Secretion of VLPs and soluble E (sE). (A and B, left panels) Schemes of VLPs encoding constructs (PrME) of ZIKV (A) and DENV2 (B), with CaZ (blue), CaW (red), CaD (black) or the nonviral sec (gray), as signal peptides. Right panels show nonreducing Western blots of cellular extracts (E), culture supernatants (S), and ultracentrifuged pellet fraction (P; shown with [+] or without [−] 0.5% NP-40) of HEK293T cells transfected with the ZIKV (A) or DENV2 (B) VLP constructs. Protein E was detected with mAb 4G2. (C and D, left panels) Schemes of constructs encoding the covalent dimer (cvD) format of ZIKV PrMsE (C) or sE (D) with CaZ (blue), CaW (red), or the nonviral sec (gray), as signal peptides. Right panels show nonreducing Western blots of cellular extracts (E) and culture supernatants (S) of transfected HEK293T cells developed with anti-SV5. Arrows indicate the position of dimeric E.