PDlim2 Selectively Interacts with the PDZ Binding Motif of Highly Pathogenic Avian H5N1 Influenza A Virus NS1

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins

MERS-CoV 4b protein interferes with the NF-κB-dependent innate immune response during infection

This work is licensed under a Creative Commons Attribution 4.0 International License.Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that emerged in 2012, causing severe pneumonia and acute respiratory distress syndrome (ARDS), with a case fatality rate of ~36%. When expressed in isolation, CoV accessory proteins have been shown to interfere with innate antiviral signaling pathways. However, there is limited information on the specific contribution of MERS-CoV accessory protein 4b to the repression of the innate antiviral response in the context of infection. We found that MERS-CoV 4b was required to prevent a robust NF-κB dependent response during infection. In wild-type virus infected cells, 4b localized to the nucleus, while NF-κB was retained in the cytoplasm. In contrast, in the absence of 4b or in the presence of cytoplasmic 4b mutants lacking a nuclear localization signal (NLS), NF-κB was translocated to the nucleus leading to the expression of pro-inflammatory cytokines. This indicates that NF-κB repression required the nuclear import of 4b mediated by a specific NLS. Interestingly, we also found that both in isolation and during infection, 4b interacted with α-karyopherin proteins in an NLS-dependent manner. In particular, 4b had a strong preference for binding karyopherin-α4 (KPNA4), which is known to translocate the NF-κB protein complex into the nucleus. Binding of 4b to KPNA4 during infection inhibited its interaction with NF-κB-p65 subunit. Thereby we propose a model where 4b outcompetes NF-κB for KPNA4 binding and translocation into the nucleus as a mechanism of interference with the NF-κB-mediated innate immune response

Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model

Analiza naprężeń jednozakładkowych połączeń klejowych z plastycznym odkształceniem łączonych materiałów

In this work the results of numerical stress analysis of single adhesive lap joints were presented. In the analysis both the linear-elastic and the elastic-plastic models of adherends materials were considered. Plastic deformation of adherends has a significant influence on the stress state in the adhesive layer. In the first part of the work the mechanical properties of adherends material obtained in experimental investigations were presented. In next part of the study the numerical model of joint was presented. The results of static analysis using the finite element method showed that in the case of joining materials with low value of yield stress the plastic deformation occurs in adherend at load much smaller than destructive force of the joint. In this kind of joints the plastic deformation of adherend has an influence on rapid stress increase in adhesive layer, in final stage of loading. This phenomenon causes a decrease of load capacity of single adhesive lap joints of elasticplastic materials.W pracy przedstawiono wyniki numerycznej analizy naprężeń jednozakładkowych połączeń klejowych. Dla łączonych blach zastosowano dwa modele materiału: liniowo-sprężysty oraz sprężysto-plastyczny. Odkształcenie plastyczne materiału klejonego wywiera znaczący wpływ na stan naprężeń w warstwie kleju. W pierwszej części pracy przedstawiono właściwości mechaniczne materiału łączonego, które otrzymano w badaniach eksperymentalnych. W następnej części pracy przedstawiono model numeryczny połączenia. Wyniki analizy statycznej metodą elementów skończonych wykazały, że w przypadku łączenia materiałów charakteryzujących się niską wartością granicy plastyczności odkształcenie plastyczne występuje w materiale łączonym przy obciążeniu znacznie mniejszym od siły niszczącej połączenie. Zjawisko to powoduje obniżenie nośności jednozakładkowych połączeń klejowych materiałów sprężysto-plastycznych

The effect of technological parameters on intensity of shot peening process of 51CrV4 steel

In the paper the effect of selected technological parameters of shot peening on process intensity of 51CrV4 steel was presented. The experiments were conducted according to statistical 3-level completed plan PS/DC 32. Technological parameters were changed in the range: shot peening time t = 1-3 min and pressure p = 0.2-0.4 MPa. In the article the analysis of experiment reproducibility, impact parameters significance and adequacy of equation were done. As the result of investigations the adequate equation was obtained describing the effect of technological parameters. Significant influence on process intensity was found in case of pressure and interaction of both analyzed technological parameters. The biggest energy of stream shots was gained at the maximum pressure of 0.4 MPa and the shot peening time of 3 minutes. As the result of analysis according to design of experiment (DOE) the adequate equation describing the dependencies between technological parameters and process intensity was found