FIG 3.

Virion morphogenesis in the absence of uncleaved NS2-3 in nonchimeric viruses. (A) Schematic drawing of the genome organizations of N7-2/EV-Ubi-3/VA and N7-2/EV-IRES-3/VA. Ubi, ubiquitin insertion. The mutations within NS2 (E1576A) and NS3 (V1721A) are indicated. (B) One microgram of the RNA genomes depicted in panel A were electroporated into MDBK cells. Viral replication was visualized by NS3-specific IF at 24 h pe. (C) At 48 h pe, viral supernatants were harvested and analyzed for the presence of infectious viruses. Viral titers were determined as TCID₅₀/ml. (D) Growth curve analysis. MDBK cells were infected with the indicated viruses at an MOI of 0.1, and viral growth was analyzed for 72 h. At the indicated time points, supernatants were collected and viral titers were determined (in TCID₅₀/ml). Analyzed viruses include N7-2/EV-Ubi-3/VA, N7-2/EV-IRES-3/VA, N7-2/EV-IRES-3/VA (passage), and the N7-2/EV-IRES-3/VA derivative, which encompasses all three titer-enhancing mutations in E2, NS2, and NS5B (E2/+K, 2/TA, and 5B/EG). NCP7 WT, wild type. Mean values from three independent experiments and standard deviations are depicted.