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.
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ABSTRACT: The mechanism of assembly of flexuous viruses, such as potyviruses, is poorly understood. Using a recombinant system, we provide evidence that disassembly and reassembly of Pepper vein banding virus (PVBV), a member of the genus potyvirus, proceeds via a ring-like intermediate, and show that electrostatic interactions may be pivotal in stabilizing the particles. Although the surface-exposed N- and C-terminal residues can be removed from the virus-like particles (VLPs) by limited trypsinization without affecting their stability, such truncated CP subunits are unable to form VLPs. To further evaluate importance of these residues, N- and C-terminal deletion mutants were generated and their assembly behavior was investigated. N-terminal 53 and C-terminal 23 amino acids were found to be crucial for the intersubunit interactions involved in the initiation of virus assembly. These segments are surface exposed in the ring-like intermediate and dispensable for further interactions that result in the formation of the VLPs.Virology 12/2003; 316(2):325-36. DOI:10.1016/S0042-6822(03)00593-2 · 3.28 Impact Factor
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ABSTRACT: Pepper vein banding virus (PVBV) is an important virus infecting chilli pepper in south India. Earlier reports suggested it to be a distinct potyvirus. The nucleotide sequence of PVBV RNA from the 3'-end (3862 nt) was determined. Analysis of the nucleotide and deduced amino acid sequence revealed that it encompasses a partial open reading frame encoding the partial sequence of VPg, NIa-protease, NIb, coat protein (CP) and 3'-untranslated region (UTR). Comparison of the amino acid sequence of CP and the nucleotide sequence of 3'-UTR with those of other potyviruses confirmed an earlier observation that PVBV is a distinct member of the Potyvirus sub-group and it had significant similarity to a recently characterized virus infecting chilli pepper, chilli vein-banding mottle virus (CVbMV), from Thailand. The analysis showed that both PVBV and CVbMV might represent strains of the same virus. Further, the PVBV CP gene was overexpressed in E. coli, which assembled into potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate the CP mRNA.Archives of Virology 02/1999; 144(9):1679-87. DOI:10.1007/s007050050696 · 2.28 Impact Factor
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ABSTRACT: The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli. To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CPDelta63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of alpha-helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.Archives of Virology 05/2004; 149(4):699-712. DOI:10.1007/s00705-003-0247-x · 2.28 Impact Factor