FIG. 7.

SubAB treatment cleaves GRP78 and reduces infectious-DENV production. (A) 293 cells were mock, SubAB, or SubA₂₇₂B mutant toxin treated for 12 h, and then the cells were lysed and subjected to Western analysis against the C-terminal region of GRP78 (rabbit anti-GRP78 [525 to 638]). Antibody complexes were detected with anti-rabbit IgG-HRP conjugate and chemiluminescence. The filters were reprobed with rabbit anti-14-3-3 zeta, and mouse anti-actin and antibody complexes were detected with anti-rabbit or anti-mouse IgG-HRP conjugate and chemiluminescence. (B and C) 293 cells were mock or DENV infected at an MOI of 3, and at 12 h p.i., they were mock, SubAB, or SubA₂₇₂B mutant toxin treated. At 24 h p.i. (after 12 h of mock, SubAB, or SubA₂₇₂B mutant toxin treatment), the supernatants were assayed for infectious-virus release by plaque assay (B), and RNA was extracted and subjected to real-time RT-PCR for DENV negative-strand RNA (C). DENV controls (bars 1 to 3) were serial 1/10 dilutions of in vitro-transcribed DENV negative-strand RNA. The results are representative of three independent infections.