , Dados de Sequência Molecular , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
OBJECTIVES: The rotavirus nonstructural protein 4 (NSP4) is responsible for the increase in cytoplasmic calcium concentration through a phospholipase C-dependent and phospholipase C-independent pathways in infected cells. It is shown that increasing of intracellular calcium concentration in rotavirus infected cells is associated with the activation of some members of protein kinases family such as calcium/calmodulin-dependent kinase II, which plays a crucial role in replication and pathogenesis of the virus. The aim of this study was to expression bovine rotavirus NSP4 gene in HEK293 cell and evaluation of its biological effect related to activation of calcium/calmodulin-dependent kinase II in cell culture. MATERIALS AND METHODS: MA104 cells was used as a sensitive cell for propagation of virus and defined as a positive control. The NSP4 gene was amplified and inserted into an expression vector, and introduced as a recombinant plasmid into HEK293T cells. Western blot analysis was performed as a confirmation test for both expression of NSP4 protein and activation of calcium/calmodulin-dependent kinase II. RESULTS: Expression of NSP4 and activated form of calcium/calmodulin-dependent kinase II were demonstrated by western blotting. CONCLUSION: It was shown that the expression of biologically active full- length NSP4 protein in HEK293T cells may be associated with some biological properties such as calcium calmodulin kinase II activation, which was indicator of rotaviruses replication and pathogenesis.
RESUMO
BACKGROUND: Hepatitis C virus (HCV) causes chronic hepatitis C in 2-3% of world population and remains one of the health threatening human viruses, worldwide. In the absence of an effective vaccine, therapeutic approach is the only option to combat hepatitis C. Interferon-alpha (IFN-alpha) and ribavirin (RBV) combination alone or in combination with recently introduced new direct-acting antivirals (DAA) is used to treat patients infected with HCV. The present study utilized feature selection methods (Gini Index, Chi Squared and machine learning algorithms) and other bioinformatics tools to identify genetic determinants of therapy outcome within the entire HCV nucleotide sequence. RESULTS: Using combination of several algorithms, the present study performed a comprehensive bioinformatics analysis and identified several nucleotide attributes within the full-length nucleotide sequences of HCV subtypes 1a and 1b that correlated with treatment outcome. Feature selection algorithms identified several nucleotide features (e.g. count of hydrogen and CG). Combination of algorithms utilized the selected nucleotide attributes and predicted HCV subtypes 1a and 1b therapy responders from non-responders with an accuracy of 75.00% and 85.00%, respectively. In addition, therapy responders and relapsers were categorized with an accuracy of 82.50% and 84.17%, respectively. Based on the identified attributes, decision trees were induced to differentiate different therapy response groups. CONCLUSIONS: The present study identified new genetic markers that potentially impact the outcome of hepatitis C treatment. In addition, the results suggest new viral genomic attributes that might influence the outcome of IFN-mediated immune response to HCV infection.
Assuntos
Algoritmos , Antivirais/uso terapêutico , Inteligência Artificial , DNA Viral/genética , Técnicas de Apoio para a Decisão , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Interferons/uso terapêutico , Nucleotídeos/análise , Ribavirina/uso terapêutico , Nucleotídeos de Adenina/análise , Distribuição de Qui-Quadrado , Biologia Computacional , Nucleotídeos de Citosina/análise , Árvores de Decisões , Quimioterapia Combinada , Genótipo , Nucleotídeos de Guanina/análise , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Hidrogênio/análise , Oxigênio/análise , Seleção de Pacientes , Resultado do Tratamento , Nucleotídeos de Uracila/análiseRESUMO
The p7 protein of hepatitis C virus (HCV) is a multifunctional, integral membrane protein that plays a critical role in virus life cycle. This study demonstrated that the p7 proteins from different HCV genotypes (GTs) were unstable and degraded via the proteasome-dependent pathway. Mutagenesis analysis of the one and only lysine in JFH1 p7 (K4), the exclusive substrate for ubiquitination, did not stabilize p7 in human hepatoma cells. Collectively, this report demonstrated that p7 is degraded via the proteasome-dependent pathway, and provided evidence suggesting that p7 degradation is an ubiquitin-independent process.
Assuntos
Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Análise Mutacional de DNA , Hepatócitos/virologia , Humanos , Mutagênese Sítio-Dirigida , ProteóliseRESUMO
The dengue virus membrane (M) protein is a key component of the mature virion. Here, we characterised the cellular behaviour of M using a recombinant protein construct to understand its inherent properties. Using confocal microscopy, we showed that M and its intracellular precursor, prM, localised to the endoplasmic reticulum. M protein was also detected on the cell surface and secreted, suggesting that M can enter the secretory pathway. In addition, cross-linking studies showed that M can form dimers and tetramers. These findings suggest that M behaves as a secretory protein analogous to the major envelope protein E.
Assuntos
Vírus da Dengue/metabolismo , Retículo Endoplasmático/metabolismo , Rim/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Membrana Celular/metabolismo , Chlorocebus aethiops , Reagentes de Ligações Cruzadas/química , Células HEK293 , Humanos , Rim/citologia , Rim/virologia , Microscopia Confocal , Peso Molecular , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Propriedades de Superfície , Células Vero , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
The transmembrane domains (TMDs) of dengue virus type-1 M protein (DENV-1M) were reported to form cation-selective channels in artificial lipid bilayers. We further explored this observation using the two-electrode voltage clamp (TEVC) method on the Xenopus laevis oocytes expressing DENV PrM and M proteins. Using myc epitope tagged M proteins, M was first shown to adopt its predicted native topology in mammalian cells when expressed on its own. The recombinant proteins were then successfully expressed on the surface of Xenopus oocytes. Using influenza A M2 (Inf A/M2) protein as a control, we measured the conductance of oocytes expressing DENV proteins under hyperpolarized or low-pH conditions. Inf A/M2 showed pH-dependent, amantadine-sensitive channel activity that was consistent with previously published reports. However, no activity was detected for DENV proteins. We conclude that DENV PrM and M proteins do not show pH-activated ion channel activity.
Assuntos
Vírus da Dengue/patogenicidade , Canais Iônicos/metabolismo , Oócitos/virologia , Proteínas Virais/metabolismo , Xenopus laevis/virologia , Animais , Feminino , Concentração de Íons de Hidrogênio , Técnicas de Patch-Clamp/métodosRESUMO
To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B(12), was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B(12) and the core protein differ, and they target different regions of the IRES.