Interferon Escape of Respiratory Syncytial Virus: Functional Analysis of Nonstructural Proteins NS1 and NS2

Respiratory syncytial virus (RSV) is recognised as the most frequent cause of severe lung infections in infants and cattle worldwide. Currently, no effective treatments are available and the development of a successful vaccine has been hampered by the fact that natural infection does not provide complete and durable protection. RSV nonstructural proteins, NS1 and NS2, are strong inhibitors of IFN α/β-production by specifically preventing interferon regulatory factor (IRF)-3 phosphorylation. However, the exact mechanisms leading to NS protein-mediated inhibition of IRF3 remain to be unravelled. One of the objectives of this study was to identify amino acid domains in the human respiratory syncytial virus (HRSV) nonstructural proteins (NS) responsible for their ability to ablate the IFN-β signalling pathway. Furthermore, I wanted to find out at which level of this signalling pathway the NS proteins exert their suppressive activity and which are their major cellular targets. HRSV strains A2 and Long differ in their ability to block interferon type I synthesis. Sequence analysis of their NS proteins revealed the presence of an amino acid residue in the NS2 protein with a potential role for RSV IFN-inhibitory functions. Two recombinant bovine respiratory syncytial (BRSV) viruses harbouring HRSV NS1 and NS2 genes were generated and tested in their ability to restrict IFN-β synthesis. These recombinant viruses differed only in the identity of the residue at position 26 of the HRSV NS2 protein: rBRSVh1/2 has a Threonine as in the Long strain, while in rBRS h1/2*T26I this amino acid was mutated into an Isoleucin similarly to A2 virus. Sets of in vitro tests revealed that IFN-β induction was impaired by rBRSVh1/2*T26I when compared to rBRSV h1/2. Analysis of the transcriptional factors (AP-1, NF-kB and IRF3) involved in the activation of IFN-β synthesis provided evidence that the inhibitory ability of rBRSVh1/2*T26I was correlated to a selective block of IRF3. The mutation (T26I) in the NS2 protein did neither effect the NF-kB activation pathway nor perturbed the IFN-resistance characteristics of the chimeric viruses. IRF3 is activated upon phosphorylation mediated by IKK-related kinases (TBK1 and IKK-ε). TBK1 was therefore cloned from a human lung cDNA library and its biological activities regarding the induction of IFN-β were compared in mock-infected and infected cells. rBRSVh1/2*T26I and HRSV A2 precluded virus-induced IRF3 activation by interfering with TBK1 functions. No direct interaction between TBK1 and NS2 protein was demonstrated indicating that the kinase TBK1 may not be the sole target involved in RSV mechanisms of evasion of the innate immune response. Recombinant IFN-inducible rabies viruses expressing HRSV Long-derived (rGFP-Ph2/1) or HRSV A2-like (rGFP-Ph2*T26I/1) NS proteins were also generated. The HRSV NS2 protein expressing an Isoleucin (NS2*T26I) at position 26 was not able to suppress IFN-β induction and to rescue the growth of the recombinant eGFP-Ph2*T26I/1 in interferon-competent cells. A low expression of the mutated NS2*T26I protein was probably the reason of this failure. In summary, these results show that the HRSV NS2 protein possesses an intrinsic IFN-β inhibitory activity, which is achieved throughout a selective inhibition of the IRF3 activation pathway. The block appears to be exerted at the level of IRF3-kinase TBK1. Interferonantagonist functions of the HRSV NS2 protein are linked to a particular amino acid motif in the N-terminus of the protein. Identification of this amino acid domain and of TBK1 as the cellular target provide a better insight of how the HRSV NS2 protein prevents the establishment of the antiviral innate immune response and therefore it might contribute to the development of an effective vaccine

Cell cycle progression or translation control is not essential for vesicular stomatitis virus oncolysis of hepatocellular carcinoma.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). Identifying key regulators in diverse transduction pathways that define VSV oncolysis in cancer cells represents a fundamental prerequisite to engineering more effective oncolytic viral vectors and adjusting combination therapies. After having identified defects in the signalling cascade of type I interferon induction, responsible for attenuated antiviral responses in human HCC cell lines, we have now investigated the role of cell proliferation and translation initiation. Cell cycle progression and translation initiation factors eIF4E and eIF2Bepsilon have been recently identified as key regulators of VSV permissiveness in T-lymphocytes and immortalized mouse embryonic fibroblasts, respectively. Here, we show that in HCC, decrease of cell proliferation by cell cycle inhibitors or siRNA-mediated reduction of G(1) cyclin-dependent kinase activities (CDK4) or cyclin D1 protein expression, do not significantly alter viral growth. Additionally, we demonstrate that translation initiation factors eIF4E and eIF2Bepsilon are negligible in sustaining VSV replication in HCC. Taken together, these results indicate that cellular proliferation and the initiation phase of cellular protein synthesis are not essential for successful VSV oncolysis of HCC. Moreover, our observations indicate the importance of cell-type specificity for VSV oncolysis, an important aspect to be considered in virotherapy applications in the future

Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogenactivated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/β). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein

Nonstructural Proteins NS1 and NS2 of Bovine Respiratory Syncytial Virus Block Activation of Interferon Regulatory Factor 3

We have previously shown that the nonstructural (NS) proteins NS1 and NS2 of bovine respiratory syncytial virus (BRSV) mediate resistance to the alpha/beta interferon (IFN)-mediated antiviral response. Here, we show that they, in addition, are able to prevent the induction of beta IFN (IFN-β) after virus infection or double-stranded RNA stimulation. In BRSV-infected MDBK cells upregulation of IFN-stimulated genes (ISGs) such as MxA did not occur, although IFN signaling via JAK/STAT was found intact. In contrast, infection with recombinant BRSVs lacking either or both NS genes resulted in efficient upregulation of ISGs. Biological IFN activity and IFN-β were detected only in supernatants of cells infected with the NS deletion mutants but not with wild-type (wt) BRSV. Subsequent analyses of IFN-β promoter activity showed that infection of cells with the double deletion mutant BRSV ΔNS1/2, but not with BRSV wt, resulted in a significant increase in IFN-β gene promoter activity. Induction of the IFN-β promoter depends on the activation of three distinct transcription factors, NF-κB, ATF-2/c-Jun, and IFN regulatory factor 3 (IRF-3). Whereas NF-κB and ATF-2/c-Jun activities were readily detectable and comparable in both wt BRSV- and BRSV ΔNS1/2-infected cells, phosphorylation and transcriptional activity of IRF-3, however, were observed only after BRSV ΔNS1/2 infection. NS protein-mediated inhibition of IRF-3 activation and IFN induction should have considerable impact on the pathogenesis and immunogenicity of BRSV

Low-Cost Devices for Three-Dimensional Cell Aggregation, Real-Time Monitoring Microscopy, Microfluidic Immunostaining, and Deconvolution Analysis

The wide use of 3D-organotypic cell models is imperative for advancing our understanding of basic cell biological mechanisms. For this purpose, easy-to-use enabling technology is required, which should optimally link standardized assessment methods to those used for the formation, cultivation, and evaluation of cell aggregates or primordial tissue. We thus conceived, manufactured, and tested devices which provide the means for cell aggregation and online monitoring within a hanging drop. We then established a workflow for spheroid manipulation and immune phenotyping. This described workflow conserves media and reagent, facilitates the uninterrupted tracking of spheroid formation under various conditions, and enables 3D-marker analysis by means of 3D epifluorescence deconvolution microscopy. We provide a full description of the low-cost manufacturing process for the fluidic devices and microscopic assessment tools, and the detailed blueprints and building instructions are disclosed. Conclusively, the presented compilation of methods and techniques promotes a quick and barrier-free entry into 3D cell biology

Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus

Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection

Effects of concomitant inhibition of mTOR and MNK on VSV proliferation.

<p>Cells were mock-treated (DMSO) or treated with rapamycin at 50 nm, MNK1 inhibitor at 20 µM, alone or together as indicated. <b>A)</b> Cell proliferation assays were performed using the MTT assay. Representative results of at least two independent experiments are shown. <b>B)</b> Western blot analysis of lysates obtained by PH5CH8 and HepG2 cell lines mock-treated (DMSO) or treated with rapamycin (RAPA), MNK inhibitor (MNK in) alone or in combination (RAPA+MNK in). The levels of S6K and eIF4E phosphorylated forms were monitored after inhibitor treatment. <b>C)</b> Cells were infected with VSV-wt at an MOI of 0.1 for 24 hours. Viral titers represent the mean ± standard deviation of three experiments.</p

IFN system analysis in PH5CH8 cell line.

<p><b>A)</b> Fold induction of IFN-β promoter-Luciferase reporter gene in HCC cell lines (HepG2 and Huh-7), immortalized hepatocytes (PH5CH8) and primary human hepatocytes (PHH). Cells were transfected with the reporter plasmid containing the firefly luciferase gene under the control of the IFN-β promoter. At 24 hours post-transfection, cultures were stimulated by a second round of transfection with Poly (I:C) (T-pIC), Poly (I:C) was added to the medium (M-pIC), or infected with VSV-wt or VSV-M51R. IFN-luciferase activities were measured and normalized to Renilla luciferase (RL) gene used as an internal control. Significance was calculated by comparison with mock-treated cultures expressing basal firefly luciferase activity (* p<0.05; ** p<0.01; ***p<0.001). <b>B)</b> Interferon protection assay in PH5CH8 compared to PHH and HepG2 and Huh-7 cells as representatives for HCC. Cells were treated overnight with 500 IU/ml of universal type I interferon (IFN) or simply mock-treated. VSV-wt infection was performed at MOI of 1 and viral titers were obtained 24 hr post-infection. Titers are the mean of at least three independent experiments (* p<0.05).</p