Fig 1: In Vitro replication and expression of RV-gp120TM. (a) Schematic of RV-gp120TM genome. The codon-optimized gp120TM (96ZM651) gene contains the native ZM96 signal sequence and FMDV 2A cleavage element at the N and C termini, respectively. (b) Maintenance of the gp120TM gene insert was assessed by 10 serial passages in helper Vero cells at MOI of 0.01. Viral titers were determined using anti-Env (goat a-gp120, Ab21179, Abcam) and anti-WN NS1 (Mab8152, Chemicon) antibodies providing titers of insert-containing and total infectious particles, respectively. VRC01 Mab was used to evaluate gp120TM conformation in the titration assay at selected passage samples 5, 7, 8, 9, and 10. (c) Stability of the gp120TM insert as evidenced by a single RT-PCR amplicon of the expected size produced from viral RNA isolated from P10 genetic stability passage using WN vector-specific primers outside of the insert. (d) Cell surface exposure and correct folding of gp120TM as shown by immunostaining of infected, formalin-fixed Vero cells using conformational VRC01 Mab; infected cells are also visualized with WN NS1-specific Mab 8152 (Chemicon).