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1.
J Virol ; 96(3): e0192321, 2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-34787460

RESUMO

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.


Assuntos
Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Macrófagos/metabolismo , Macrófagos/virologia , Norovirus/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Replicação Viral , Animais , Infecções por Caliciviridae/imunologia , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Ativação de Macrófagos , Macrófagos/imunologia , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Especificidade da Espécie
2.
Curr Protoc ; 3(7): e828, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37478303

RESUMO

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells Support Protocol 3: Purification of the norovirus NS1-2 protein Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents.


Assuntos
Caliciviridae , Norovirus , Camundongos , Humanos , Animais , Norovirus/genética , Antivirais/farmacologia , Caliciviridae/genética , Genoma , Mamíferos/genética
3.
J Virol ; 85(15): 7900-11, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632757

RESUMO

Iridoviruses (IV) are nuclear cytoplasmic large DNA viruses that are receiving increasing attention as sublethal pathogens of a range of insects. Invertebrate iridovirus type 9 (IIV-9; Wiseana iridovirus) is a member of the major phylogenetic group of iridoviruses for which there is very limited genomic and proteomic information. The genome is 205,791 bp, has a G+C content of 31%, and contains 191 predicted genes, with approximately 20% of its repeat sequences being located predominantly within coding regions. The repeated sequences include 11 proteins with helix-turn-helix motifs and genes encoding related tandem repeat amino acid sequences. Of the 191 proteins encoded by IIV-9, 108 are most closely related to orthologs in IIV-3 (Chloriridovirus genus), and 114 of the 126 IIV-3 genes have orthologs in IIV-9. In contrast, only 97 of 211 IIV-6 genes have orthologs in IIV-9. There is almost no conservation of gene order between IIV-3, IIV-6, and IIV-9. Phylogenetic analysis using a concatenated sequence of 26 core IV genes confirms that IIV-3 is more closely related to IIV-9 than to IIV-6, despite being from a different genus of the Iridoviridae. An interaction between IIV and small RNA regulatory systems is supported by the prediction of seven putative microRNA (miRNA) sequences combined with XRN exonuclease, RNase III, and double-stranded RNA binding activities encoded on the genome. Proteomic analysis of IIV-9 identified 64 proteins in the virus particle and, when combined with infected cell analysis, confirmed the expression of 94 viral proteins. This study provides the first full-genome and consequent proteomic analysis of group II IIV.


Assuntos
Genoma Viral , Iridovirus/metabolismo , Proteoma , Spodoptera/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Iridovirus/genética , MicroRNAs/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Spodoptera/citologia , Espectrometria de Massas em Tandem , Proteínas Virais/química , Proteínas Virais/genética
4.
mBio ; 13(2): e0017522, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35404121

RESUMO

Human norovirus (HNoV) is a global health and socioeconomic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3 days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next, we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27- IgD+, memory-switched CD27+ IgD-, memory-unswitched CD27+ IgD+, and double-negative CD27- IgD- cells). While the susceptibilities of the subsets were similar, changes in the B cell subset distribution upon infection were observed, which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly, primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro, which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, the elderly, and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover, HNoV does not elicit lifelong immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen, and lymph node specimens, while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses.


Assuntos
Infecções por Caliciviridae , Gastroenterite , Norovirus , Idoso , Humanos , Imunoglobulina D , Ativação Linfocitária , Norovirus/fisiologia
5.
J Virol ; 84(11): 5836-41, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20335264

RESUMO

Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to approximately 8-A resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.


Assuntos
Microscopia Crioeletrônica/métodos , Vírus da Doença Hemorrágica de Coelhos/química , Norovirus/química , Receptores Virais/química , Animais , Sítios de Ligação , Imageamento Tridimensional , Camundongos , Maleabilidade , Conformação Proteica , Coelhos
6.
Viruses ; 13(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209211

RESUMO

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.


