Herpes simplex type1:lacZ recombinant viruses. II. Microtiter plate-based colorimetric assays for the discovery of new antiherpes agents and the points at which such agents disrupt the viral replication cycle.
A panel of microtiter plate-based colorimetric assays for monitoring HSV-1 growth has been made. The panel consists of 4 different HSV-1 (strain KOS) lacZ recombinant viruses which express beta-galactosidase under the control of different HSV-1 promoters derived from each class of herpes simplex gene expression: immediate-early (ICP4), early (TK), delayed early (gD) and late (gC). Inhibitors of HSV-1 growth were evaluated using differential effects on each of the reporter viruses as a measure of which points in the viral replication cycle an inhibitor was acting. Aphidicolin (DNA synthesis inhibitor) was studied as a model compound. At an m.o.i. of 0.05, at 24 h postinfection (h p.i.), aphidicolin inhibited 80% of viral growth at 1 micrograms/ml, as determined by a reduction in ICP4-driven activity within the second cycle of infection. At m.o.i. 5, within the first infectious cycle, aphidicolin had no effect on the signals from either the ICP4 or TK viruses at 3 micrograms/ml, while largely suppressing gD and fully inhibiting gC-driven signals at 2 micrograms/ml. This profile is consistent with the behavior expected of a DNA synthesis inhibitor. Five inhibitors of unknown mechanism were evaluated. Two compounds inhibited ICP4-driven activity within the first infectious cycle and were classified as potential inhibitors of viral entry, uncoating or IE gene expression (XF884, BT318). One compound inhibited gD and gC-driven activity without inhibiting signal from the ICP4 and TK viruses, and was classified as a potential DNA synthesis inhibitor (DS810). Two compounds (S5193, ER622) had effects on gD- and gC-driven activity which were somewhat different from aphidicolin and DS810, but which could be interpreted as inhibition of viral assembly and/or egress. The potency of XF884 varied with the time postinfection at which it was added to cells (IC50 3.7 to > 10 micrograms/ml) while the effects of BT318 were independent of time of addition (IC50 11.4 micrograms/ml). These results suggest XF884 inhibits viral entry while BT318 is acting after viral entry, possibly as a direct inhibitor of ICP4 gene expression. Together, these results suggest the panel of recombinant herpes viruses has utility in aiding in the identification of the points in the herpes life cycle at which antiherpes drug candidates, of unknown mechanisms, are acting.