Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of IFN-β. Thus, phosphorylation of eIF4E has a key role in antiviral host defense by selectively stimulating the translation of mRNA that encodes a critical suppressor of the innate antiviral response.

Oncolytic viruses constitute a promising therapy against malignant gliomas (MGs). However, virus-induced type I IFN greatly limits its clinical application. The kinase mammalian target of rapamycin (mTOR) stimulates type I IFN production via phosphorylation of its effector proteins, 4E-BPs and S6Ks. Here we show that mouse embryonic fibroblasts and mice lacking S6K1 and S6K2 are more susceptible to vesicular stomatitis virus (VSV) infection than their WT counterparts as a result of an impaired type I IFN response. We used this knowledge to employ a pharmacoviral approach to treat MGs. The highly specific inhibitor of mTOR rapamycin, in combination with an IFN-sensitive VSV-mutant strain (VSV ΔM51 ), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSV ΔM51 selectively killed tumor, but not normal cells, in MG-bearing rats treated with rapamycin. These results demonstrate that reducing type I IFNs through inhibition of mTORC1 is an effective strategy to augment the therapeutic activity of VSV ΔM51 .innate antiviral immunity | malignant gliomas | mTORC1 | oncolytic viruses M alignant gliomas (MGs) are by far the most frequent, aggressive, and lethal primary brain tumor variants (1, 2). Patients with MGs have a median survival time of approximately 1 y and respond poorly to most available therapeutic modalities (3-5). Thus, more effective treatments are needed.Recent evidence implicates the PI3K/mammalian target of rapamycin (mTOR) signaling pathway as one of the main oncogenic signaling pathways whose deregulation may underlie gliomagenesis (6, 7). mTOR exists in two complexes: mTOR complex 1 (mTORC1), which is sensitive to the drug rapamycin and regulates mRNA translation, and mTORC2, which is rapamycininsensitive and regulates the organization of the actin cytoskeleton (reviewed in refs. 8-10). mTORC1 stimulates type I IFN production via phosphorylation of its target proteins 4E-BPs and S6K1/2 (11). Evidence for the critical role of the mTORC1 signaling pathway in innate immunity emerged from the findings that the mTORC1 inhibitor rapamycin suppresses type I IFN in plasmacytoid dendritic cells (pDCs), which are the major producers of systemic type I IFN (12). In addition, genetic deletion of the mTOR downstream target S6K1/2 leads to impaired type I IFN response (see Results). In contrast, we recently found that the lack of the translational repressors 4E-BP1/2 leads to enhanced type I IFN production (13).Oncogenic transformation is associated with a deficient type I IFN response, which constitutes the first line of defense against virus infection (14,15). Oncolytic viruses are studied as effective anticancer agents because they exploit this selective defect (16)(17)(18)(19). One of the best characterized oncolytic viruses, whose replication is extremely sensitive to the inhibition by IFN, is vesicular stomatitis virus (VSV) (20). However, there are several reasons that limit the use of oncolytic viruses for the treatment of MGs. First, some MGs exhibit a robust type I I...

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis, pancreatitis, and encephalitis. Much of what is known about the coxsackievirus intracellular replication cycle is based on the information already known from a well-studied and closely related virus, poliovirus. Like that of poliovirus, the 5′ noncoding region (5′ NCR) of CVB3 genomic RNA contains secondary structures that function in both viral RNA replication and cap-independent translation initiation. For poliovirus IRES-mediated translation, the interaction of the cellular protein PCBP2 with a major secondary structure element (stem-loop IV) is required for poliovirus gene expression. Previously, the complete secondary structure of the coxsackievirus 5′ NCR was determined by chemical structure probing and overall, many of the RNA secondary structures bear significant similarity to those of poliovirus; however, the functions of the coxsackievirus IRES stem-loop structures have not been determined. Here we report that a CVB3 RNA secondary structure, stem-loop IV, folds similarly to poliovirus stem-loop IV and like its enterovirus counterpart, coxsackievirus stem-loop IV interacts with PCBP2. We used RNase foot-printing to identify RNA sequences protected following PCBP2 binding to coxsackievirus stem-loop IV. When nucleotide substitutions were separately engineered at two sites in coxsackievirus stem-loop IV to reduce PCBP2 binding, inhibition of IRES-mediated translation was observed. Both of these nucleotide substitutions were engineered into full-length CVB3 RNA and upon transfection into HeLa cells, the specific infectivities of both constructs were reduced and the recovered viruses displayed small plaque phenotypes and slower growth kinetics compared to wild type virus.

No abstract

Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5′ noncoding region (5′ NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5′ NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.