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1.
Nat Commun ; 10(1): 3048, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296855

RESUMO

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas Virais/metabolismo , Xanthomonas/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Oryza/microbiologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Fatores de Elongação da Transcrição/isolamento & purificação , Fatores de Elongação da Transcrição/ultraestrutura , Proteínas Virais/isolamento & purificação , Proteínas Virais/ultraestrutura , Xanthomonas/virologia
2.
Mol Biotechnol ; 61(8): 622-630, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31165966

RESUMO

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 µg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.


Assuntos
DNA Topoisomerases Tipo I/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Vírus Vaccinia , Proteínas Virais/isolamento & purificação , Animais , Baculoviridae/genética , Bombyx/metabolismo , Bombyx/virologia , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Magnésio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus Vaccinia/enzimologia , Vírus Vaccinia/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Nat Commun ; 10(1): 2104, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068591

RESUMO

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.


Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
4.
J Immunol Res ; 2019: 2075803, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30723748

RESUMO

Aim: The aim of this study was to evaluate the expression of persistence of mumps virus and some cells that interact with viral infection in the focus of the autoimmune epithelitis and peripheral blood of Sjögren's syndrome patients in comparison to patients with rheumatoid arthritis (RA) and nonautoimmune sicca syndrome (nSS). Materials and Methods: 126 patients (119 women and 7 men) were grouped into four groups: (1) patients with primary Sjögren's syndrome (pSS), (2) patients with secondary Sjögren's syndrome due to rheumatoid arthritis (sSS), (3) patients with rheumatoid arthritis (RA), and (4) patients with nonautoimmune sicca syndrome (nSS). Immunohistochemical analysis of immune response to the suggested silent persistence of mumps virus in the minor labial salivary gland biopsies and flow cytometric analysis of blood cells was done. Results: Immunohistochemical signs of mumps virus persistence were found in the minor salivary glands of all study groups. Also, a significantly different immune response to virus infection (protein IFI16, interferons gamma and beta, dendritic cells, and receptor for natural killers) was revealed in the minor salivary glands of the study groups. Cytometric analysis of the blood cells revealed a dropping amount of circulating natural killers and dendritic cells in patients with SS. Significant correlations between immunohistochemical staining and serological findings were revealed. Conclusions: Abundant immunohistochemical signs of mumps virus protein in the salivary glands and depletion of circulating immune cells make a background for thought of presumable mumps or/and other virus participation in epithelial damage causing sicca syndrome in predisposed patients.


Assuntos
Vírus da Caxumba/imunologia , Glândulas Salivares/virologia , Síndrome de Sjogren/imunologia , Idoso , Artrite Reumatoide/imunologia , Biópsia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Glândulas Salivares/citologia , Glândulas Salivares Menores/citologia , Glândulas Salivares Menores/virologia , Síndrome de Sjogren/virologia , Proteínas Virais/isolamento & purificação
5.
Arch Virol ; 164(4): 1049-1058, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30778744

RESUMO

Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is ~ 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.


Assuntos
Antígenos Virais/análise , Antígenos Virais/genética , Capripoxvirus/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Doenças das Cabras/virologia , Infecções por Poxviridae/veterinária , Proteínas Virais/análise , Proteínas Virais/genética , Animais , Antígenos Virais/isolamento & purificação , Antígenos Virais/metabolismo , Capripoxvirus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Doenças das Cabras/diagnóstico , Cabras , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/virologia , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
6.
Microb Pathog ; 128: 414-422, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30597256

RESUMO

Exosomes are micro messengers encapsulating RNA, DNA, and proteins for intercellular communication associated with various physiological and pathological reactions. Several viral infection processes have been reported to pertain to exosomal pathways. However, because of the difficulty in obtaining avian-sourced exosomes, avian virus-related exosomes are scarcely investigated. In this study, we developed a protein A/G-correlated method and successfully obtained the Newcastle disease virus-related exosome (NDV Ex). These exosomes promoted NDV propagation, proven by both GW4869-mediated deprivation and exosomal supplementation. Viral structural proteins NP and F were detected in the NDV Ex and further investigation indicated that the NP protein can be transferred to DF-1 cells through exosomes. The intracellular NP protein exhibited viral replication-promoting and cytokine-suppressing abilities. Therefore, NDV infection produces exosomes, which transfer viral NP protein and promote NDV infection, emphasizing the importance of exosomes in an NDV infection.