Assuntos
Norovirus/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Alanina/genética , Aminoácidos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Norovirus/genética , Ligação Proteica , Sondas RNA
7.
Vaccines (Basel) ; 9(3)2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802376

RESUMO

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w/v) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

8.
Vaccines (Basel) ; 9(5)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066318

RESUMO

Breast cancer (BC) is the most frequently diagnosed cancer in women, with many patients experiencing recurrence following treatment. Antigens delivered on virus-like particles (VLPs) induce a targeted immune response and here we investigated whether the co-delivery of multiple antigens could induce a superior anti-cancer response for BC immunotherapy. VLPs were designed to recombinantly express murine survivin and conjugated with an aberrantly glycosylated mucin-1 (MUC1) peptide using an intracellular cleavable bis-arylhydrazone linker. Western blotting, electron microscopy and UV absorption confirmed survivin-VLP expression and MUC1 conjugation. To assess the therapeutic efficacy of VLPs, orthotopic BC tumours were established by injecting C57mg.MUC1 cells into the mammary fat pad of mice, which were then vaccinated with surv.VLP-SS-MUC1 or VLP controls. While wild-type mice vaccinated with surv.VLP-SS-MUC1 showed enhanced survival compared to VLPs delivering either antigen alone, MUC1 transgenic mice vaccinated with surv.VLP-SS-MUC1 showed no enhanced survival compared to controls. Hence, while co-delivery of two tumour antigens on VLPs can induce a superior anti-tumour immune response compared to the delivery of single antigens, additional strategies must be employed to break tolerance when targeted tumour antigens are expressed as endogenous self-proteins. Using VLPs for the delivery of multiple antigens represents a promising approach to improving BC immunotherapy, and has the potential to be an integral part of combination therapy in the future.

9.
J Immunol Res ; 2019: 5364632, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729137

RESUMO

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Melanoma/terapia , Proteínas Virais/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais/administração & dosagem
10.
Oncoimmunology ; 5(3): e1112941, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27141366

RESUMO

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

11.
Viruses ; 7(7): 3361-79, 2015 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-26114474

RESUMO

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.


Assuntos
Actinidia/microbiologia , Bacteriófagos/genética , Genoma Viral , Doenças das Plantas/microbiologia , Podoviridae/genética , Proteoma/metabolismo , Pseudomonas syringae/virologia , Proteínas Virais/genética , Bacteriófagos/química , Bacteriófagos/isolamento & purificação , Bacteriófagos/metabolismo , Frutas/microbiologia , Podoviridae/química , Podoviridae/isolamento & purificação , Podoviridae/metabolismo , Proteoma/química , Proteoma/genética , Pseudomonas syringae/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo
12.
J Immunother ; 36(1): 11-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23211625

RESUMO

Virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) can be used as a scaffold to facilitate the delivery of antigens to induce cell-mediated immune responses. In this study, we investigated the immune response to lymphocytic choriomeningitis virus-derived peptide antigen (gp33) delivered by RHDV VLP. The gp33 peptides were incorporated into the VLP in 2 different forms, either recombinantly expressed inside the VLP (VLP-gp33r) or chemically coupled to the surface of the VLP (VLP-gp33c). We showed that VLP-gp33r induced a greater level of cytotoxicity than VLP-gp33c against gp33-coated target cells in vivo. Both VLP, when delivered as prophylactic vaccines, inhibited the growth of Lewis' lung carcinoma tumors expressing gp33 (LL-LCMV) in mice to a similar degree. Studies to investigate the mechanism induced by these VLP showed that 2 CD11c DC subsets, CD8α and CD8α, acquired VLP in vivo and in vitro, and VLP-gp33r were cross-presented by both these subsets to prime CD8 T cells through a TAP-independent, endosomal recycling pathway. Depletion of Langerin DC in vivo before and after vaccination with VLP-gp33r, lead to reduced cytotoxicity implicating these cells in the induction of cytotoxic effector cells. These results suggest that recombinant VLP expressing tumor peptides targeted to Langerin DC may have clinical application. Finally we found that VLP-gp33r were more effective antitumor vaccines than VLP-gp33c when delivered therapeutically. The findings of this study suggest the potential of VLP as a platform for delivery of tumor-associate antigen and elicit protective immunity against tumors.


