Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis.

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.

Deficiency in either 4E-BP1 or 4E-BP2 augments innate antiviral immune responses.

Genetic deletion of both 4E-BP1 and 4E-BP2 was found to protect cells against viral infections. Here we demonstrate that the individual loss of either 4E-BP1 or 4E-BP2 in mouse embryonic fibroblasts (MEFs) is sufficient to confer viral resistance. shRNA-mediated silencing of 4E-BP1 or 4E-BP2 renders MEFs resistant to viruses, and compared to wild type cells, MEFs knockout for either 4E-BP1 or 4E-BP2 exhibit enhanced translation of Irf-7 and consequently increased innate immune response to viruses. Accordingly, the replication of vesicular stomatitis virus, encephalomyocarditis virus, influenza virus and Sindbis virus is markedly suppressed in these cells. Importantly, expression of either 4E-BP1 or 4E-BP2 in double knockout or respective single knockout cells diminishes their resistance to viral infection. Our data show that loss of 4E-BP1 or 4E-BP2 potentiates innate antiviral immunity. These results provide further evidence for translational control of innate immunity and support targeting translational effectors as an antiviral strategy.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E.

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 eukaryotic 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 interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Vesicular stomatitis virus oncolysis is potentiated by impairing mTORC1-dependent type I IFN production.

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(DeltaM51)), dramatically increased the survival of immunocompetent rats bearing MGs. More importantly, VSV(DeltaM51) 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(DeltaM51).

Altered interactions between stem-loop IV within the 5' noncoding region of coxsackievirus RNA and poly(rC) binding protein 2: effects on IRES-mediated translation and viral infectivity.

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 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.

The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

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.