FIG 3.

(A) Effect of RSV604 on intracellular viral replication in HeLa cells. HeLa cells were infected with RSV A2 at an MOI of 0.1 in the presence of DMSO, RSV604 (1× EC₉₀), or AZ-27 (1× EC₉₀). The quantity of RSV RNA in the infected cells was determined by RT-qPCR at the indicated times postinfection. (B and C) Effect of RSV604 on viral RNA synthesis in RSV replicon cells. Following compound treatment at the same concentrations as for panel A, percent inhibition of the replicon luciferase reporter activity (B) and intracellular viral replicon RNA copy numbers (C) was determined by luciferase assay at 48 h and by qRT-PCR assay at the indicated time points, respectively. The data shown are means and SD (n ≥ 2). (D) Effect of RSV604 on viral RNA synthesis in the RNP assay. Viral RNA synthesis in the RSV RNP replication complex was visualized using [³²P]UTP substrate in a 2-h reaction in the presence of DMSO (control), anti-N monoclonal antibody (MAb), or RSV604 (10 μM), followed by 6% PAA-urea gel electrophoresis. The RSV transcripts are labeled based on their expected sizes (lengths in nucleotides). The crude RSV RNP fraction used in this reaction was isolated from RSV A2-infected HEp-2 cells.