IMPROVED CELL-SURVIVAL BY THE REDUCTION OF IMMEDIATE-EARLY GENE-EXPRESSION IN REPLICATION-DEFECTIVE MUTANTS OF HERPES-SIMPLEX VIRUS TYPE-1 BUT NOT BY MUTATION OF THE VIRION HOST SHUTOFF FUNCTION

Derivatives of herpes simplex virus type 1 (HSV 1) have elicited consi derable interest as gene transfer vectors because of their ability to infect a wide range of cell types efficiently, including fully differe ntiated neurons. However, it has been found that infection of many typ es of cell with vectors derived from replication-defective mutants of HSV-I is associated with cytopathic effects (CPE). We have previously shown that viral gene expression played an important role in the induc tion of CPE caused by an HSV-1 mutant deleted for the essential immedi ate-early gene 3 (IE 3) (P. A. Johnson, A. Miyanohara, F. Levine, T. C ahill, and T. Friedmann, J. Virol. 66:2952-2965, 1992). We have invest igated which viral genes might be responsible for CPE by comparing the ability of each of the individual genes expressed by an IE 3 deletion mutant during a nonproductive infection to inhibit biochemical transf ormation after cotransfection of BHK or CV-1 cells with a selectable m arker gene. Transfection of IE genes 1, 2, and 4 individually all caus ed a marked inhibition of colony formation, while transfection of IE 5 and the large subunit of ribonucleotide reductase had little effect. These results suggested that it would be necessary to mutate or reduce the expression of nearly all HSV-1 IE genes to reduce virus-induced C PE. Therefore, we have used VP16 mutants, which are unable to transind uce IE gene expression (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Came ron, and C. M. Preston, J. Virol. 63:2260-2269, 1989), to derive two r eplication-defective strains: 14H Delta 3, which is deleted for both c opies of IE 3, and in1850 Delta 42, which has a-deletion in the essent ial early gene UL42. The IE 3-VP16 double mutant, 14H Delta 3, is sign ificantly less toxic than a single IE 3 deletion mutant over a range o f multiplicities of infection, as measured in a cell-killing assay, an d has an enhanced ability to persist in infected cells in a biological ly retrievable form. In contrast, the UL42-VP16 double mutant, in 1850 Delta 42, showed reduced toxicity only at low multiplicities of infec tion. To test the role of the virion host shutoff function as an addit ional candidate to influence virus-induced CPE, we have introduced a l arge insertion mutation into the virion host shutoff gene of an IE 3 d eletion mutant and the double mutant 14H Delta 3. Mutation of this gen e did not reduce the cytotoxicity of either strain. These results demo nstrate that long-term survival of cells infected with replication-def ective HSV-I mutants can be enhanced through genetic manipulations tha t reduce viral gene expression.