RESUMO
CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L(-/-) and CD4(-/-)) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8(+) T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L(-/-) and CD4(-/-) mice revealed that the protection was indeed CD40L mediated but CD4(+) T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.
Assuntos
Imunidade Adaptativa/imunologia , Ligante de CD40/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Imunidade Adaptativa/genética , Adenoviridae/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/deficiência , Ligante de CD40/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cães , Feminino , Vetores Genéticos/genética , Células HEK293 , Humanos , Imunização , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Nucleoproteínas/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sobrevida , Linfócitos T Citotóxicos/imunologiaRESUMO
All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.
Assuntos
Anticorpos Monoclonais/imunologia , Sequência Conservada , Farmacorresistência Viral/imunologia , Epitopos/imunologia , Vírus da Influenza B/enzimologia , Influenza Humana/prevenção & controle , Neuraminidase/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/imunologia , Cães , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Epitopos/química , Humanos , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/crescimento & desenvolvimento , Vírus da Influenza B/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Neuraminidase/antagonistas & inibidores , Neuraminidase/químicaRESUMO
The only universally conserved sequence amongst all influenza A viral neuraminidase (NA) is located between amino acids 222-230 and plays crucial roles in viral replication. However, it remained unclear as to whether this universal epitope is exposed during the course of infection to allow binding and inhibition by antibodies. Using a monoclonal antibody (MAb) targeting this specific epitope, we demonstrated that all nine subtypes of NA were inhibited in vitro by the MAb. Moreover, the antibody also provided heterosubtypic protection in mice challenged with lethal doses of mouse-adapted H1N1 and H3N2, which represent group I and II viruses, respectively. Furthermore, we report amino acid residues I222 and E227, located in close proximity to the active site, are indispensable for inhibition by this antibody. This unique, highly-conserved linear sequence in viral NA could be an attractive immunological target for protection against diverse strains of influenza viruses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteção Cruzada , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Modelos Animais de Doenças , Epitopos de Linfócito B/imunologia , Feminino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologiaRESUMO
The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.
Assuntos
Epitopos/metabolismo , Vírus da Influenza A/enzimologia , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise , Domínio Catalítico , Linhagem Celular , Embrião de Galinha , Estabilidade Enzimática/genética , Epitopos/química , Epitopos/genética , Células HEK293 , Humanos , Vírus da Influenza A/genética , Cinética , Modelos Moleculares , Mutação , Neuraminidase/química , Neuraminidase/genética , Estrutura Terciária de Proteína , Especificidade por Substrato , Temperatura , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Vaccination is the most effective prophylactic method for preventing influenza. Quantification of influenza vaccine antigens is critically important before the vaccine is used for human immunization. Currently the vaccine antigen quantification relies on hemagglutinin content quantification, the key antigenic component, by single radial immunodiffusion (SRID) assay. Due to the inherent disadvantages associated with the traditional SRID; i.e. low sensitivity, low throughput and need for annual reagents, several approaches have been proposed and investigated as alternatives. Yet, most alternative methods cannot distinguish native hemagglutinin from denatured form, making them less relevant to antigenic analyses. Here, we developed a quantitative immunoassay based on the sialic acid binding property of influenza vaccine antigens. Specifically, we chemically synthesized human and avian influenza virus receptors analogues, N-acetylneuraminic acid-2,6-lactose and N-acetylneuraminic acid-2,3-lactose derivatives with an azidopropyl aglycon, using α-2,6- and α-2,3-sialyltransferases, respectively. The azido group of the two sialyllactose-derivatives was reduced and conjugated to mouse serum albumin through a squarate linkage. We showed that the synthetic α-2,6- and α-2,3-receptors selectively bound to human and avian-derived hemagglutinins, respectively, forming the basis of a new, and robust assay for hemagglutinin quantification. Hemagglutinin treated at high temperature or low pH was measured differentially to untreated samples suggesting native conformation is dependent for optimal binding. Importantly, this receptor-based immunoassay showed excellent specificity and reproducibility, high precision, less turnaround time and significantly higher sensitivity and throughput compared with SRID in analyzing multiple influenza vaccines.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vacinas contra Influenza/análise , Ácido N-Acetilneuramínico/síntese química , Animais , Antígenos Virais/análise , Antígenos Virais/imunologia , Azidas/química , Aves , Glicosídeos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Imunodifusão , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Desnaturação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Sialiltransferases/metabolismo , Especificidade da Espécie , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
We identified the interaction between HBV X (HBx) protein and the oncogene AIB1 (amplified in breast cancer 1). A serine/proline motif (SSPSPS) in HBx was found to be required for the interaction. Two LXD motifs [LLXX(X)L, X means any amino acids], LLRNSL and LLDQLHTLL in AIB1 were also found to be involved in the HBx-AIB1 interaction. The HBx-AIB1 interaction was important for the activation of NFκB signal transduction, the HBx mutant that did not interact with AIB1showed dramatically lower NFκB activation activity than the WT HBx. These findings contribute to the new understanding on signal transduction activation mechanisms of HBx.
