Replication-defective adenovirusp259Acaused a 400-fold increase in the sequence-specific antiherpetic activity of oligo(nucleoside methylphosphonate) (ONMP) IE4,5SA. Herpes simplex virus type 1 (HSV-1) growth was not inhibited in cells exposed top259Ain the absence of IE4,5SA or in cells cotreated with I E4,5SA and heated (10 minutes, 90°C)p259Avirus. Fluorescent microscopy of Vero cells treated with BODIPY-conjugated IE4,5SA revealed intracellular localization within endocytic-like vesicles with minimal cytoplasmic and intranuclear distribution. Diffuse staining over the entire cell was observed in cell cotreated with the BODIPY-conjugated IE4,5SA andp259Avirus. This effect was not observed in cells cotreated with the BODIPY-conjugated ONMP and heatedp259Avirus. We interpret these findings to indicate thatp259Aaugments IE4,5SA antiherpetic activity presumably via its ability to increase ONMP uptake and release from endocytic-like vesicles