FIG. 5.

Secretion of prM-E particles is not affected by NS2A-I59N mutation in a cell line stably expressing both a structural gene cassette and replicon RNA. (A) Schematic representation of production of secreted prM-E particles and replicon RNA-containing VLPs in tetKUNCprMErepZeo cells. Stably expressing cells were generated as described in Materials and Methods. (B) Northern blot analysis of RNA replication in tetKUNCprME-packaging BHK-21 cells stably expressing WT and NS2A-I59N-mutated repZeo replicon RNAs using ³²P-labeled probes specific for the KUN 3′ UTR and β-actin. CMV, cytomegalovirus; DOX, doxycycline; PAC, puromycin; SV40, simian virus 40; TRE, tetracycline-responsive element. (C) Production of VLPs containing repZeo RNAs in culture fluids harvested every 24 h for 5 days after induction of structural protein expression. VLP titers were determined by infectivity assay on VERO cells using immunofluorescence staining with anti-NS1 antibodies, as described in Materials and Methods. Error bars indicate standard deviations. (D) Sucrose gradient separation of prM-E particles and VLPs. Culture fluids were collected from tetKUNCprME-packaging BHK-21 cells stably expressing WT or NS2A-I59N-mutated repZeo RNAs 3 days postinduction of structural proteins, concentrated, and placed on a 10 to 40% continuous sucrose gradient. Fractions were taken from the top to the bottom of the gradient and analyzed by E capture ELISA. The detection of infectious VLPs in gradient fractions was performed by an infectivity assay of VERO cells using immunofluorescence staining with anti-NS1 antibodies.