Replication-competent herpes simplex virus (HSV-1) mutants are used in clinical trials in the experimental treatment of cancer. Mutants G207, HSV1716, NV1020, and Oncovex GM-CSF share in common a defect in one or both copies of the gene encoding the neurovirulence factor, ICP34.5, and are thus neuroattenuated. These viruses are acknowledged to differ from one another (a) in the specific types of mutations intentionally introduced during their derivation and (b) in the inherent genetic differences retained from the different parent strains used in their construction. Unintended mutations are expected to emerge at some low frequency during the selection for and passage of mutant viruses. Here we demonstrate that during the construction of the oncolytic virus R3616, a nonsense mutation arose in an untargeted region of the HSV-1 genome that resulted in a substantial truncation of the viral protein known as UL3. This report is the first published documentation that oncolytic herpesviruses developed and used in clinical trials contain adventitious mutations. The implications of these findings for the characterization and development of vectors proposed for use in clinical trials are discussed.

The herpes simplex virus 1 U L 3 and U L 4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U L 4 protein colocalizes with the products of the ␣22/U S 1.5 genes in small nuclear dense bodies. Here we report that the U L 3 protein also colocalized in these small nuclear dense bodies and the localization of U L 3 and U L 4 proteins in these bodies required the presence of ␣22/U S 1.5 genes. In cells infected with a mutant lacking intact ␣22/U S 1.5 genes, U L 3 was diffused throughout the nucleus even though the overall accumulation of the ␥2 U L 3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U L 3 accumulation and as the structural component and anchor of these small dense nuclear bodies.

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Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the ␥ 1 34.5 ؊ gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (