Fig. 4.

Compounds 10 (L-731,988), 40, and 41 inhibit viral RNA synthesis besides affecting the virus entry process. Light gray bars: the test compounds L-742,001 (20 μM), 10 (200 μM), 40 (150 μM), 41 (150 μM), or chloroquine (80 μM) were added to MDCK cells, and after 30 minutes incubation at 35°C, influenza virus A/PR/8/34 was added. Dark gray bars: virus was added first and allowed to enter during 1 hour incubation, after which the compounds were added at the same concentrations as above. In both conditions, total cellular RNA was extracted at 10 hours postinfection (VC, untreated virus control). The number of vRNA copies was quantified by two-step real-time RT-PCR. On the y-axis, the fold increase in vRNA copies is shown relative to the viral copy number added at time zero. L-742,001 remains fully effective when added after virus entry. In contrast, the reported entry inhibitor chloroquine (Vanderlinden et al., 2012) is inactive when added at 1 hour postinfection. Compounds 10, 40, and 41 have a dual effect by acting both upon virus entry and viral RNA synthesis. Data shown are the mean ± S.E.M. of two independent tests.