Assuntos
Exossomos/metabolismo , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/patogenicidade , Estruturas Virais/isolamento & purificação , Estruturas Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Galinhas , Citocinas/metabolismo , Humanos , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Nucleoproteínas/isolamento & purificação , Nucleoproteínas/metabolismo , Proteínas Recombinantes , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Proteínas Virais de Fusão/isolamento & purificação , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
7.
Arch Virol ; 164(3): 657-665, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30498963

RESUMO

The genome of Chilo iridescent virus (CIV) has two open reading frames (ORFs) with matrix metalloprotease (MMP) domains. The protein encoded by ORF 136R contains 178 amino acids with over 40% amino acid sequence identity to hypothetical metalloproteases of other viruses, and the protein 165R contains 264 amino acids with over 40% amino acid sequence identity to metalloproteases of a large group of organisms, primarily including a variety of Drosophila species. These proteins possess conserved zinc-binding motifs in their catalytic domains. In this study, we focused on the functional analysis of these ORFs. They were cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf9 cells with an N-terminal His tag, and purified to homogeneity at 72 hours postinfection using Ni-NTA affinity chromatography. Western blot analyses of purified 136R and 165R proteins with histidine tags resulted in 24- and 34-kDa protein bands, respectively. Biochemical assays with the purified proteins, performed using azocoll and azocasein as substrates, showed that both proteins have protease activity. The enzymatic activities were inhibited by the metalloprotease inhibitor EDTA. Effects of these proteins were also investigated on Galleria mellonella larvae. Insecticidal activity was tested by injecting the larvae with the virus derived from the AcMNPV bacmid carrying 136R or 165R ORFs. The results showed that the baculoviruses harbouring the iridoviral metalloproteases caused early death of the larvae compared to control group. These data suggest that the CIV 136R and 165R ORFs encode functional metalloproteases. This study expands our knowledge about iridoviruses, describes the characterization of CIV matrix metalloproteinases, and might ultimately contribute to the use of this virus as a research tool.


Assuntos
Iridovirus/enzimologia , Metaloproteases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Genoma Viral , Iridovirus/química , Iridovirus/genética , Lepidópteros , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Células Sf9 , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
8.
Int J Biol Macromol ; 124: 810-818, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500497

RESUMO

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.


Assuntos
Bacteriófagos/química , Endopeptidases/química , Histidina/química , Metaloproteínas/química , Oligopeptídeos/química , Proteínas Virais/química , Zinco/química , Bacteriófagos/enzimologia , Cálcio/química , Cálcio/metabolismo , Domínio Catalítico , Cátions Bivalentes , Cromatografia de Afinidade , Clonagem Molecular , Diálise/métodos , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Imidazóis/química , Metaloproteínas/genética , Metaloproteínas/isolamento & purificação , Metaloproteínas/metabolismo , Níquel/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo , Zinco/metabolismo
9.
Methods Mol Biol ; 1918: 67-86, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30580400

RESUMO

A critical component of bacterial detection assays is choosing a suitable affinity molecule that retains sensitivity and specificity for the target pathogen over a wide range of in situ applications. Bacteriophages (phages) are bacterial viruses that bind and infect their host cells with unmatched specificity. Phage host range is often determined by their long tail fibers (LTFs) that mediate adsorption of the virus particle to potential bacterial host cells, by binding to specific cell surface receptors. The inherent specificity of the LTFs for distinct bacterial species makes them ideal candidates for development into recombinant affinity molecules. In this chapter, we describe the development of the Salmonella phage S16 LTF (S16 LTF) into an affinity molecule as part of a novel assay to detect Salmonella cells. The enzyme-linked long tail fiber assay (ELLTA) involves two steps: (1) Immobilization and separation of Salmonella cells using S16 LTF-coated paramagnetic beads (LTF-MBs), and (2) Labeling of bead-captured Salmonella using horseradish peroxidase-conjugated S16 LTF (HRP-LTF). Rapid HRP-mediated conversion of a chromogenic substrate provides visual confirmation for the presence of Salmonella. Overall, the ELLTA assay requires as little as 2 h to detect as few as 102 cfu/ml Salmonella cells from liquid culture. The absorbance of the enzyme-generated color substrate is largely proportional to the present bacterial concentrations between 102 and 107 cfu/ml, providing semiquantitative determination of Salmonella cell counts. The methodology described in this chapter can be adapted for other phage receptor-binding proteins, to develop ELLTAs for the detection of other relevant bacterial pathogens.


