Figure 3

Indomethacin and curcumine inhibit MHV replication at the level of RNA synthesis. Caco-MHVR cells were incubated with and maintained in culture medium containing DMSO, 20 μM indomethacin, or 30 μM curcumine as described in figure legend 1. After 1 h, the cells were inoculated with MHV-A59 (m.o.i. = 1). At 6 and 9 h p.i., supernatants were collected and cells were harvested to isolate infectious viral particles, proteins and total RNA. (A) Caco-MHVR cells were inoculated with serial dilutions of combined supernatants and cleared cell homogenates from mock-treated (black bars), indomethacin-treated (grey bars) and curcumine-treated (white bars) cultures collected at 6 and 9 h p.i. The amount of ffu in the samples was determined with an indirect IFA as described in the legend of Figure 1 (n = 3). (B) Protein samples were analyzed on a SDS-15% polyacrylamide gel followed by Western blotting using the polyclonal anti-MHV serum. The N protein levels and the percentage of reduction (normalized for β-tubulin expression (data not shown)) in drug-treated cells compared to mock-treated cells are indicated. (C) The size of the observed syncytia was measured by counting the number of nuclei per syncytium of MHV-infected cells in the absence or presence of 20 μM indomethacin. (D) The expression levels of the N gene of MHV were determined by Taqman RT-PCR using primers 2915 (5'-GCCTCGCCAAAAGAGGACT-3') and 2916 (5'-GGGCCTCTCTTTCCAAAACAC-3') and a dual labeled probe (5'-6-FAM-CAAACAAGCAGTGCCCAGTGCAGC-TAMRA-3'). The relative amount of viral RNA in the drug-treated cells was expressed as a percentage of the average amount of viral RNA in the mock-treated cells.