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[摘要]：The ICP34.5 protein of herpes simplex virus (HSV) is involved in many aspects of viral pathogenesis; promoting neurovirulence, inhibiting interferon-induced shutoff of protein synthesis, interacting with PCNA and TBK1, inhibiting dendritic cell (DC) maturation, and binding to Beclin 1 to interfere with autophagy. Because of its key role in neuropathogenicity, the gamma 34.5 gene is deleted in all oncolytic HSVs (oHSVs) currently in clinical trial for treating malignant gliomas. Unfortunately, deletion of gamma 34.5 attenuates virus replication in cancer cells, especially human glioblastoma stem cells (GSCs). To develop new oHSVs for use in the brain and that replicate in GSCs, we explored the effect of deleting the gamma 34.5 Beclin 1 binding domain (BBD). To ensure cancer selectivity and safety, we inactivated the ICP6 gene (UL39, large subunit of ribonucleotide reductase), constructing ICP6 mutants with different gamma 34.5 genotypes: Delta 68HR-6, intact gamma 34.5; Delta 68H-6, gamma 34.5 BBD deleted; and 1716-6, gamma 34.5 deleted. Multimutated Delta 68H-6 exhibited minimal neuropathogenicity in HSV-1-susceptible mice, as opposed to Delta 68H and Delta 68HR-6. It replicated well in human glioma cell lines and GSCs, effectively killing cells in vitro and prolonging survival of mice bearing orthotopic brain tumors. In contrast, 1716 and 1716-6 barely replicated in GSCs. Infection of glioma cells with Delta 68H-6 and 1716-6 induced autophagy and increased phosphorylation of eIF2 alpha, while inhibition of autophagy, by Beclin 1 short hairpin RNA (shRNA) knockdown or pharmacological inhibition, had no effect on virus replication or phosphorylated eIF2 alpha (p-eIF2 alpha) levels. Thus, Delta 68H-6 represents a new oHSV vector that is safe and effective against a variety of brain tumor models. |
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