Assuntos
Antígenos Virais/imunologia , Vacinas Anticâncer/administração & dosagem , Carcinoma Pulmonar de Lewis/terapia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células Dendríticas/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia
13.
J Control Release ; 159(3): 338-45, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22386518

RESUMO

Virus-like particles (VLP) are effective vehicles for delivery of heterologous antigen to antigen-presenting cells. However VLP alone are insufficiently stimulatory to generate the signals required to facilitate effective priming of naïve T cells. We show that the VLP derived from rabbit hemorrhagic disease virus can bind the galactose-containing adjuvant α-galactosylceramide to form a composite particle for co-delivery of antigen and adjuvant to the same antigen-presenting cell. Vaccination with VLP and α-galactosylceramide activated splenic iNKT cells to produce IFN-γ and IL-4, led to the generation of antigen-specific T cells that protected prophylactically against subcutaneous tumor challenge, and was more effective at generating anti-tumor immune responses than either component individually. These data demonstrate a novel method for immunopotentiating VLP to increase their efficacy in the generation of anti-tumor responses via the innate ligand recognition properties of calicivirus-derived nanoparticles.


Assuntos
Adjuvantes Imunológicos , Vacinas Anticâncer/imunologia , Galactosilceramidas/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Galactosilceramidas/administração & dosagem , Galactosilceramidas/uso terapêutico , Vírus da Doença Hemorrágica de Coelhos/genética , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Linfócitos T/imunologia , Vírion/genética , Vírion/imunologia
14.
Bioprocess Biosyst Eng ; 31(5): 469-75, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18175154

RESUMO

The yield of two proteins, avidin and green fluorescent protein (GFP), expressed from a modified Autographa californica nucleopolyhedrovirus (AcMNPV), was compared in Sf9 cell culture monolayer, Sf21 cell suspension culture and intact Spodoptera litura larvae. GFP expressed from the p10 promoter yielded up to 1.5% of total soluble protein in larvae, 20-fold higher than that in monolayer suspension culture. Avidin, expressed from the polh promoter, yielded up to 2.3% of total soluble protein in larvae, 10-fold higher than that in suspension culture and 40-fold higher than that in monolayers. Avidin expression did not affect amounts of GFP in dual-expressing baculovirus compared with those detected from a GFP-only expressing AcMNPV. A biotin-binding assay showed that all avidin expressed in larvae was fully active. Glycosylation patterns of chicken-avidin and Spodoptera-avidin were very similar, though the latter showed a proportion of partially glycosylated material.


Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Spodoptera/fisiologia , Animais , Regulação da Expressão Gênica/fisiologia
15.
J Gen Virol ; 86(Pt 12): 3253-3261, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16298970

RESUMO

Baculovirus chitinases and other family 18 glycohydrolases have been shown to possess both exo- and endochitinase activities when assayed against fluorescent chito-oligosaccharides. Homology modelling of the chitinase of Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) against Serratia marcescens chitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and an alpha/beta TIM barrel catalytic domain characteristic of a family 18 glycohydrolase. EppoNPV chitinase has many features in common with other baculovirus chitinases, including high amino acid identity, an N-terminal secretion signal and a functional C-terminal endoplasmic reticulum-retention sequence. EppoNPV chitinase displayed exo- and endochitinolytic activity against fluorescent chito-oligosaccharides, with K(m) values of 270+/-60 and 240+/-40 microM against 4MU-(GlcNAc)2 and 20+/-6 and 14+/-7 microM against 4MU-(GlcNAc)3 for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc)(2-6) chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc)2, indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidal beta-chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc)2 complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc)(3-6) substrates, but not (GlcNAc)2, acted as inhibitors of EppoNPV chitinase. Short-chain substrates are unlikely to interact with the aromatic residues of the PKD1 substrate-feeding mechanism and hence may not accurately reflect the activity of these enzymes against native substrates. Based upon these results, the chitinase of the baculovirus EppoNPV is an exochitinase.


Assuntos
Baculoviridae/enzimologia , Quitinases/química , Quitinases/metabolismo , Acetilglucosamina/isolamento & purificação , Domínio Catalítico , Quitina/metabolismo , Dissacaridases/isolamento & purificação , Inibidores Enzimáticos , Espectrometria de Massas , Modelos Moleculares , Peso Molecular , Oligossacarídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismo
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