, 1999, Mazumder et al., 2002 and Zhu et al., 2004). Among the steps of HSV infection and replication, attachment and entry have been considered as potential targets. The findings presented in Table 2 are in agreement Tenofovir in vitro with those published by other authors, who stated that the mechanism underlying the antiherpes activity of polysaccharides, especially sulfated ones, may be related to the inhibition of HSV adsorption (Carlucci et
al., 1999, Eo et al., 2000 and Zhang et al., 2007). Since there was no detectable loss of HSV residual infectivity at 4 °C in the presence of MI-S, the virucidal mechanism in the adsorption assays was dismissed. Table 2 shows that MI-S and DEX-S displayed IC50 values lower than 1.21 μg/mL, whereas HEP showed values higher than 13.34 μg/mL. Since HEP was the only tested sulfated polysaccharide with a linear chain, it can be suggested that the presence of lateral branches could be important for the inhibition of the herpes virus penetration. HCS assay The lack of inhibition of viral adsorption and penetration by the non-sulfated polysaccharide (MI) confirmed that the presence of sulfate groups is required for such activities. In addition to the inhibition of HSV replication at 1 h p.i. treatment, MI-S presented inhibitory activity even when added at longer times after infection (Fig. 2), suggesting an action in post-entry events.
This hypothesis was investigated by Western blotting analyses, in which a considerable reduction of α (ICP27), β (UL42), and γ (gB) HSV-1 proteins expression was found when MI-S
was added at 1, 4, and 8 h p.i., respectively. Differently, infected cells treated with MI-S resulted in a slight reduction of gD expression. As for now, considering the performed experiments, the authors are unable to point out the reason for differences observed in reduction of expression of the late proteins gB and gD. Furthermore, the detected general reduction of protein production by MI-S could be associated with a secondary effect on another step of the viral cycle, as observed for ACV, for which inhibition of protein expression was due to an indirect effect on suppression of viral DNA replication. Although we are not aware of previous reports indicating the inhibition of HSV protein expression by sulfated polysaccharides, one study described the reduction of HIV Y-27632 2HCl protein expression by a sulfated oligosaccharide as well as by dextran sulfate (Artan et al., 2010). Since an efficient dissemination of virus has an important role in its infectivity, the inhibition of viral intercellular diffusion is an attractive target for new antiviral drugs. In the plaque size reduction assay, MI-S significantly reduced plaque areas. Recently, Ekblad and colleagues (2010) have shown the inhibition of HSV cell-to-cell spread by a sulfated tetrasaccharide. Here, a synergistic effect of MI-S with ACV was also found, supporting the results of their combination by Western blotting assay.