HERPES-SIMPLEX TYPE 1-LACZ RECOMBINANT VIRUSES .1. CHARACTERIZATION AND APPLICATION TO DEFINING THE MECHANISMS OF ACTION OF KNOWN ANTIHERPES AGENTS

Recombinant viruses with the lacZ gene placed under the control of the HSV-1 ICP4, TK and go regulatory regions were constructed by recombin ation into the TK locus of HSV-1. Difficulty in isolating ICP4 and gD recombinant viruses with high level, regulated expression of beta-gala ctosidase was overcome by the use of HSV-1 translational initiation se quences of these genes in place of vector-derived sequences. beta-Gala ctosidase expression displayed the kinetics particular to each viral c lass, The maximal expression of beta-galactosidase from the recombinan t viruses within a 22-h period (m.o.i. 5) (relative to the ICP4 virus) was gD(3)> gC(2)> ICP4(1)> TK(0.5). The ICP4 virus produces easily qu antifiable levels of beta-galactosidase activity for multiplicities of infection from 5 x 10(-4) through 5 over 48 h postinfectoon At multip licities of infection between 2 and 5, ICP4-driven activity was measur able within 2 h postinfection from a monolayer of 3 x 10(4) Vero cells in microtiter wells. Mechanisms of inhibition of several antivirals w ere probed by using the regulated expression of beta-galactosidase fro m the ICP4 virus as a marker for viral growth, An experimental antivir al (E3925, IC50 1 mu g/ml) and a neutralizing gD MAb (DUP55306, IC50 0 .6 mu g/ml) acted prior to immediate early synthesis, consistent with inhibition of viral entry or uncoating. IFN-gamma inhibited expression of immediate-early synthesis, while having no effect on viral entry. IC50 values for E3925 obtained using either the ICP4 or gD viruses at m.o.i. 0.005, were in good agreement with those obtained by standard p laque assays, but were determined in only 1 day, using a microtiter pl ate format. Thus, these reporter viruses are useful tools for defining the mechanisms of action of antiherpes agents, while quantitatively r eproducing the results for IC50 determinations from standard plaque as says within 24 h in a microtiter plate format.