Amino acid substitutions associated with treatment failure of hepatitis C virus infection

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022.Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of patients do not achieve such a response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. By deep sequencing analysis of 220 subtyped HCV samples from infected patients who failed therapy, collected from 39 Spanish hospitals, we determined amino acid sequences of the DAA-target proteins NS3, NS5A and NS5B, by UDS of HCV patient samples, in search of resistanceassociated substitutions (RAS). Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide RAS. They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the threedimensional structures of the proteins harboring them. The information on HRSs that will be gathered during sequencing should be relevant not only to help predict treatment outcomes and disease progression but also to further understand HCV population dynamics, which appears much more complex than thought prior to the introduction of deep sequencing.The work at CBMSO was supported by grants SAF2014-52400-R from MINECO, SAF2017-87846-R and BFU2017-91384-EXP MICIU, PI18/00210 from ISCIII, S2013/ABI-2906 (PLATESA) and S2018/BAA-4370 (PLATESA2) from Comunidad de Madrid/FEDER. C.P. is supported by the Miguel Servet program of the ISCIII (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd is funded by ISCIII. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by ISCIII, cofinanced by ERDF grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).Peer reviewe

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT

Role of Azathioprine in steroid resistant non infectious ocular inflammatory diseases Eficacia de la azatioprina en la enfermedad ocular inflamatoria no infecciosa resistente a tratamiento esteroidal sistémico

Background: Topical and systemic steroids are the first line of treatment of non infectious inflammatory ocular disease. Immunosuppresants are reserved as a second line treatment. Aim: To evaluate the role of Azathioprine (AZA) as a coadyuvant immunosuppressive treatment for non infectious ocular inflammatory diseases (OIDs) resistant to systemic steroid therapy in a retrospective, noncomparative interventional case series. Patients and methods: Patients using oral Prednisone due to an active or recurrent OID, without clinical response, and not receiving any other immunosuppressive treatment were studied. A standard protocol of oral Prednisone (0.5 mg/kg/day) and oral AZA (2-3 mg/kg/day) during one year was used. Ocular and systemic monthly evaluations were done including relapse rate, steroid dosage, inflammatory score and visual acuity. Results: Thirty patients (10 male) aged 18-75 years (mean 44 years) were studied. Three had bilateral anterior uveitis, one had pars planitis, four

Synthesis of bis(pentachlorophenyl) silanediol and related compounds. Crystal structure of diethoxybis(pentachlorophenyl) silane

Bis(pentachlorophenyl) silanediol (3) has been prepared by neutral hydrolysis of perchlorodiphenylsilane (2). Reduction of 2 with LiAlH4 gives bis(pentachlorophenyl) silane (4) which upon treatment with bromine gives dibromobis(pentachlorophenyl)silane (5). Reaction of 2 with methanol and ethanol leads to dimethoxybis(pentachlorophenyl)silane (6) and diethoxybis(pentachlorophenyl)silane (7) respectively. The IR spectra of 2-7, as well as the UV and 1H NMR spectra of some of these compounds, are reported. The results of thermogravimetric and differential scanning calorimetric studies on silanediol 3 are discussed. The structure of the silane 7 has been established by X-ray crystallography. © 1992.Peer Reviewe

Retinoic acid binds to the C2-domain of protein kinase Cα

Protein kinase Cα (PKCα) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKCα, a regulatory module in the protein that binds Ca2+ and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 Å resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca2+-binding pocket, where Ca2+ ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in β-strands three and four. These observations are strongly supported by [3H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca2+ coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKCα activity.Peer Reviewe

Comparison of the clinical efficacy of two different immunosuppressive regimens in patients with chronic Vogt-Koyanagi-Harada disease

Purpose: To prospectively compare 2 immunosupressive regimens in patients with active Vogt-Koyanagi-Harada disease in spite of systemic glucocorticoid treatment. Methods: Forty-four patients were diagnosed between 1998 and 2005. Twenty-one developed chronic intraocular inflammation in spite of glucocorticoid treatment and were randomized to receive either prednisone and azathioprine (AZA) (n = 12) or prednisone and cyclosporine (CyA) (n = 9). Results: In the AZA group Tyndall score decreased from 1.21±1.10 to 0.29±0.62 (p<.01), and visual acuity (LogMAR) improved from 0.32±0.35 to 0.09±0.16 (p<.001). In the CyA group Tyndall score decreased from 1.67±1.08 to 0.16±0.51 (p<.001), and visual acuity improved from 0.41±0.40 to 0.25±0.42 (p<.001). Patients in the AZA group needed a significantly higher average prednisone dose and total cumulative dose than those in the CyA group, p<.01 for each comparison. Conclusions: Both regimens showed a good clinical efficacy, but CyA seems to be a bet

