Oncolytic viruses used for gene therapy have been genetically modified to selectively target tumor cells while sparing normal host tissue. The multimutant virus G207 has been attenuated by inactivation of viral ribonucleotide reductase and by deletion of both viral gamma134.5 genes. Deletion of gamma134.5 greatly decreases the neurovirulence of this mutant virus but also reduces its antitumor efficacy. The mammalian homologue to the gamma134.5 gene product is the GADD34 protein. This protein can functionally substitute for the gamma134.5 gene and is also up-regulated during DNA damage. We postulated that combining use of the chemotherapy agent mitomycin C (MMC) with G207 will selectively up-regulate GADD34 in tumor that may complement the gamma134.5 gene deletion and augment viral antitumor efficacy. This hypothesis was tested in human gastric cells in vitro and in vivo. Using both the isobologram method and combination-index method of Chou-Talalay, significant synergism was demonstrated between MMC and G207. As a result of such synergism, a dose-reduction for each agent can be accomplished over a wide range of drug-effect levels without sacrificing tumor cell kill. Northern blot analysis confirmed that expression of GADD34 mRNA was increased by MMC treatment. SiRNA directed at GADD34 decreased MMC-associated enhancement of viral proliferation and resulted in decreased viral synergy with MMC. These data indicate that induction of GADD34 selectively restores the virulent phenotype of the deleted gene in G207 and thus provides a cellular basis for the combined use of DNA-damaging agents and gamma134.5 HSV mutants in the treatment of cancer.