Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VP1.

Abstract

Poliovirus type 1 cDNA sequences coding for viral capsid polypeptide VP1 were inserted into the fi-lactamase sequence of Escherichia coli plasmid pBR322. Resulting recombinant plasmid pSWll9 expressed in Escherichia coli a VPI-filactamase fusion protein that reacted with antibodies raised against poliovirus capsid polypeptide VP1 and with a monoclonal poliovirus type 1 neutralizing antibody, C3. Deletions of various lengths were generated within the VP1 sequence. The hybrid proteins expressed by the deleted plasmids did not react any more with C3 when the region of VP1 amino acids 95-110 (poliovirus nucleotides 2,754-2,806) was deleted. Therefore, the C3 epitope responsible for virus neutralization is most probably located in this region of the capsid polypeptide.Poliovirus, the causative agent of poliomyelitis, is a picornavirus. These are small viruses with a RNA genome of plus-strand polarity enclosed in a capsid composed of 60 copies each of four structural polypeptides, VP1, VP2, VP3, and VP4. Antibodies that neutralize virus infectivity are the major factor in protection against disease. The purified capsid polypeptides from several picornaviruses have been shown to induce a neutralizing antibody response in animals (1-4). In the case of foot-and-mouth disease virus (FMDV), for example, purified capsid polypeptide VP1 isolated from virions or prepared by DNA recombinant technology (1, 2, 5) was shown to protect susceptible animals against the disease. Moreover, synthetic peptides representing the sequence of the VP1 neutralization epitope have been used successfully as "subunit vaccine" (6, 7).In the case of poliovirus, however, the induction of neutralizing antibodies and the virus neutralization process are still poorly understood processes. Neutralizing sera raised against native poliovirions (D particles) and neutralizing monoclonal antibodies generated from mice immunized with native virions fail to react with the isolated viral capsid polypeptides (8)(9)(10). This suggests that the epitope(s) they recognize result, at least in part, from the three-dimensional arrangement of the proteins in the virion. Attempts have been made to raise neutralizing antisera by using single isolated capsid polypeptides, and a neutralizing antibody response was obtained by inoculating animals with purified VP1 but not with purified VP2 or VP3 (11,12). In a more recent study, however, another neutralization epitope was identified on VP3 or VP4 (13). The identification of the poliovirus capsid polypeptide recognized by neutralizing antibodies was also approached through chemical crosslinking of the antibodies to their virion binding sites. Two distinct neutralization epitopes were localized in this fashion to VP1 (10).Recently, we generated a hybridoma cell line producing neutralizing monoclonal antibodies from mice injected with heatinactivated virions (C particles) (14). In addition to binding to heat-denatured and to native virus, this antibody, C3, was found to selectively immunoprecipitate VP1. The fact that ...

Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12

“…Strains GC069, GC082, GC128, GC135, and GC440 all demonstrated complete resistance to microcin 25 (22,35); the rfaQ mutation in CS2774 results in a partial deep rough phenotype (22,35 flzA(pBB440), a strain which expressed diminished amounts of the chimeric protein ( Fig. 3 and 4) terized determinant (19,42,43) against which MAbs are available (3,12). In addition, its utility as a reporter epitope for insertion into outer membrane proteins is well documented (8,10,11,41).…”

Permissive sites and topology of an outer membrane protein with a reporter epitope

“…The sequencing procedure was derived from the dideoxynucleotide method (2) by using rapid preparations of the plasmid DNA (26) (45).…”

Immunogenicity of poliovirus B and T cell epitopes presented by hybrid porcine parvovirus particles

“…C3:B is one of the four neutralization antigenic sites of the poliovirus and is located in positions 93-103 of the VPI capsid protein (Van der Werf et al, 1983). It has been inserted into different vectors, including HBsAg (Delpeyroux et al, 1986(Delpeyroux et al, , 1988, diphtheria toxin protein (Phalipon et aI., 1989) and membrane and periplasmic Escherichia coli proteins (Charbit et al, 1988;Leclerc et al, 1990).…”