Assuntos
Bactérias/virologia , Bacteriófagos/fisiologia , Proteínas Virais/metabolismo , Bactérias/imunologia , Infecções Bacterianas/microbiologia , Biotinilação , Imunoensaio , Plasmídeos/genética , Proteínas Recombinantes , Fagos de Salmonella/fisiologia , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
10.
Appl Microbiol Biotechnol ; 103(2): 833-842, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30421111

RESUMO

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.


Assuntos
Infecções por Circoviridae/prevenção & controle , Circovirus/metabolismo , Kluyveromyces/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais/metabolismo , Virossomos/metabolismo , Animais , Anticorpos Antivirais/sangue , Reatores Biológicos , Cromatografia , Infecções por Circoviridae/patologia , Infecções por Circoviridae/virologia , Circovirus/genética , Códon , Meios de Cultura/química , Modelos Animais de Doenças , Kluyveromyces/genética , Fígado/virologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Baço/virologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Virossomos/genética
11.
J Biochem ; 165(2): 167-176, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30371907

RESUMO

Interactions of phosphorylated eIFiso4E binding to VPg as a function of temperature and ionic strength were assessed employing fluorescence spectroscopic. Phosphorylation increased the binding affinity ∼3.5-fold between VPg and eIFiso4E under equilibrium conditions. Binding affinity of VPg for eIFiso4Ep correlates with the ability to enhance in vitro protein synthesis. Addition of VPg and eIFiso4Ep together to Dep WGE enhances the translation for both uncapped and capped mRNA. However, capped mRNA translation was inhibited with addition of eIFiso4Ep alone in dep WGE, suggesting that phosphorylation prevents the cap binding and favours the VPg binding to promotes translation. Temperature dependence showed that the phosphorylated form of the eIFiso4E is preferred for complex formation. A van't Hoff analysis reveals that eIFiso4Ep binding to VPg was enthalpy driven (ΔH = -43.9 ± 0.3 kJ.mol-1) and entropy-opposed (ΔS = -4.3 ± 0.1 J.mol-1K-1). Phosphorylation increased the enthalpic contributions ∼33% for eIFiso4Ep-VPg complex. The thermodynamic values and ionic strength dependence of binding data suggesting that phosphorylation increased hydrogen-bonding and decreased hydrophobic interactions, which leads to more stable complex formation and favour efficient viral translation. Overall these data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/isolamento & purificação , Fosforilação , Proteínas Virais/química , Proteínas Virais/isolamento & purificação
12.
Vet Microbiol ; 228: 252-258, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593375

RESUMO

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes upper respiratory tract disease in chickens and significant losses to the poultry industry worldwide. Both antibody and cell-mediated responses are generated against ILTV infection; however, the correlation of humoral immune response with protection against ILTV infection is debatable. To examine if whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, four ILTV glycoproteins (gD, gE, gG and gJ) were expressed as recombinant proteins and used in conjunction with commercially available recombinant gC and gI in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-ILTV vaccination sera found that gE was the most antigenic glycoprotein and that gC ODs were strongly correlated with those of gI and gJ, while ODs to gG had a relatively poor correlation with those of other glycoproteins. Moderate to poor correlations were found between microscopic tracheal lesion scores and ODs to individual glycoproteins. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation with protective immunity as measured by the severity of clinical signs, gross lesions, and tracheal viral load. Results from this study demonstrated that systemic antibody titers to individual ILTV glycoproteins C, D, E, G, I and J had a relatively poor correlation to protective immunity.


