Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells |
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Authors: | Boura E Liebl D Spísek R Fric J Marek M Stokrová J Holán V Forstová J |
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Affiliation: | Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicná 5, 128 44 Prague 2, Czech Republic. |
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Abstract: | A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion. |
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Keywords: | AcNPV, Autographa californica nuclear polyhedrosis virus BMDC, bone marrow-derived dendritic cells CFSE, carboxyfluorescein diacetate succinimidyl ester CM, complete culture medium DC, immature human monocyte-derived dendritic cells EEA1, early endosomal antigen EGFP, enhanced green fluorescent protein FCS, foetal calf serum FSC and SSC, forward and side scatter characteristics EM, electron microscopy GM-CSF, granulocyte macrophage-colony stimulating factor LAMP2, lysosomal associated membrane protein LPS, lipopolysaccharide NMuMG, normal murine mammary gland cells p.a., post adsorption p.i., post infection PFU, plaque forming unit poly(I:C), inosine-cytosine dsRNA polynucleotide PyV, mouse polyomavirus t-VP3, truncated, C-terminus of VP3 minor capsid protein VLPs, empty artificial virus-like particles VP1-VLPs, pseudocapsids composed of VP1 major capsid protein EGFP-VLPs, pseudocapsids composed of VP1 major capsid protein and EGFP-t-VP3 fusion protein |
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