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

AbstractCoxsackievirus 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

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.</div

Deficiency in Either 4E-BP1 or 4E-BP2 Augments Innate Antiviral Immune Responses

<div><p>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 <i>Irf-7</i> 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.</p></div

Lack of 4E-BP1 or 4E-BP2 renders MEFs refractory to VSV infection.

<p>(A) Western blotting analysis of 4E-BP1 or 4E-BP2 expression in WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs. β-actin served as a loading control. (B) WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs were mock-infected or infected at a MOI of 1 PFU per cell with VSV-GFP. Twenty-four hpi, viral infection was assessed by GFP fluorescence and CPE. (C) Western blotting analysis for the detection of VSV proteins at the defined time points post-infection of WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs with VSV-GFP at a MOI of 1 PFU/cell. β-actin was used as a loading control. (D) Viral titer quantified by plaque assay at 24 hpi in WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs with VSV-GFP at a MOI of 1 PFU/cell. (E) Photomicrograph of CPE resulting from infections of WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs with FLU, Sindbis and EMCV virus at 1MOI 12 hpi. (F) Cell viability in experiment in (E) was assessed by MTT assay.</p

Silencing 4E-BP1 or 4E-BP2 inhibits VSV replication in MEFs.

<p>(A) Western blotting analysis of 4E-BP1 or 4E-BP2 expression following transduction of WT MEFs with lentiviruses containing a non-specific shRNA (scrambled), or a shRNA targeting <i>4E-BP1</i> or <i>4E-BP2</i> mRNA. β-actin served as a loading control. (B) Scrambled MEFs and MEFs knockdown on 4E-BP1 or 4E-BP2 were infected with VSV-GFP at a MOI of 1 PFU/cell and virus replication was assessed by GFP fluorescence and cytopathic effect (CPE). (C) Western blotting analysis for the detection of VSV proteins at the defined time points post-infection with VSV-GFP at a MOI of 1 PFU/cell. β-actin was used as a loading control. (D) Viral titer quantified by plaque assay at 24 hpi with VSV-GFP at a MOI of 1 PFU/cell.</p

Exogenous expression of 4E-BP1 or 4E-BP2 in respective 4E-BP1^−/− or 4E-BP2^−/− MEFs increases susceptibility to VSV infection.

<p>(A) 4E-BP1^−/− and 4E-BP2^−/− MEFs were transduced with retroviruses carrying an empty vector (control), or retroviruses expressing either 4E-BP1 or 4E-BP2. Western blotting analysis for exogenous expression of 4E-BP1 and 4E-BP2 in their respective single knockout MEFs. (B) Control and transduced MEFs were infected with VSV-GFP at a MOI of 1 PFU/cell and viral infection was assessed by GFP fluorescence and by (C) Western blotting analysis for VSV viral protein expression.</p

Exogenous expression of 4E-BP1 or 4E-BP2 in MEFs knockout for both translation repressors augments their susceptibility to VSV infection.

<p>(A) 4E-BP1/2 DKO MEFs were transduced with retroviruses expressing either 4E-BP1, 4E-BP2 or an empty vector as control. Western blotting analysis for the expression levels of exogenous 4E-BP1 or 4E-BP2 in DKO MEFs. (B) Control and 4E-BP1- or 4E-BP2-expressing DKO MEFs were infected with VSV-GFP at a MOI of 1 PFU/cell for 24 hours and infection was monitored by GFP fluorescence and CPE. (C) Western blotting analysis for the detection of VSV proteins at the defined time points post-infection with VSV-GFP at a MOI of 1 PFU/cell. β-actin was used as a loading control.</p

Deficiency of 4E-BP1 or 4E-BP2 enhances type-I interferon production.

<p>(A) Protection assay: diagram of experimental protocol: after stimulation with poly(I:C) (6 hours) the medium of WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs were collected and transferred to three different plates containing WT MEFs. (B) WT MEFs from (A) were then infected with VSV-GFP. The protective effects of the different media were assessed 24 hpi by fluorescence microscopy, cytopathic effect and (C) virus titration. (D) WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs were infected with VSV at a MOI of 1 PFU/cell for 6 hours and the induction of a type-I IFN response (<i>Irf-7</i>, <i>Ifn-α</i> and <i>Ifn</i>-β mRNA levels) was determined by RT-PCR (*longer exposure). (E) Luciferase assays showing the ratio of expression of the different luciferase constructs harbouring either 5′UTR-IRF-7 (translational level), IFN-α promoter or IFN-β promoter (transcriptional level), normalized to the transfection control (<i>Renilla</i> luciferase). Fluc activity/Rluc activity in WT MEFs was set as 1.</p