Assuntos
Anticorpos Antivirais/sangue , Galinhas/imunologia , Herpesvirus Galináceo 1/imunologia , Glicoproteínas de Membrana/imunologia , Doenças das Aves Domésticas/imunologia , Vacinas Virais/imunologia , Animais , Galinhas/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Células HEK293 , Humanos , Imunidade Humoral , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Doenças das Aves Domésticas/virologia , Vacinação/veterinária , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificação
13.
Artigo em Inglês | MEDLINE | ID: mdl-30392576

RESUMO

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and animals. In bacteria, these enzymes are rare and have been introduced via horizontal gene transfer. Since viruses function as horizontal gene transfer vectors and since lipoxygenases may be helpful for releasing assembled virus particles from host cells we explored whether these enzymes may actually occur in viruses. For this purpose we developed a four-step in silico screening strategy and searching the publically available viral genomes for lipoxygenase-like sequences we detected a single functional gene in the genome of a mimivirus infecting Acantamoeba polyphaga. The primary structure of this protein involved two putative metal ligand clusters but the recombinant enzyme did neither contain iron nor manganese. Most importantly, it did not exhibit lipoxygenase activity. These data suggests that this viral lipoxygenase-like sequence does not encode a functional lipoxygenase and that these enzymes do not occur in viruses.


Assuntos
Expressão Gênica , Lipoxigenase , Mimiviridae , Proteínas Virais , Acanthamoeba/virologia , Lipoxigenase/química , Lipoxigenase/genética , Lipoxigenase/isolamento & purificação , Mimiviridae/enzimologia , Mimiviridae/genética , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
14.
Vet Res Commun ; 42(4): 289-295, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30219981

RESUMO

Bovine mastitis causes severe economic losses to dairy farmers. Staphylococcus aureus, is one of the most important pathogen implicated in etiology of clinical and subclinical mastitis in bovines. In view of increasing antimicrobial resistance alternatives to antibiotic therapy are much needed. The present decade has witnessed a renewed interest in phage based therapeutics and diagnostics. The present study, describes isolation and characterization of two lytic phages SAJK-IND and MSP against Staphylococcus aureus having a potential to be used in therapy against mastitis. SAJK-IND and MSP phages belonged to Myoviridae and Podoviridae families, respectively. TEM imaging of the two phages revealed an iscosahedral head. MSP phage has a short non contractile tail. SAJK-IND and MSP have a burst size of 44 ± 3 and 25 ± 5 PFU/ infected cell, respectively. SAJK-IND and MSP phages revealed Ì´ 12 and Ì´16 proteins, respectively on SDS-PAGE analysis. The lytic activity of the phages was specific for Staphylococcus aureus. SAJK-IND revealed 100% lytic activity against several strains of Staphylococcus aureus isolated from mastitis milk samples whereas, MSP had only 40% lytic activity. SAJK-IND phage genome was sequenced, assembled and deposited in Genbank under accession no MG010123.


Assuntos
Bacteriófagos , Mastite Bovina/terapia , Terapia por Fagos/veterinária , Infecções Estafilocócicas/veterinária , Animais , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bovinos , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Genoma Viral/genética , Índia , Microscopia Eletrônica de Transmissão/veterinária , Myoviridae/genética , Myoviridae/isolamento & purificação , Terapia por Fagos/métodos , Podoviridae/genética , Podoviridae/isolamento & purificação , Proteoma/genética , Infecções Estafilocócicas/terapia , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Sequenciamento Completo do Genoma/veterinária
15.
Artigo em Inglês | MEDLINE | ID: mdl-30245048

