FIG 4.
Cleavage by NS2B/NS3 viral protease and signal peptidase. (A) Schematic representation of packaging construct carrying the SV5 tag at the C N terminus. Ca sequence derived from WNV (CaW) is shown in red. (B) Western blot analysis of cellular extracts of HEK293T cells transfected with the constructs shown in panel A in the absence (−) or presence (+) of the WNV-rep. The migration of C, CPrM, and CPrME are indicated. (C) Representative cytofluorimetry profile of cells infected with pseudoviruses produced with the packaging constructs shown in panel A. (D) Scheme of the Ca from ZIKV, WNV, and DENV2 (CaZ, blue; CaW, red; and CaD, black) used as signal peptides on an irrelevant secretory reporter protein. The nonviral signal peptide sec (gray) is also shown. The vertical line indicates signal peptidase cleavage site. All constructs contain the first 5 amino acids of viral Pr indicated as reported previously (5) using the same color code. (E) Western blot of cell extracts (E) and culture supernatants (S) of cells transfected with constructs shown in panel D. Reporter protein was developed with anti-mouse IgG. Actin was used as a loading control.