FIG 7.
Tripartite production of pseudoviruses. (A) Schematic representation of the tripartite system of transfection for the production of pseudoviruses. (B) Packaging constructs used. ZIKV C was expressed alone (C) or fused to its own anchor CaZ (blue, C-CaZ), while ZIKV PrME was expressed with the indicated Ca (or sec), as signal peptides. (C) Representative cytofluorimetry profiles of Vero cells infected with ZIKV pseudoviruses produced with the indicated set of trans packaging constructs. (D) Quantification of ZIKV pseudoviruses production determined by cytofluorimetry of infected Vero cells at 24 hpi (n = 6 in all cases). (E) Nonreducing Western blot (upper panels) of cell extracts and pellets of ultracentrifuged supernatants of HEK293T cells, transfected with the indicated packaging constructs and developed with mAb 4G2. The bottom panel shows an agarose gel of RT-PCR products of the same supernatants stained with ethidium bromide. (F) Western blot analysis of cellular extracts of HEK293T cells cotransfected with ZIKV SV5-tagged constructs C and C-Ca (left panel) or C-Ca and C-PrME (right panel) and without (−) or with (+) WNV-rep. Cleaved and uncleaved C are indicated by red and black arrows, respectively.