RESUMO

Fowl adenovirus-4 (FAdV-4) causes hydropericardium syndrome (HPS) in poultry worldwide. An understanding of viral structural protein composition is important for developing novel immunodiagnostics and immunoprophylactics. Here we report isolation, culture, molecular and protein profile of FAdV-4 isolates recovered from HPS outbreaks in chicken in the states of Himachal Pradesh and Tamil Nadu in India. We performed a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting-based protein profiling of FAdV-4 isolates against a reference FAdV-1 or Chicken Embryo Lethal Orphan (CELO) virus. SDS-PAGE analysis showed that seven protein bands in FAdV-4 isolates were similar to CELO expect an additional band of 110 kDa in CELO virus. On Western blotting, two protein fractions of 43 kDa and 78 kDa size were observed in FAdV-4 isolates. Overall, results show that FAdV-4 isolates recovered from different regions of the country had similar protein profile and possibly a common source of origin.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/química , Galinhas , Doenças das Aves Domésticas/virologia , Proteínas Virais/análise , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/genética , Aviadenovirus/crescimento & desenvolvimento , Aviadenovirus/isolamento & purificação , Western Blotting/métodos , DNA Viral/química , Surtos de Doenças/veterinária , Eletroforese em Gel de Poliacrilamida/métodos , Adenovirus A das Aves/química , Soros Imunes/imunologia , Índia/epidemiologia , Masculino , Filogenia , Reação em Cadeia da Polimerase , Aves Domésticas , Doenças das Aves Domésticas/epidemiologia , Coelhos , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
16.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976672

RESUMO

Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivoIMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.


Assuntos
DNA Helicases/química , DNA Helicases/fisiologia , DNA Primase/química , DNA Primase/fisiologia , Herpesvirus Humano 1/patogenicidade , Proteínas Virais/química , Proteínas Virais/fisiologia , Liberação de Vírus , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Primase/genética , DNA Primase/isolamento & purificação , Olho/virologia , Células HEK293 , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases , Células Vero , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Vírion/fisiologia , Virulência , Montagem de Vírus
17.
Sci Rep ; 8(1): 10589, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002425

RESUMO

The anaerobic spore-forming bacterium Clostridium perfringens is a source of one of the most common food-borne illnesses in the United States and Europe. The costs associated with disease management are high and interventions are limited; therefore, effective and safe antimicrobials are needed to control food contamination by C. perfringens. A viable solution to this problem could be bacteriophage lysins used as food additives or food processing aids. Such antimicrobials could be produced cost-effectively and in ample supply in green plants. By using edible plant species as production hosts the need for expensive product purification can be reduced or obviated. We describe the first successful expression in plants of C. perfringens-specific bacteriophage lysins. We demonstrate that six lysins belonging to two different families (N-acetylmuramoyl-L-alanine amidase and glycosyl hydrolase 25) are active against a panel of enteropathogenic C. perfringens strains under salinity and acidity conditions relevant to food preparation environments. We also demonstrate that plant-expressed lysins prevent multiplication of C. perfringens on cooked meat matrices far better than nisin, the only currently approved bacteriocin food preservative to control this pathogen.


Assuntos
Bacteriófagos/metabolismo , Clostridium perfringens/efeitos dos fármacos , Conservantes de Alimentos/farmacologia , Proteínas Virais/farmacologia , Bacteriófagos/genética , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/isolamento & purificação , Contagem de Colônia Microbiana , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos , Conservantes de Alimentos/isolamento & purificação , Conservantes de Alimentos/metabolismo , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Carne/microbiologia , Testes de Sensibilidade Microbiana , Nisina/farmacologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Tabaco/genética , Tabaco/metabolismo , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
18.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29950422

