Construction and characterization of herpes simplex virus type 1 mutants with conditional defects in immediate early gene expression

The herpes simplex virus type 1 (HSV-1) mutant in1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICPO, to yield mutant in1820. In almost all cell types tested, in1820 initiated infection less efficiently than in1814, behaving as if lacking both VP16 and ICPO functions, but in BHK cells in1820 was less impaired than in1814. A rescuant of in1820 at the VP16 locus, in1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types. In1825 was unable to complement an ICPO null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICPO protein in functionally significant amounts. The novel host range properties of in1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-I mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-I tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, into in1820 to produce in1820K. This mutant could be propagated effectively in BHK cells at 31 degrees but was effectively devoid of the major regulators ICPO, ICP4, and VP16 in other cells types at 38.5 degrees. Cultures could withstand infection with 5 PFU of in1820K per cell without detectable cytopathology and could be reseeded to form colonies al approximately 90% efficiency. A derivative of in1820K containing the Escherichia coli lacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of beta-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5 degrees. Cultures infected with 5 PFU per cell of an in1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based on in1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-I-derived vectors. (C) 1997 Academic Press.