Mapping of the Low pH‐Sensitive Conformational Epitope of Rabies Virus Glycoprotein Recognized by a Monoclonal Antibody #1‐30‐44

Abstract

Monoclonal antibody (mAb) #1‐30‐44 recognized an acid‐sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP‐Flury strain and the epitope‐negative CVS strain as well as the mAb‐resistant escape mutants, two distant sites that contained Lys‐202 and Asn‐336 were shown to be involved in the epitope formation. Lys‐202 is located in the so‐called neurotoxin‐like sequence, while Asn‐336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP‐Flury strain, in which Gln‐333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1‐30‐44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin‐like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1‐30‐44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH‐dependent function of G protein.

Further Studies on the Soluble Form (Gs) of Rabies Virus Glycoprotein (G): Molecular Structure of Gs Protein and Possible Mechanism of the Shedding

“…2 and Table 1, Gs proteins were recognized by most of our conformational epitope-specific anti-G mAbs except for one mAb, #1-30-44 (Fig. 2, lane 2), the mAb that recognized an acid-sensitive conformational epitope that is formed by two separate regions (Lys-202-containing and Asn-336-containing regions) of G protein (10). These observations indicate that the Gs protein took a 1-30-44-epitope-negative conformation (referred to as the Gc form), while the 1-30-44 epitope-positive conformation (referred to as the G B form) was detected in the authentic G protein (Table 1; 11).…”

“…As to the 1-30-44 epitope, we previously reported that at least two amino acids located in two separate regions (i.e., Lys-202 and Asn-336) are involved in the epitope formation (10,11). The Lys-202 is included in the neurotoxin-like region (17), while Asn-336 is located close to Arg-333, substitution of which affects the neurotropic nature of rabies virus in vivo (24,26).…”

“…These deletion mutants were inserted into pCDM8 vector (inserted downstream of the T7 promoter to be expressed by transcription with T7 RNA polymerase; reconstructed expression vectors were referred to as pCDM8-G, pCDM8-G∆TC and pCDM8-CT1). G gene expression was performed by transfecting plasmid DNAs to BHK-21 cells according to a protocol of the calcium phosphate method (1) with the help of recombinant vaccinia virus (RVV-T7) which provided T7 RNA polymerase as described previously (10).…”

“…All anti-G monoclonal antibodies (mAbs) used in this study were prepared and some were characterized in our previous studies (9)(10)(11)22). Most studies were performed by using mAbs #1-30-44 (specific for a low pHsensitive conformational epitope of G protein; 10), #1-46-12 (specific for a highly conserved conformational epitope; 9), and #7-1-9 (linear epitope-specific; 22).…”

“…For instance, one (#1-30-44) of those mAbs recognizes the acid-sensitive epitope of mature G protein, and two amino acids in separate regions, Lys-202 and Asn-336, have been suggested to be involved in the epitope formation (10). Using this mAb, rabies virus G protein was shown to take either of two conformations under neutral pH conditions, the 1-30-44 epitope-positive (referred to as G B ) and the negative (referred to as G C ) ones (11).…”