Herpes simplex type 1:lacZ recombinant viruses. I. 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 gD regulatory regions were constructed by recombination into the TK locus of HSV-1. Difficulty in isolating ICP4 and gD recombinant viruses with high level, regulated expression of beta-galactosidase was overcome by the use of HSV-1 translational initiation sequences of these genes in place of vector-derived sequences. beta-Galactosidase expression displayed the kinetics particular to each viral class. The maximal expression of beta-galactosidase from the recombinant 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 quantifiable levels of beta-galactosidase activity for multiplicities of infection from 5 x 10(-4) through 5 over 48 h postinfection. At multiplicities of infection between 2 and 5, ICP4-driven activity was measurable within 2 h postinfection from a monolayer of 3 x 10(4) Vero cells in microtiter wells. Mechanisms of inhibition of several antivirals were probed by using the regulated expression of beta-galactosidase from the ICP4 virus as a marker for viral growth. An experimental antiviral (E3925, IC50 1 microgram/ml) and a neutralizing gD MAb (DUP55306, IC50 0.6 microgram/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 plaque assays, but were determined in only 1 day, using a microtiter plate format. Thus, these reporter viruses are useful tools for defining the mechanisms of action of antiherpes agents, while quantitatively reproducing the results for IC50 determinations from standard plaque assays within 24 h in a microtiter plate format.
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DuPont Merck Pharmaceutical Co., Experimental Station, Wilmington, DE 19880-0400, USA.
Seetharam
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D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D015152:Blotting, Northern; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008969:Molecular Sequence Data; D010087:Oxides / Q000494:pharmacology; D010957:Plasmids; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; C099443:E 3925; D010087:Oxides; D007371:Interferon-gamma; D001616:beta-Galactosidase