JNK-deficiency enhanced oncolytic vaccinia virus replication and blocked activation of double-stranded RNA-dependent protein kinase

Abstract

Vaccinia virus has recently been used as an expression vector for gene delivery and an oncolytic agent for cancer therapy. Although it has been established that interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) and RNase L interfere with viral replication, little else is known about the other host factors that might affect viral replication and virus-mediated host cell killing. In this study, we evaluated the roles of c-Jun NH2-terminal kinase (JNK) in oncolytic vaccinia virus replication and vaccinia virus-mediated host cell killing. We found that JNK knockout mouse embryonic fibroblasts (MEFs) were more susceptible to oncolytic vaccinia virus infection than wild-type MEFs. Moreover, viral replication and the production of infectious viral progeny were up to 100-fold greater in JNK-deficient MEFs than in wild-type MEFs. A similar result was observed for wild-type vaccinia virus. The increased killing of infected cells and the production of viral progeny was also observed in wild-type MEFs that had been treated with JNK inhibitors and in human colon cancer cells that had been transfected with dominant-negative JNK constructs. Moreover, testing on several human lung cancer cell lines and HeLa cells showed an inverse correlation between levels of JNK expression and susceptibility to oncolytic vaccinia virus. Our study also revealed that oncolytic virus infection-mediated PKR activation was blocked or diminished in JNK-deficient MEFs. The adenovirus-mediated ectopic expression of human PKR in JNKdeficient MEFs reduced vaccinia virus replication to the levels observed in wild-type MEFs, indicating that JNK is required for vaccinia virus to efficiently activate PKR. Our results demonstrated that the cellular status of JNK function can dramatically affect oncolytic vaccinia virus replication and vaccinia virus-mediated host cell killing. This finding may enable further improvements in oncolytic virotherapy using vaccinia virus.

A Vaccinia Virus-Driven Interplay between the MKK4/7-JNK1/2 Pathway and Cytoskeleton Reorganization

“…These observations, however, are in apparent contrast to those of Hu and colleagues (12). These authors have shown that by separately infecting either JNK1 or JNK2-KO cells with VACV WR to quantitate viral titers, a 40-fold increase was observed with JNK1-KO cells, which was paralleled by the infection of JNK2-KO cells, though to a lesser extent than for the WT cells.…”

“…Indeed, we were not able to find any evidence of apoptotic cell death in VACV-infected JNK1/2-KO cells on the basis of activation of the executioner caspase-3 or its substrate, poly(ADPribose) polymerase (data not shown). On the contrary, the data presented by the authors mentioned above (12) did not exclude the possibility that infection of either JNK1-KO cells (which still express JNK2) or JNK2-KO cells (which still express JNK1) is enough to stimulate the apoptotic pathway. It is conceivable that the presence of one of the isoforms in the single-KO cells could, in turn, account for the enhanced cell death associated with the enhanced virus replication.…”

“…Although activation of JNK upon VACV infection has been the focus of recent studies (12,45), the role that JNK1/2 may have in VACV-stimulated cytoskeleton reorganization has not been elucidated. In the present study, we demonstrated that the MKK4/7-JNK1/2 pathway is stimulated in an early-late manner during VACV infection and this event plays a role in both early cell contractility and cell migration.…”

Prognostic Significance of RNA-Dependent Protein Kinase on Non–Small Cell Lung Cancer Patients

“…In recent years, we have studied PKR pathways and have found them to be clearly necessary for induction of cell death in some cancer cells including lung cancer after certain treatments (20–25). In the present study, we tested the hypothesis that differences in the levels of expression of PKR proteins could act as a prognostic marker for clinical outcomes in NSCLC patients.…”

Engineering Oncolytic Vaccinia Viruses for Non-Invasive Optical Imaging of Tumors

“…The importance of these signal-ing pathways for virus survival are supported by unique strategies acquired by poxviruses to prevent activation of the host anti-viral response targeting TLRs [77,78], expression of type I and II IFN viroceptors, TNF-, IL-1 or CC chemokines [79][80][81][82]. The cellular status of c-Jun NH2-terminal kinase (JNK) function also dramatically affected oncolytic VV replication and vaccinia virus-mediated host cell killing [83]. Other oncolytic viruses such as VSV showed preferential replication in cells with an activated Ras-ERK pathway and defective IFN pathway [84].…”