If the glucoevatromonoside inhibit the Na+K+ATPase, a reversion of the viral inhibition by cells exposure to culture medium containing an increased amount of K+ would be expected. Therefore, MEM was modified to contain 27 mM of potassium (5 times more than in the usual MEM = 5.4 mM). When Vero cells were infected and remained in this modified

MEM (lane 6), the HSV replication occurred characteristically suggesting that the K+ supplementation GW786034 order did not cause any alterations to the host cells, and consequently to the virus replication. In the same way, when the infected cells were treated with glucoevatromonoside and remained in the modified medium (lane 7), the viral protein levels were not inhibited. Furthermore, the treatment with glucoevatromonoside at 0.26 μM (IC100)

using the plaque reduction assay performed with K+ supplementation reestablished 22% of the viral replication capacity (data not this website shown), so these results indicated that the ion K+ is required to HSV-1 replication confirming the results obtained by Nagai et al. (1972). Still striving to elucidate the mechanism of antiherpes activity of glucoevatromonoside, its ability to interfere on virus release was investigated through the determination of intracellular and extracellular HSV-1 titers. Digitoxin, an inhibitor of this stage (Su et al., 2008), was used as a positive control. Every glucoevatromonoside tested concentrations significantly (p < 0.0001) reduced the extracellular

and intracellular virus titers confirming the inhibition of this step of virus replication (data not shown). Farnesyltransferase For example, the lower tested concentration (0.065 μM) reduced the extracellular and intracellular virus titers more than 99.99% (a reduction of 4.8 Log) and 90% (a reduction of 1.6 Log), respectively. In the same way, the percentages of virus release in the presence of different concentrations of glucoevatromonoside and digitoxin were calculated. These compounds inhibited HSV-1 release in a concentration-dependent manner. However, the glucoevatromonoside at its IC50/2 (0.065 μM) was more effective (99.7% of viral release inhibition) than the digitoxin at its IC50/2 (0.17 μM. (76% of viral release inhibition). It is well known that HSV infect epidermis and mucosae cells, and a rapid viral cell-to-cell spread is very important to the establishment of productive primary or recurrent infections in humans (Nyberg et al., 2004). The effect of different concentrations (0.015–0.25 μM) of glucoevatromonoside on HSV-1 cell-to-cell spread was evaluated through a viral plaque size reduction assay, and the results showed a significant (p < 0.001) reduction in the areas of formed viral plaques (from 56% to 98%), when compared to those formed in viral control (data not shown). This effect could be a consequence of the inhibition of viral release.