Dec rvkr cmk

To produce a more homogeneous population of RVPs with reduced uncleaved prM (referred to as DENV furin), RVPs were created by transfection of a HEK-293T cell line that expresses very high levels of human furin (HEK-293T-FIRB) (33 (link)). Transfected HEK-293T-FIRB cells were incubated at 30 °C and DENV furin RVPs harvested on days 3 to 6 posttransfection, clarified through a 0.22-μM filter, and frozen at −80 °C until further use. RVPs that retain significant uncleaved prM (DENV prM⁺) were produced including an inhibitor of furin-like protease activity during RVP production in WT HEK-293T cells. Briefly, transfection of replicon and structural gene plasmids was performed as described above. At 4 h after transfection, the media of transfected cells were exchanged with media containing 25 μM furin inhibitor Dec-RVKR-CMK (Enzo Life Sciences). Transfected cells were incubated at 37 °C and DENV prM⁺ RVPs harvested on days 1 to 2 posttransfection, clarified through a 0.22-μM filter, and frozen at −80 °C until further use. The efficiency of virion maturation was characterized functionally via neutralization studies with the maturation state–sensitive E DII fusion loop (FL)–reactive mAb E60 (25 (link)).