Expression of the potyvirus coat protein mediated by recombinant vaccinia virus and assembly of potyvirus-like particles in mammalian cells.

CSIRO, Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Archives of Virology (Impact Factor: 2.39). 02/1998; 143(7):1433-9. DOI: 10.1007/s007050050387
Source: PubMed

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.

5 Reads
  • Source
    • "A " ring " -like aggregate was also observed and it was suggested to be an intermediate in the reassembly of potyviruses . Potyviral CP has been expressed in Escherichia coli, yeast, insect, and mammalian systems (Edwards et al., 1994; Hammond et al., 1998; Jagadish et al., 1991; Joseph and Savithri, 1999) and in all the systems the formation of heterogeneous length filamentous virus-like particles (VLPs) were reported. One interesting feature of the potyviruses and their VLPs in general is that the amino-and carboxy-terminal regions of the CPs are surface exposed and they can be removed by limited trypsin treatment without affecting the assembly status (Jagdish et al., 1993). "
    [Show abstract] [Hide abstract]
    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 · 3.32 Impact Factor
  • [Show abstract] [Hide abstract]
    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 · 2.39 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: From the Introduction: Designed genetic reconstruction of natural proteins started in the early 1980s when Alan R. Fersht and his colleagues conceived the basic idea of protein engineering, i.e., a mutational intervention into the structure of proteins, based on spatial knowledge and oriented toward the creation of some kind of artificially improved proteins with desired functions. Since then, protein engineering as a specific branch of gene engineering has achieved much success, constructing new forms of enzymes and their inhibitors and elucidating the basic rules of protein folding and changing their specific activities...
    Artificial DNA: Methods and Applications, 1st edited by Y.E.Khudyakov, H.A.Fields, 09/2002: chapter Chapter 8. Artificial genes for chimeric virus-like particles: pages 249-327; CRC Press LLC, Boca Raton., ISBN: ISBN-10: 0849314267 ISBN-13: 978-0849314261
Show more