RESUMO

Goatpox virus (GTPV) is an important member of the Capripoxvirus genus of the Poxviridae Capripoxviruses have large and complex DNA genomes encoding many unknown proteins that may contribute to virulence. We identified that the 135 open reading frame of GTPV is an early gene that encodes an ∼18-kDa protein that is nonessential for viral replication in cells. This protein functioned as an inhibitor of NF-κB activation and apoptosis and is similar to the N1L protein of vaccinia virus. In the natural host, sheep, deletion of the 135 gene from the GTPV live vaccine strain AV41 resulted in less attenuation than that induced by deletion of the tk gene, a well-defined nonessential gene in the poxvirus genome. Using the 135 gene as the insertion site, a recombinant AV41 strain expressing hemagglutinin of peste des petits ruminants virus (PPRV) was generated and elicited stronger neutralization antibody responses than those obtained using the traditional tk gene as the insertion site. These results suggest that the 135 gene of GTPV encodes an immunomodulatory protein to suppress host innate immunity and may serve as an optimized insertion site to generate capripoxvirus-vectored live dual vaccines.IMPORTANCE Capripoxviruses are etiological agents of important diseases in sheep, goats, and cattle. There are rare reports about viral protein function related to capripoxviruses. In the present study, we found that the 135 protein of GTPV plays an important role in inhibition of innate immunity and apoptosis in host cells. Use of the 135 gene as the insertion site to generate a vectored vaccine resulted in stronger adaptive immune responses than those obtained using the tk locus as the insertion site. As capripoxviruses are promising virus-vectored vaccines against many important diseases in small ruminants and cattle, the 135 gene may serve as an improved insertion site to generate recombinant capripoxvirus-vectored live dual vaccines.


Assuntos
Apoptose/genética , Capripoxvirus/genética , NF-kappa B/antagonistas & inibidores , Proteínas Virais/genética , Vacinas Virais/genética , Animais , Capripoxvirus/imunologia , Capripoxvirus/patogenicidade , Vetores Genéticos , Células HEK293 , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Imunidade Inata , Fatores Imunológicos/imunologia , Mutagênese Insercional , NF-kappa B/genética , Fases de Leitura Aberta/genética , Vírus da Peste dos Pequenos Ruminantes/química , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/imunologia , Ovinos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Vacinas Virais/imunologia
19.
FEBS Lett ; 592(15): 2572-2581, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29933499

RESUMO

PB1F2 is a proapoptotic protein encoded by an alternative reading frame in the influenza A virus. Its accumulation accelerates mitochondrial fragmentation by decreasing the mitochondrial membrane potential following translocation into the mitochondrial inner membrane space, but the mechanistic underpinnings remain unclear. Herein, the PB1F2 from HK97 was expressed and purified in soluble form. The interaction between PB1F2 and the mitochondrial membrane were investigated using three membrane mimics, liposomes, bicelles, and nanodiscs. We show that the interactions between PB1F2 and membrane mimics depend on lipid type and are time- and dose-dependent. The primary membrane target of PB1F2 is phosphatidylcholine, the lipid that forms the major component of mitochondrial inner membranes. PB1F2 disrupts the integrity of lipid membranes by forming micelle-like PB1F2-lipid assemblies.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Vírus da Influenza A , Lipídeos de Membrana/química , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Apoptose , Membrana Celular/virologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Ligação Proteica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificação , Internalização do Vírus
20.
Br Poult Sci ; 59(4): 402-407, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29798683

RESUMO

1. Infectious laryngotracheitis is a respiratory disease that affects the poultry industry worldwide. It is common in flocks with high-bird density, causing major economic losses. 2. In this study, a SYBR® FAST polymerase chain reaction (PCR) double-strand DNA intercalating agent assay was performed for the detection of infectious laryngotracheitis virus (ILTV) in clinical samples in comparison with a conventional nested-PCR, both based on the glycoprotein E encoding gene. This assay amplified 56 bp and was capable of detecting 19 to 1 copies of virus. 3. In total, 164 clinical samples were obtained from birds with respiratory problems from the period of 2009-2016. In the nested-PCR, there were 45.12% positive samples and 54.88% negative samples, while in the real-time PCR (qPCR), there were 81.1% positive samples and 18.9% negative samples. 4. In conclusion, qPCR from the DNA double-strand intercalating agent SYBR® GREEN FAST was useful for the diagnosis of ILTV because it detected samples that were negative in nested-PCR. This assay has advantages, such as a shortened processing-time, and no need for post-amplification processing (electrophoresis) with additional reagents, such as MgCl2 and agarose. Hence, qPCR proved to be useful, rapid and low cost for use with clinical samples.


Assuntos
Galinhas , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/isolamento & purificação , Glicoproteínas de Membrana/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Virais/isolamento & purificação , Animais , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Glicoproteínas de Membrana/genética , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Virais/genética
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