Oncolytic Reovirus Induces Intracellular Redistribution of Ras to Promote Apoptosis and Progeny Virus Release

Abstract

Ras is a dynamic protein capable of interacting with plasma membrane microdomains and intracellular membranes. Its activity, as controlled by post-translational modifications, is essential in regulating cell growth, differentiation, and death. Consequently, mutations that result in constitutive Ras activity lead to cell transformation, and are associated with over 30% of all human cancers. Reovirus is a naturally oncolytic virus that preferentially replicates in Ras-transformed cells and is currently undergoing clinical trials as a cancer therapeutic. It was previously demonstrated that Ras transformation promotes uncoating of the parental virion during entry, production of infectious viral progeny, and virus release through apoptosis; however, the mechanism behind the latter is not well understood. This study set out to determine whether reovirus alters the intracellular location of oncogenic Ras to induce apoptosis of H-RasV12-transformed fibroblasts. Here, I show that reovirus decreases palmitoylation levels of H-RasV12 and causes accumulation of the oncogenic protein in the Golgi body through Golgi fragmentation. With the Golgi body being the site of Ras palmitoylation, treatment of target cells with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts a greater accumulation of H-RasV12 in the Golgi body, as well as a dose-dependent increase in reovirus release and spread. Use of 2BP on cell lines expressing H, N, or K-Ras oncogenic isoforms causes an increase in reovirus release that correlates with the palmitoylation status of the respective Ras protein. Alternatively, tethering H-RasV12 to the plasma membrane, and preventing its movement back to the Golgi, allows for efficient virus production, but results in basal levels of reovirus-induced cell death. Furthermore, treatment of H-RasV12 cells with the FKBP12 inhibitor, FK506, which is known to retain Ras at the plasma membrane, reduces virus-related death and plaque size. Analysis of Ras downstream signalling reveals that cells expressing cycling H-RasV12 have elevated levels of phosphorylated JNK, and that Ras retained at the Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK signalling pathway to promote cell death. Collectively, the data suggests that reovirus induces Golgi fragmentation of target cells, and the subsequent accumulation of oncogenic Ras in the Golgi body initiates apoptotic signalling events required for virus release.