Fig 4.

Expression of lytic RRV genes but not episome delivery to the nucleus is dependent on ERK activation. (A) RhF were infected in the absence or presence of U0126. At 4 h p.i, nuclei were isolated and DNA was extracted and analyzed by qPCR using RRV ORF45 primers. The RRV genome copy number was normalized using the human GAPDH copy number (see Materials and Methods). (B) RhF were infected in the absence or presence of U0126 with or without phosphonoacetic acid (PAA). At 48 h p.i., total RNA was extracted from cells with Tri-Reagent. RNA was treated with DNase and converted to first-strand cDNA. Diluted cDNA (5 to 20 ng) was assayed by real-time quantitative PCR (SYBR green) using primers for RRV ORF50 (left columns), ORF37 (middle columns), and ORF25 (right columns). Relative numbers of viral transcripts were normalized to GAPDH mRNA.