Expression of the potyvirus coat protein mediated by recombinant vaccinia virus and assembly of potyvirus-like particles in mammalian cells.
ABSTRACT The coat protein of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed using a recombinant vaccinia virus (VV) system. Ultra-thin section electron microscopy demonstrated that the coat protein assembled into potyvirus-like particles (PVLPs) in recombinant VV infected cells. Infection of cells with two additional VV recombinants expressing coat protein plus N-terminal and N- and C-terminal extensions also resulted in the formation of PVLPs. These results suggest that the ability of VV to express the potyvirus coat protein at sufficient levels to allow PVLP formation in vitro, could make VV a suitable vector for the delivery of PVLPs displaying vaccine antigens in vivo without the need for particle purification and/or inclusion of adjuvant. Use of such a vaccine strategy would also benefit from the proven advantages of poxviruses as vaccines such as stability in a freeze dried form, resistance to environmental factors and the potential for oral administration.
- SourceAvailable from: Adrianna Loniewska-LwowskaProgress in Nucleic Acid Research and Molecular Biology 02/2005; 80:135-68. · 0.31 Impact Factor
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ABSTRACT: Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.Acta biochimica Polonica 02/2007; 54(3):523-36. · 1.19 Impact Factor
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ABSTRACT: Vaccine strategies for the treatment of human papillomavirus-induced cervical cancer are based mainly on the human papillomavirus 16 E7 (HPV16 E7) oncoprotein. The immunogenicity of the E7 gene has been enhanced by its fusion to many different genes. Here, we linked a short sequence coding for the E7 peptide (aa 44-60) containing immunodominant epitopes for B and T cells to the 3' end of the gene coding for the whole coat protein (CP) of the poty-virus, potato virus A (PVA), and its deleted form (CPdel) with a short C-terminal deletion of 5 amino acids (LGVKG). CP-E7 and CPdel-E7 fusion proteins, just like CP alone, spontaneously assembled into virus-like particles in both procaryotic and eucaryotic cells. The CP-E7 and CPdel-E7 fusion genes induced slightly stronger E7-specific cytotoxic T-lymphocyte responses than the whole E7 gene, although they were still lower than those elicited by the previously constructed fusion gene, Sig/E7GGG/LAMP-1. The E7- and CP-specific antibody responses were not detected in mice vaccinated with CP-E7 and CPdel-E7 fusion genes. The CP-E7 and CPdel-E7 fusion genes protected mice against the development of tumors induced by TC-1 cells producing the E7 antigen and were also effective in the therapeutic setting, i.e. when the vaccination was performed after tumor cell administration. Their antitumor effect was comparable to those of the whole E7 gene and Sig/E7GGG/LAMP-1 fusion gene. There was no relevant difference between immune responses elicited by CP-E7 and CPdel-E7 DNA vaccination.Oncology Reports 11/2005; 14(4):1045-53. · 2.30 Impact Factor