Assuntos
Carcinógenos , NF-kappa B/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Dados de Sequência Molecular , Mutação , Coativador 3 de Receptor Nuclear/genética , Domínios e Motivos de Interação entre Proteínas , Serina/genética , Serina/metabolismo , Transdução de Sinais , Transativadores/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais Reguladoras e AcessóriasRESUMO
Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before.
Assuntos
Adenoviridae/genética , Portadores de Fármacos/administração & dosagem , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas de Ligação a RNA/imunologia , Vacinação/métodos , Proteínas do Core Viral/imunologia , Animais , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo , Infecções por Orthomyxoviridae/mortalidade , Plasmídeos , Proteínas de Ligação a RNA/administração & dosagem , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Core Viral/administração & dosagem , Proteínas do Core Viral/genéticaRESUMO
The potency of varicella vaccines is currently determined by a plaque assay technique, which usually takes seven days and is laborious and has considerable inter- and intra-assay variability. Here, we report a new potency assay for varicella vaccine based on quantitative polymerase chain reaction in conjunction with a much more efficient virus infection step. Potency results can be obtained within 24h of infection and demonstrates acceptable accuracy and reproducibility when compared with the plaque assay, which relies on manual counting of plaques formed one week after viral infection. Using multiple vaccine lots from 7 manufacturers, we found no significant difference in infectivity determined between the new assay and plaque assay. The optimized conditions for viral infection and polymerase chain reaction are of significant value for the potency determination of the vaccine due to its rapidity, accuracy and the high throughput capacity of the assay.
Assuntos
Vacina contra Varicela/imunologia , Vacina contra Varicela/normas , Reação em Cadeia da Polimerase/métodos , Tecnologia Farmacêutica/métodos , Vacina contra Varicela/genética , Humanos , Controle de QualidadeRESUMO
BACKGROUND: Many proteins contain conserved sequence patterns (motifs) that contribute to their functionality. The process of experimentally identifying and validating novel protein motifs can be difficult, expensive, and time consuming. A means for helping to identify in advance the possible function of a novel motif is important to test hypotheses concerning the biological relevance of these motifs, thus reducing experimental trial-and-error. RESULTS: GOmotif accepts PROSITE and regular expression formatted motifs as input and searches a Gene Ontology annotated protein database using motif search tools. The search returns the set of proteins containing matching motifs and their associated Gene Ontology terms. These results are presented as: 1) a hierarchical, navigable tree separated into the three Gene Ontology biological domains - biological process, cellular component, and molecular function; 2) corresponding pie charts indicating raw and statistically adjusted distributions of the results, and 3) an interactive graphical network view depicting the location of the results in the Gene Ontology. CONCLUSIONS: GOmotif is a web-based tool designed to assist researchers in investigating the biological role of novel protein motifs. GOmotif can be freely accessed at http://www.gomotif.ca.
Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteínas/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Humanos , Internet , Estrutura Terciária de Proteína , Proteínas/genética , SoftwareRESUMO
There are few reports in the literature of hepatitis as a manifestation of parvovirus B19 infection. We describe a case of parvovirus B19-associated acute hepatitis diagnosed based on a positive serologic test (IgM) and molecular detection of parvovirus B19 DNA in a liver biopsy specimen. Parvovirus B19 infection should be considered in the differential diagnosis of patients presenting with acute hepatitis.
Assuntos
Hepatite Viral Humana/diagnóstico , Hepatite Viral Humana/virologia , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/isolamento & purificação , Adulto , Anticorpos Antivirais/sangue , Biópsia , DNA Viral/isolamento & purificação , Feminino , Histocitoquímica , Humanos , Imunoglobulina M/sangue , Fígado/patologia , Fígado/virologia , MicroscopiaRESUMO
Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses(1,2). As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)(3). However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity(4), the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method(5). Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines. Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses(6-7). One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide(6), while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)(7). Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera(7,8). Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.
