Fig 3.

Tr and E reduce HSV-2 binding/attachment, preferentially acting through cells rather than the virus, and lower viral replication, even when added after HSV-2 infection. In panel A, HEC-1A cells and HSV-2 were either treated with medium alone (−) or with 1 μg/ml of Tr or E protein for 1 h at 37°C before HSV-2 at an MOI of 1 was added to cells and allowed to attach at 4°C for 5 h. After viral challenge, cells were repeatedly washed, and whole-cell lysates were prepared and assessed for attached virus by immunoblotting using anti-gD antibodies and GAPDH as a loading control. (B) Relative intensities of WB bands were quantified using MBF_ImageJ for Microscopy Software. In panel C, HEC-1A cells (C lanes) were either pretreated with medium (med) or Tr and E as described above, repeatedly washed, and received HSV-2 at an MOI of 1 (V) for 5 h at 4°C as described above. Alternatively, virus was either left untreated or pretreated for 1 h with Tr or E before being added to medium-treated cells for 5 h at 4°C. WB in panel C and its quantification in panel D were performed as described above. In panels E and F, virus was either left untreated or preincubated with Tr or E for 1 h either at 4°C (E) or 37°C (F) and then added onto Vero cells for plaque assay as described in Materials and Methods. Data show viral titers as PFU/ml and are representative of two experiments, means ± SD. In panel G, Tr and E (1 μg/ml) were added in serum-free medium for 1 h at 37°C to HEC-1A cells 2 h after infection with HSV-2 at an MOI of 1 and cell washing. After 1 h, serum-containing medium was added, and cells were incubated for 24 h, after which viral titers were determined in cell supernatants by plaque assay as described in Materials and Methods. Data represent an average of three experiments with similar results, done in triplicate; means ± SD of PFU/ml, with significance shown in the graph.