Fig. 1: Design and in vitro characterization of mRNA constructs expressing the three different versions of Varicella Zoster Virus (VZV) glycoprotein E (gE).

A Schematic representation of the various domains of VZV gE. The N-terminal domain contains disulfide bonds, N-glycans, and O-glycans (O-glycans not shown). Three versions of the VZV gE protein that we designed are shown: The soluble gE protein (gE soluble) contains only the signal sequence (SS) and the extracellular N-terminal domain. The truncated gE protein (gE truncated) contains the signal sequence, extracellular N-terminal domain, transmembrane (TM) domain and part of the C-terminal domain with one point mutation in the trans-Golgi network (TGN) localization motif, wherein AYRV sequence was modified to AARV (Y569A). The full-length gE protein (gE full-length) contains the entire wild-type open reading frame of the glycoprotein. B Western blot of cell lysates (left) and cell supernatants (right) after transfection with the indicated gE-expressing mRNA constructs. gE-FL – gE full-length, gE-T – gE truncated, and gE-S–gE soluble protein. Red bands correspond to the gE protein, green bands correspond to beta actin that is used as loading control. The observed molecular weight sizes for gE-FL (90 kDa) and gE-T (80 kDa) correspond to their partially glycosylated forms. gE-S (observed at 75 kDa) is not detected in the cell lysates, as expected, but is instead detected in the supernatant (right). C, D Detection of gE expression on cell surface (gE full-length and gE truncated) and in supernatant (gE soluble) by ELISA. EC50 values (in nM) are shown as Mean ± SD; R square values are shown to highlight the goodness of fit. The detection is performed using anti-gE antibodies. E, F Binding of human Fc fragment to cell surfaces expressed gE (gE full-length and gE truncated) (E) and gE in supernatant (F) to demonstrate the appropriate conformation and functional nature of the gE proteins. EC50 values (in nM) are shown as Mean ± SD; R square value is shown to highlight the goodness of fit.