PERSISTENCE AND EXPRESSION OF THE HERPES-SIMPLEX VIRUS GENOME IN THE ABSENCE OF IMMEDIATE-EARLY PROTEINS

The immediate-early (IE) proteins of herpes simplex virus (HSV) functi on on input genomes and affect many aspects of host tell metabolism to ensure the efficient expression and regulation of the remainder of th e genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, th eir expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system . In this study, we describe a series of ICP4 mutants that are defecti ve in different subsets of the remaining IE genes. One mutant, d109, d oes not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegaloviru s IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was ca pable of establishing persistent infections in both of these cell type s. Paradoxically, the genetic manipulations that were required to elim inate toxicity and allow the genome to persist in cells for long perio ds of time also dramatically lowered the level of transgene expression . Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the le vel of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks afte r the initial infection, demonstrating the functionality of the persis ting genomes.