Molecular characterization of proteolytic processing of the human spumaretrovirus (HSRV) Gag proteins and the precise determination of cleavage sites was performed. For in vitro processing of recombinant HSRV Gag proteins, a recombinant enzymatically active HSRV protease was employed. Recombinant Gag proteins and protease were cloned and expressed as hexa-histidine-tagged proteins in pET-32b and pET-22b vectors, respectively, in the E. coli BL21 expression strain. The recombinant proteins were purified by affinity chromatography on an immobilized metal ion matrix. To determine the precise processing sites, recombinant Gag proteins or synthetic peptides derived from Gag sequences were cleaved in vitro by the recombinant protease. Proteolytic processing reactions were carried out under optimal reaction conditions of HSRV protease in sodium phosphate buffer, pH 6.0, supplied with 2 M NaCl at 37 degrees C. The cleavage sites were determined by amino-terminal amino acid sequencing as well as by matrix-assisted laser desorption/ionization mass spectrometry analysis of the reaction products. Fluorescence spectrophotometry was used to determine cleavage kinetics of peptides mimicking different cleavage sites within the HSRV Gag proteins.
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http://dx.doi.org/10.1385/1-59259-907-9:435 | DOI Listing |
Viruses
December 2024
Institute of Virology and Immunology, Länggass-Str. 122, CH-3001 Bern, Switzerland.
Bovine viral diarrhea virus (BVDV), a pestivirus in the family , is a major livestock pathogen. Horizontal transmission leads to acute transient infections via the oronasal route, whereas vertical transmission might lead to the birth of immunotolerant, persistently infected animals. In both cases, BVDV exerts an immunosuppressive effect, predisposing infected animals to secondary infections.
View Article and Find Full Text PDFPathogens
December 2024
Immunology and Infectious Diseases Program, Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, NL A1B 3V6, Canada.
Natural killer (NK) and CD8 T cell function is compromised in human immunodeficiency virus type 1 (HIV-1) infection by increased expression of inhibitory receptors such as TIGIT (T cell immunoreceptor with Ig and ITIM domains). Blocking inhibitory receptors or their ligands with monoclonal antibodies (mAb) has potential to improve antiviral immunity in general and facilitate HIV eradication strategies. We assessed the impact of TIGIT engagement and blockade on cytotoxicity, degranulation, and interferon-gamma (IFN-γ) production by CD8 T cells from persons living with HIV (PLWH).
View Article and Find Full Text PDFInt J Mol Sci
December 2024
Faculty of Nursing, Tokai University School of Medicine, Isehara 259-1193, Japan.
Retrotransposon Gag-like 4 (), a gene acquired from a retrovirus, is a causative gene in autism spectrum disorder. Its knockout mice exhibit increased impulsivity, impaired short-term spatial memory, failure to adapt to novel environments, and delayed noradrenaline (NA) recovery in the frontal cortex. However, due to its very low expression in the brain, it remains unknown which brain cells express RTL4 and its dynamics in relation to NA.
View Article and Find Full Text PDFHIV-1 assembly is initiated by the binding of Gag polyproteins to the inner leaflet of the plasma membrane, mediated by the myristylated matrix (MA) domain of Gag. Subsequent to membrane binding, Gag oligomerizes and buds as an immature, non-infectious virus particle, which, upon cleavage of the Gag precursor by the viral protease, transforms into a mature, infectious virion. During maturation, the MA lattice underlying the viral membrane undergoes a structural rearrangement and the newly released capsid (CA) protein forms a mature capsid that encloses the viral genome.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
January 2025
Grup d'Enginyeria de Bioprocessos i Biocatàlisi Aplicada, ENG4BIO, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Campus de Bellaterra, Cerdanyola del Vallès, 08193, Barcelona, Spain.
Several strategies have been developed in recent years to improve virus-like particle (VLP)-based vaccine production processes. Among these, the metabolic engineering of cell lines has been one of the most promising approaches. Based on previous work and a proteomic analysis of HEK293 cells producing Human Immunodeficiency Virus-1 (HIV-1) Gag VLPs under transient transfection, four proteins susceptible of enhancing VLP production were identified: ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ).
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