Likewise,

incubation of PG545 with cells for a 2 h period

Likewise,

incubation of PG545 with cells for a 2 h period occurring just after 2 h period of inoculation of cells with RSV resulted in ∼60% reduction of RSV infectivity suggesting that PG545 could affect some steps occurring after virus penetration into the cells FG-4592 price or target the virus particles remaining on the cell surface since the cell entry rate of RSV is known to be relatively slow (Techaarpornkul et al., 2001). To clarify which event of the early RSV-cell interaction is targeted by PG545, the effect of this compound on the virus attachment to cells was tested. PG545, at a concentration range of 0.8–100 μg/ml, reduced the binding to cells of purified and radiolabeled RSV particles by ∼50% (Fig. 3A). In contrast muparfostat at 4–100 μg/ml prevented ∼75% of RSV virions from their binding to cells. Due to partial reduction of RSV binding to cells by PG545, we sought to investigate whether this compound could interfere with the events of RSV

cycle occurring after the virus attachment to cells. To this end, the virus was adsorbed to cells for 2 h at 4 °C prior to the addition of PG545 ��-catenin signaling in warm medium to trigger the entry of preadsorbed virus into the cells. Under these conditions PG545, and to lesser degree muparfostat, inhibited infection of cells by the pre-adsorbed virus (Fig. 3B) indicating that this compound could either displace the cell-attached virus or block the virus entry into the cells. Altogether, these data suggest that PG545 acts, at least in

part, through inhibition of RSV attachment to and entry into the cells. Furthermore, presence of PG545 in culture medium throughout the development of viral plaques reduced their aminophylline size by ∼42% (P < 0.005). In particular, the mean area of viral plaques (n = 28) developed 6 days after inoculation in mock-treated cells and in the presence of PG545 (4 μg/ml) was 0.31 ± 0.13 and 0.18 ± 0.06 mm2, respectively (data not shown). To identify which component of RSV particles is targeted by PG545, we attempted to select viral variants resistant to this compound. For comparative purposes, we also attempted to select viral variants resistant to muparfostat. To this end, plaque purified RSV A2 strain was subjected to 10 passages in HEp-2 cells in the presence of muparfostat (50 μg/ml) or to 13 passages in the presence of increasing concentrations (1–4.5 μg/ml) of PG545. The virus was also mock-passaged in the absence of the test compounds to serve as controls. However, the PG545 resistance of RSV generated in this way was not apparent. In particular, this virus could resist a maximum 4.5 μg/ml of the compound.

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