Studies on the Conditions Required for Structural and Functional Maturation of Rabies Virus Glycoprotein (G) in G cDNA‐Transfected Cells

Abstract

The spike projections of the rabies virion are composed of homotrimers of a single type of virus-coded glycoprotein (G) composed of 505 amino acids (1,11,35). The spike G proteins are involved in both the receptor recognition and binding to receptor molecules as well as in the low pH-dependent membrane fusion that occurs in the endosomes for releasing the viral ribonucleoprotein into the cytoplasm (30,31,34). As suggested from the experiments in which an amino acid substitution at position 333 of G protein strongly affected the viral neurovirulent nature (4,28,32), certain viral G protein function(s) might be involved in the selective viral invasion into and/or replication in the limited regions and/or the limited cell species in the brain. But, molecular and cellular mechanisms of viral neurovirulence also remain obscure yet.Precise mechanisms of the receptor recognition by rabies virus G protein as well as the low pH-dependent envelope fusion remain unclear yet (6, 7). By using Abstract: When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV-T7), functional G proteins were produced in terms of their ability to induce low pH-dependent syncytium formation and the formation of conformational epitopes, including the acid-sensitive epitope recognized by mAb #1-30-44. Such an ability and the 1-30-44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV-T7 using a tetracycline-regulated expression vector (pTet-G), although they were normally transported to the surface of established G protein-producing BHK-21 (G-BHK) cells. But, when the G-BHK cells were treated with 2.5 mM sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein-producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 mM as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1-30-44 epitope, while NaP at a higher concentration as 5.0 mM did induce similarly the increased production and enhanced maturation of G protein, including the 1-30-44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1-30-44 epitope formation, and 2.5 mM NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.

Two Different Conformations of Rabies Virus Glycoprotein Taken under Neutral pH Conditions

“…In this study, we first confirmed the presence of two forms of rabies virus G protein that are detected under neutral pH conditions on the surface of the virion and infected cells, the 1-30-44 epitope-positive and -negative forms, which were tentatively termed the B and C forms, respectively. (In other words, the difference between the B and C forms is based on their reactivity to mAb #1- [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44].) The ratio between the B and C forms was pH-dependent, and was the highest at pH 8.0.…”

“…In our previous study, maturation of the G protein to a 1-30-44 epitope-positive B form was not easily accomplished in the G cDNA-transfected BHK cells, but was achieved in a few days' treatment with 2.5 mM sodium butyrate in the absence of other viral factors (32). In addition, as suggested in this study, the 1-30-44 epitope-positive conformation is not stable, whereby the G protein is prone to undergo structural changes to lose the 1-30-44 epitope-positive conformation even under neutral pH conditions, which is not yet enough, however, for acquiring the membrane fusion activity.…”

“…(In other words, the difference between the B and C forms is based on their reactivity to mAb #1- [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44].) The ratio between the B and C forms was pH-dependent, and was the highest at pH 8.0.…”