Interferon induction by avian reovirus

We have previously shown that the replication of avian reovirus (ARV) in chicken embryo fibroblasts (CEF) is more resistant to the antiviral action of interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study we examined the capacity of these three viruses to induce the expression of IFN when infecting avian cells. Efficient expression of both type-α and type-β IFNs, as well as of the double-stranded RNA (dsRNA)-activated protein kinase (PKR), takes place in ARV-infected CEF, but not in cells infected with VSV or VV. PKR expression is not directly induced by ARV infection, but by the IFN secreted by ARV-infected cells. IFN induction in ARV-infected cells requires viral uncoating, but not viral gene expression, a situation similar to that reported for apoptosis induction by ARV-infected cells. However, our results demonstrate that IFN induction by ARV-infected CEF occurs by a caspase-independent mechanismThis work was funded by grants from the Spanish Ministerio de Economia y Competitividad (BFU2010-22228 and BFU2013-43513-R) and from the Xunta de Galicia (CN 2012/018). I.L.S. was a recipient of a predoctoral FPU fellowship (Ministerio de Educación y Ciencia) and a Research Fellowship (Bolsa de Investigación) (Deputación Provincial da Coruña)S

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cellsThe Spanish Ministerio de Economía y Competitividad provided funding to Javier Benavente and José Martínez-Costas under grant numbers BFU2010-22228 and BFU2013-43513-R. Support was also provided by the Xunta de Galicia (CN 2012/018). Irene Lostalé-Seijo was a recipient of a predoctoral FPU fellowship (Ministerio de Educación y Ciencia) and a Research Fellowship (Bolsa de Investigación; Deputación Provincial da Coruña)S

Avian reovirus-triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muNS protein

Avian reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of avian reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particlesThis work was funded by grants from the Ministerio de Economia Competitividad (BFU2010-22228) and from the Xunta de Galicia (CN 2012/018)S

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cellsThis research was supported by grants from the Spanish Ministerio de Ciencia y Tecnología (BFU2007-61330/BMC) and from the Xunta de Galicia (08CSA009203PR). L. V-I. and I. L-S. were recipients of predoctoral fellowships from the FPI and FPU programs of the Spanish Ministerio de Ciencia y TecnologíaS

Straightforward access to bisbenzamidine DNA binders and their use as versatile adaptors for DNA-promoted processes

Bisbenzamidines are an important family of minor groove DNA-binding agents. We present a one-step synthesis of aromatic aza-bisbenzamidines that allows straightforward and versatile access to a large number of these molecules. One of them, the azide-aza-bisbenzamidine 13, can be readily modified via click-chemistry with a variety of functionalities that can, therefore, be delivered to the vicinity of an A/T-rich DNA minor groove. This strategy, therefore, provides a simple means for triggering site selective, DNA-promoted biochemical and physicochemical processesS

In Vivo Light-Driven DNA Binding and Cellular Uptake of Nucleic Acid Stains

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Chemical Biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see: https://pubs.acs.org/doi/10.1021/cb300100rChemical derivatization of nucleic stains such as ethidium bromide or DAPI with tailored, photoresponsive caging groups, allows for “on demand” spatiotemporal control of their in vivo nucleic acid binding, as well as for improving their cellular uptake. This effect was particularly noteworthy for a nitro-veratryloxycarbonyl-caged derivative of ethidium bromide that, in contrast with the parent stain, is effectively internalized into living cells. The activation strategy works in light-accessible, therapeutically relevant settings, such as human retinas, and can even be applied for the release of active compounds in the eyes of living miceWe are thankful for support given by the Spanish grants SAF2007-61015, SAF2010-20822-C02, CTQ2009-14431/ BQU, Consolider Ingenio 2010 CSD2007-00006, and the Xunta de Galicia INCITE09 209 084PR, GRC2010/12, PGIDIT08CSA-047209PR. M.I.S. thanks the Spanish Ministry of Education for FPU Ph.D. fellowshipsS

Microspheres-prime/rMVA-boost vaccination enhances humoral and cellular immune response in IFNAR(−/−) mice conferring protection against serotypes 1 and 4 of bluetongue virus

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(−/−) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challenges with the homologous serotype 4 and the heterologous serotype 1 of BTV. All these results support the strategy based on microspheres in combination with rMVAs as a promising multiserotype vaccine candidate against BTVThis work was supported by grants from the Spanish Ministerio de Economía y Competitividad (AGL2011-23506, AGL-2014-57430-R and BFU2013-43513-R). Financial support from the Consellería de Cultura, Educación e Ordenación Universitaria (Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09) and the European Regional Development Fund (ERDF), is also gratefully acknowledgedS

