Mittur N. Jagadish

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus.

The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.

Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles.

When the full-length coat protein (CP) of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed in Escherichia coli or yeast, it assembled to form potyvirus-like particles. The particles were heterogeneous in length with a stacked-ring appearance and resembled JGMV particles in their flexuous morphology and width. This cell-free assembly system should permit analysis of the mechanisms of particle assembly and genome encapsidation. Two mutant forms of CP produced by site-directed mutagenesis failed to assemble into virus-like particles.

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the worlds poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

Site-directed mutagenesis of a potyvirus coat protein and its assembly in Escherichia coli.

Multiple copies of the Johnsongrass mosaic virus coat protein synthesized in Escherichia coli can readily assemble to form potyvirus-like particles. This E. coli expression system has been used to identify some of the key amino acid residues, within the core region of the coat protein, required for assembly. The two charged residues R194 and D238 previously proposed theoretically to be involved as a pair in the construction of a salt bridge crucial for the assembly process were targeted for site-directed mutagenesis. The results from our experiments suggest that the two residues are required for the assembly process but are not necessarily involved as a pair in a common salt bridge.

Chimeric Potyvirus-Like Particles as Vaccine Carriers

Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.

Versatile cassettes designed for the copper inducible expression of proteins in yeast.

A series of yeast expression vectors and cassettes utilizing the CUP1 gene of Saccharomyces cerevisiae have been constructed. The cassettes contain multiple cloning sites for gene fusions and were created by inserting a 27-bp polylinker at the +14 position of the CUP1 gene. The cassettes are portable as restriction fragments and enable copper-regulated expression of foreign proteins in S. cerevisiae. In copper sensitive yeast, multiple copies of the CUP1 cassettes confer copper resistance due to the production of the copper metallothionein. Genes cloned into the CUP1 cassettes, however, usually prevent translation of the metallothionein leading to a loss of resistance. This could be useful for one-step cloning into yeast.

Localization of a VP3 epitope of infectious bursal disease virus

An immunodominant region of VP3, one of the two structural proteins of infectious bursal disease virus (IBDV strain 002-73), has been mapped by restriction site-specific deletion analysis and subcloning in Escherichia coli, followed by immunoblot analysis of the synthesized products. The epitope located within 58 amino acids reacted very strongly with a mouse monoclonal antibody (MAb 17/80) raised against IBDV 002-73. This immunodominant region may be useful in serodiagnosis of IBDV infection in poultry.

Expression and characterization of infectious bursal disease virus polyprotein in yeast.

Various expression vectors containing a cDNA fragment encoding all but the first five amino acids (aa) of the large polyprotein (N-VP2-VP4-VP3-C) of infectious bursal disease virus were transformed into yeasts. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, co- or post-translational processing of the unfused large polyprotein occurred, generating a stable C-terminal product (VP3) or correct size, but without any detectable N-terminal product (VP2). Furthermore, when the processing of the polyprotein was interrupted, because of an engineered in-frame site-specific insertion of 4 aa, even VP3 (as part of the unprocessed polyprotein) was undetected. VP2 was detected in S. cerevisiae only when fused to yeast pre-sequences at the N terminus, suggesting that in yeast, VP2 or the unprocessed polyprotein, in the absence of its native N terminus or proper protection of its N-terminal aa residues is susceptible to proteolytic degradation. The first 8 aa of a modified pre-sequence of the CUP1 gene product and the pre-pro sequence of MF alpha 1 gene product have been used for stable intra- and extra-cellular production of VP2, respectively.

Stable synthesis of viral protein 2 of infectious bursal disease virus in Saccharomyces cerevisiae

Viral protein 2 (VP2) from infectious bursal disease virus and its precursor polyprotein (N-VP2-VP4-VP3-C), in the absence of their native N-terminal region (19 amino acids), required fusion of yeast presequences for their stable synthesis in Saccharomyces cerevisiae [Jagadish et al., Gene 95 (1990) 179-186]. Restoration of the missing 19 aa resulted in stable synthesis of VP2, indicating the significance of the N-terminal region in protein stability.