Assuntos
Anticorpos Antivirais/análise , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Immunoblotting/métodos , Vírus da Influenza A/imunologia , Neuraminidase/análise , Animais , Biologia Computacional , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/imunologia , Neuraminidase/genética , Neuraminidase/imunologia , CoelhosRESUMO
The traditional antiviral assays for the determination of interferon potency are reported to have considerable variability between and within assays. Although several reporter gene assays based on interferon-inducible promoter activities have been reported, data from comprehensive validation studies are lacking and few studies have been conducted to analyze the variant forms of interferons, which could have undesirable clinical implications. Here, a reporter gene assay employing a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter was developed and validated. The assay was found to be more sensitive, with a larger detection range than the antiviral assay. Several cytokines tested did not interfere with the test, suggesting the assay possesses a certain degree of selectivity. Moreover, the robustness of the assay was demonstrated by minimal variations in the results generated by different analysts and cell passage number (up to 52 passages). Finally, the method was employed to analyze several interferon variants (interferon-α 2a) and we found that the aggregated form has completely lost its potency; while a modest loss of bioactivity in oxidized interferon was observed (approx. 23%), the deamidated form essentially retained its activity.
Assuntos
Antivirais/farmacologia , Genes Reporter/efeitos dos fármacos , Interferons/farmacologia , Elementos de Resposta/efeitos dos fármacos , Antivirais/uso terapêutico , Bioensaio , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Interferons/uso terapêutico , Luciferases/genética , TransfecçãoRESUMO
The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Hemaglutininas Virais/imunologia , Vírus da Influenza A/imunologia , Animais , Linhagem Celular , Cães , Humanos , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A/fisiologia , Internalização do Vírus , Replicação ViralRESUMO
Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Ácidos Carboxílicos , Nigericina , Infecções por Poxviridae/tratamento farmacológico , Vaccinia virus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Bioterrorismo/prevenção & controle , Ácidos Carboxílicos/farmacologia , Ácidos Carboxílicos/uso terapêutico , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/análise , Células HeLa , Humanos , Concentração Inibidora 50 , Nigericina/análogos & derivados , Nigericina/farmacologia , Nigericina/uso terapêutico , Infecções por Poxviridae/virologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Vaccinia virus/fisiologiaRESUMO
Neuraminidase-induced immune responses are correlated with protection of humans and animals from influenza. However, the amounts of neuraminidase in influenza vaccines are yet to be standardized. Thus, a simple method capable of quantifying neuraminidase would be desirable. Here we identified two universally conserved sequences in all influenza A and B neuraminidases, one representing a novel finding of nearly 100% conservation near the enzymatically active site. Antibodies generated against the two highly conserved sequences bound to all nine subtypes of influenza A neuraminidase and demonstrated remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Importantly, employing these antibodies for the analyses of vaccines from eight manufacturers using the same vaccine seeds revealed marked variations of neuraminidase levels in addition to considerable differences between lots from the same producer. The reasons for the absence or low level of neuraminidase in vaccine preparations are complex and could be multi-factorial. The antibody-based assays reported here could be of practical value for better vaccine quality control.