Urinary tract physiological conditions promote ciprofloxacin resistance in low-level-quinolone-resistant Escherichia coli

Escherichia coli isolates carrying chromosomally encoded low-level-quinolone-resistant (LLQR) determinants are frequently found in urinary tract infections (UTIs). LLQR mutations are considered the first step in the evolutionary pathway producing high-level fluoroquinolone resistance. Therefore, their evolution and dissemination might influence the outcome of fluoroquinolone treatments of UTI. Previous studies support the notion that low urine pH decreases susceptibility to ciprofloxacin (CIP) in E. coli. However, the effect of the urinary tract physiological parameters on the activity of ciprofloxacin against LLQR E. coli strains has received little attention. We have studied the activity of ciprofloxacin under physiological urinary tract conditions against a set of well-characterized isogenic E. coli derivatives carrying the most prevalent chromosomal mutations (ΔmarR, gyrA-S83L, gyrA-D87N, and parC-S80R and some combinations). The results presented here demonstrate that all the LLQR strains studied became resistant to ciprofloxacin (according to CLSI guidelines) under physiological conditions whereas the control strain lacking LLQR mutations did not. Moreover, the survival of some LLQR E. coli variants increased up to 100-fold after challenge with a high concentration of ciprofloxacin under UTI conditions compared to the results seen with Mueller-Hinton broth. These selective conditions could explain the high prevalence of LLQR mutations in E. coli. Furthermore, our data strongly suggest that recommended methods for MIC determination produce poor estimations of CIP activity against LLQR E. coli in UTIs.Fondo de Investigación Sanitaria PI13/0006

A dendrimer–hydrophobic interaction synergy improves the stability of polyion complex micelles

This work was financially supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) (CTQ2015‐ 69021‐R), the Consellería de Cultura, Educación e Ordenación Universitaria (GRC2014/040 and Centro Singular de Investigación de Galicia Accreditation 2016‐2019, ED431G/09), and the European Regional Development Fund (ERDF). M.F.‐V. thanks the Spanish Government for a FPU FellowshipPolyion complex (PIC) micelles incorporating PEG-dendritic copolymers display an unprecedented stability towards ionic strength that is amplified via hydrophobic interactions. The tridimensional orientation of peripheral hydrophobic linkers between charged groups and the globular/rigid dendritic scaffold maximizes this stabilization compared to PIC micelles from linear polymers. As a result, micelles stable at concentrations higher than 3 M NaCl are obtained, which represents the highest saline concentration attained with PIC micelles. Advantages of this stabilizing dendritic effect have been taken for the design of a robust, pH-sensitive micelle for the controlled intracellular release of the anticancer drug doxorubicin. This micelle displays a slightly higher toxicity, and distinctive mechanisms of cell uptake and intracellular trafficking relative to the free drug. The preparation of mixed PIC micelles by combining differently functionalized PEG-dendritic block copolymers has allowed the fine-tuning of their stability, paving the way towards the facile modulation of properties like biodegradability, drug loading, or the response to external stimuliS

Membrane-disrupting iridium(III) oligocationic organometallopeptides

NOTICE: This is the peer reviewed version of the following article: Salvadó, I, Gamba, I, Montenegro J, Martínez-Costas J, Brea JM, Loza MI, Vázquez López M, Vázquez M.E. Membrane-disrupting iridium(III) oligocationic organometallopeptides. Chem. Commun., 2016,52, 11008-11011. DOI: 10.1039/C6CC05537K. This article may be used for non-commercial purposes in accordance with RSC Terms and Conditions for self-archivingA series of oligoarginine peptide derivatives containing cyclometallated iridium(III) units display remarkable cytotoxicity, comparable to that of cisplatin. In vitro studies with unilamellar vesicles support a membrane-disrupting mechanism of actionWe are thankful for the support given by the Spanish grants SAF2013-41943-R, CTQ2015-70698-R, CTQ2013-49317-EXP,CTQ2014-59646-R, and BFU2013-43513-R, and the Xunta de Galicia GRC2013-041. Support from COST Action CM1105 and the orfeo-cinqa network (CTQ2014-51912-REDC) is kindly acknowledgedS