Structure of the C2 domain from novel protein kinase Cε. A membrane binding model for Ca2+-independent C2 domains

Protein kinase Cε (PKCε) is a member of the novel PKCs which are activated by acidic phospholipids, diacylglycerol and phorbol esters, but lack the calcium dependence of classical PKC isotypes. The crystal structures of the C2 domain of PKCε, crystallized both in the absence and in the presence of the two acidic phospholipids, 1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS) and 1,2-dicaproyl-sn-phosphatidic acid (DCPA), have now been determined at 2.1, 1.7 and 2.8 Å resolution, respectively. The central feature of the PKCε-C2 domain structure is an eight-stranded, antiparallel, β-sandwich with a type II topology similar to that of the C2 domains from phospholipase C and from novel PKCδ. Despite the similar topology, important differences are found between the structures of C2 domains from PKCs δ and ε, suggesting they be considered as different PKC subclasses. Site-directed mutagenesis experiments and structural changes in the PKCε-C2 domain from crystals with DCPS or DCPA indicate, though phospholipids were not visible in these structures, that loops joining strands β1-β2 and β5-β6 participate in the binding to anionic membranes. The different behavior in membrane-binding and activation between PKCε and classical PKCs appears to originate in localized structural changes, which include a major reorganization of the region corresponding to the calcium binding pocket in classical PKCs. A mechanism is proposed for the interaction of the PKCε-C2 domain with model membranes that retains basic features of the docking of C2 domains from classical, calcium-dependent, PKCs. © 2001 Academic Press.This research was supported by grant 1FD97-1558 from DGESIC (Spain). Research at the IBMB was also supported by grant PB95-0218, and research at Universidad de Murcia by grant PB98-0389 from DGESIC (Spain). W.F.O. and J.G.G. are the recipients of fellowships from Ministerio de Educación y Cultura (Spain).Peer Reviewe

Structure of the C2 domain from novel protein kinase Cε. A membrane binding model for Ca2+-independent C2 domains

Protein kinase Cε (PKCε) is a member of the novel PKCs which are activated by acidic phospholipids, diacylglycerol and phorbol esters, but lack the calcium dependence of classical PKC isotypes. The crystal structures of the C2 domain of PKCε, crystallized both in the absence and in the presence of the two acidic phospholipids, 1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS) and 1,2-dicaproyl-sn-phosphatidic acid (DCPA), have now been determined at 2.1, 1.7 and 2.8 Å resolution, respectively. The central feature of the PKCε-C2 domain structure is an eight-stranded, antiparallel, β-sandwich with a type II topology similar to that of the C2 domains from phospholipase C and from novel PKCδ. Despite the similar topology, important differences are found between the structures of C2 domains from PKCs δ and ε, suggesting they be considered as different PKC subclasses. Site-directed mutagenesis experiments and structural changes in the PKCε-C2 domain from crystals with DCPS or DCPA indicate, though phospholipids were not visible in these structures, that loops joining strands β1-β2 and β5-β6 participate in the binding to anionic membranes. The different behavior in membrane-binding and activation between PKCε and classical PKCs appears to originate in localized structural changes, which include a major reorganization of the region corresponding to the calcium binding pocket in classical PKCs. A mechanism is proposed for the interaction of the PKCε-C2 domain with model membranes that retains basic features of the docking of C2 domains from classical, calcium-dependent, PKCs. © 2001 Academic Press.This research was supported by grant 1FD97-1558 from DGESIC (Spain). Research at the IBMB was also supported by grant PB95-0218, and research at Universidad de Murcia by grant PB98-0389 from DGESIC (Spain). W.F.O. and J.G.G. are the recipients of fellowships from Ministerio de Educación y Cultura (Spain).Peer Reviewe

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T=3 capsid protein leads to larger T=4 capsids

et al.Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution. © 2012, American Society for Microbiology.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2009-09331 to R.M., BFU2008-02328/BMC to J.L.C., BIO2008-02556 to N.V., AGL2010-22200-C02-02 to J.B., and BIO2008-02361 and BFU2011-25902 to J.R.C.), NADIR-UE-228394 (to J.B.), and the NIH Intramural Research Program with support from the Center for Information Technology (to B.L.T.).Peer Reviewe