Figure 5. Cyclic AMP stimulation potentiates SNV infectivity.
A. Pertussis toxin (PTX) treatment of CHO-A24 cells suggests that Gαi and Gαs acting through cyclic adenosine monophosphate (cAMP) regulate SNV infectivity. Gαs signaling up-regulates adenylate cyclase (AC) upstream of cAMP. cAMP stimulates the exchange protein activated by cAMP (Epac)27 a guanine nucleotide exchange factor (GEF) for Rap1.27 Rap 1 is a small GTP binding protein, which is an effector for integrin activation.39 Gαi signaling blocks Gαs activity upstream of AC. Pertussis toxin (PTX) inhibits Gαi inhibitory function. B. NF449, a Gαs specific antagonist, inhibits SNV infectivity. CHO-A24 cells were treated with 65 μM NF449 for 10 min before infection. C. PTX treatment induces a nearly two-fold increase in progeny SNV in CHO- A24 cells. 150,000 CHO A24 cells were plated onto a 48 well plate for 24 hours and then treated with 100 ng/ml PTX and incubated for ~18hrs at 37°C and then used in infection. CHO-A24 cells were treated with 100ng/ml PTX for 18 hours before infection. Viral N-protein was measured using a Western blot as previously described.9, 12 Quantitative analysis of the gel was performed using a BioRad Molecular Imager, ChemiDoc XRS+ equipped with Image Lab Software 4.1. We verified equal loading by detecting of β-actin on the same membrane with the anti-β-actin (clone AC-74 used at 1:2000 from Sigma). D. Panel inserts show fluorescence histograms of active Rap1 measured in wild-type and PTX treated cells. E. A ratiometric plot of SNV activated GTPases in PTX-treated cells and WT untreated cells measured at 5 min after activation. Values are means ± SD of triplicate measurements.