Assuntos
Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Neuraminidase/análise , Anticorpos Monoclonais/imunologia , Sequência Conservada , Reações Cruzadas , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Homologia de Sequência de AminoácidosRESUMO
The fusion peptide of influenza virus hemagglutinin (HA) has a critical role in mediating the entry of the virus into the cells and is also the only universally conserved sequence in the HAs of all strains of influenza A and influenza B viruses. Therefore, it could be an attractive target for new vaccine development and a potency marker for existing influenza vaccines. The fusion peptide epitope is hidden inside the HA proteins, making it inaccessible for quantitative antibody binding. Our simple slot blot protocol highlights pre-treatment of HA samples with moderate concentrations of denaturant to maximally expose the fusion peptide on the protein surface, followed by detection using universal antibodies targeting the fusion peptide. The method is highly reliable, inexpensive and easy to follow. The entire procedure takes only 5 h and can be applied to the quantitative determination of virtually all influenza viral HAs using a single antibody targeting the fusion peptide.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Immunoblotting/métodos , Vírus da Influenza A/química , Anticorpos Antivirais , Sequência Conservada , Epitopos/análise , Epitopos/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Immunoblotting/estatística & dados numéricos , Imunodifusão/métodos , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologiaRESUMO
The T:G mismatch specific DNA glycosylase (TDG) is known as an important enzyme in repairing damaged DNA. Recent studies also showed that TDG interacts with a p160 protein, steroid receptor coactivator 1 or nuclear receptor coactivator 1 (SRC1), and is involved in transcriptional activation of the estrogen receptor. However, whether other members of the p160 family are also involved in TDG-interaction and signal transduction regulation remains to be seen. In this study, we employed the mammalian two-hybrid system to investigate the interaction between TDG and another member of the p160 family, nuclear receptor coactivator 3 (NCoA-3). We found that a DXXD motif from aa 294-297 within TDG was responsible for the TDG-NCoA-3 interaction, we also found that a LLXXXL motif (X means any amino acid) from aa 1029-1037 (LLRNSL) and a merged LLXXL motif (LLDQLHTLL) from aa 1053-1061 in NCoA-3 were important for the TDG-NCoA-3 interactions. Mutation of the two aspartic acids (aa 294 and 297) into two alanines in TDG significantly affected the interaction and subsequent transcriptional activation of several steroid hormone receptors including, estrogen-, androgen- and progesterone- receptors in Huh7 cells. We also identified that mutations of NCoA-3 at either leucines 1029-1030 or 1053-1054 (replaced by alanines) also reduced the interaction activity between TDG and NCoA1. These data indicated that the TDG-NCoA-3 interaction is important for broad range activation of steroid hormone nuclear receptors, and may also contribute significantly to further understanding of TDG-related nuclear receptor regulation.
Assuntos
Coativador 3 de Receptor Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Timina DNA Glicosilase/metabolismo , Ativação Transcricional , Motivos de Aminoácidos , Linhagem Celular Tumoral , Humanos , Mutagênese Sítio-Dirigida , Coativador 3 de Receptor Nuclear/fisiologia , Mapeamento de Interação de Proteínas , Timina DNA Glicosilase/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus. CONCLUSIONS/SIGNIFICANCE: Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.
Assuntos
Ácido Aurintricarboxílico/farmacologia , Betainfluenzavirus/efeitos dos fármacos , Betainfluenzavirus/enzimologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Neuraminidase/antagonistas & inibidores , Amantadina/farmacologia , Animais , Linhagem Celular , Meios de Cultura , Citoproteção/efeitos dos fármacos , Cães , Farmacorresistência Viral/efeitos dos fármacos , Sinergismo Farmacológico , Corpos de Inclusão Viral/efeitos dos fármacos , Corpos de Inclusão Viral/ultraestrutura , Vírus da Influenza A/fisiologia , Vírus da Influenza A/ultraestrutura , Betainfluenzavirus/fisiologia , Betainfluenzavirus/ultraestrutura , Oseltamivir/farmacologia , RNA Viral/análise , Inativação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The multimerization/self-interaction of viral proteins is an important step in the process of viral assembly and maturation. Our previous study indicated that the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid protein forms self-multimers through a serine-arginine (SR)-rich motif (SSRSSSRSRGNSR) by using a mammalian two-hybrid system. To determine the biological relevance of this motif, we constructed a SARS-CoV reverse genetic construct by using a bacterial artificial chromosome (BAC)-based vector controlled by a T7 promoter; and subsequently deleted the SR-rich motif from the N gene. The mutated infectious clone showed reduced level of genome transcription and significantly reduced levels of the infectious virions. These results strongly suggest that the SR-rich motif is critical for effective virus replication.
Assuntos
Motivos de Aminoácidos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Replicação Viral , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Arginina/química , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Serina/química , Transfecção , Células VeroRESUMO
The fusion peptide is the only universally conserved sequence in the hemagglutinins of all 16 subtypes of influenza A and two genetic lineages of influenza B viruses. Here, peptides selected by bioinformatics approach were modified and conjugated to overcome serious technical hurdles such as the high hydrophobicity and weak immunogenicity of the viral fusion peptides. Antibodies generated against fusion peptides demonstrated remarkable specificity against the viral sequences and robustness of quantitatively analyzing the viral hemagglutinins even under stringent conditions. As quantitatively revealed by antibody-binding experiments, the fusion peptides of diverse hemagglutinins are exposed to the same degree upon unfolding at neutral pH to the physiologically fusogenic state. To our knowledge, this is the first report on the quantitative determination of virtually all influenza vaccines using a single universal antibody.