Publications by authors named "Antonio Alcami"

97 Publications

Identification of the Cleavage Domain within Glycoprotein G of Herpes Simplex Virus Type 2.

Viruses 2020 12 12;12(12). Epub 2020 Dec 12.

Institute of Virology, Hannover Medical School, 30625 Hannover, Germany.

Glycoprotein G (gG) from herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) functions as a viral chemokine binding protein (vCKBP). Soluble recombinant forms of gG of HSV-1 and HSV-2 (SgG1 and SgG2, respectively) enhance chemokine-mediated leukocyte migration, in contrast to most known vCKBPs, including those from animal alpha-herpesviruses. Furthermore, both proteins bind to nerve growth factor (NGF), but only SgG2 enhances NGF-dependent neurite outgrowth. The basis and implications of this functional difference between the two proteins are still unknown. While gG1 and gG2 are positional homologues in the genome, they share very limited sequence homology. In fact, , the open reading frame encoding gG is the most divergent genetic locus between these viruses. Full-length gG1 and gG2 are type I transmembrane proteins located on the plasma membrane of infected cells and at the viral envelope. However, gG2 is larger than gG1 and is cleaved during protein maturation, secreting the N-terminal domain to the supernatant of infected cells, whereas gG1 is not. The enzyme involved in gG2 cleavage and the functional relevance of gG2 cleavage and secretion are unknown. We aim to identify the gG2 sequence required for cleavage to determine its functional role in future experiments. Our results prove the existence of at least two cleavage motifs in gG2 within the amino acid region 314-343. Transfer of this sequence to a fusion protein results in cleavage. Finally, we show that propeptide convertases like furin are responsible for gG2 cleavage.
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http://dx.doi.org/10.3390/v12121428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763493PMC
December 2020

Poxvirus-encoded TNF receptor homolog dampens inflammation and protects from uncontrolled lung pathology during respiratory infection.

Proc Natl Acad Sci U S A 2020 10 12;117(43):26885-26894. Epub 2020 Oct 12.

Infection and Immunity Group, Department of Immunology, The John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia;

Ectromelia virus (ECTV) causes mousepox, a surrogate mouse model for smallpox caused by variola virus in humans. Both orthopoxviruses encode tumor necrosis factor receptor (TNFR) homologs or viral TNFR (vTNFR). These homologs are termed cytokine response modifier (Crm) proteins, containing a TNF-binding domain and a chemokine-binding domain called smallpox virus-encoded chemokine receptor (SECRET) domain. ECTV encodes one vTNFR known as CrmD. Infection of ECTV-resistant C57BL/6 mice with a CrmD deletion mutant virus resulted in uniform mortality due to excessive TNF secretion and dysregulated inflammatory cytokine production. CrmD dampened pathology, leukocyte recruitment, and inflammatory cytokine production in lungs including TNF, IL-6, IL-10, and IFN-γ. Blockade of TNF, IL-6, or IL-10R function with monoclonal antibodies reduced lung pathology and provided 60 to 100% protection from otherwise lethal infection. IFN-γ caused lung pathology only when both the TNF-binding and SECRET domains were absent. Presence of the SECRET domain alone induced significantly higher levels of IL-1β, IL-6, and IL-10, likely overcoming any protective effects that might have been afforded by anti-IFN-γ treatment. The use of TNF-deficient mice and those that express only membrane-associated but not secreted TNF revealed that CrmD is critically dependent on host TNF for its function. In vitro, recombinant Crm proteins from different orthopoxviruses bound to membrane-associated TNF and dampened inflammatory gene expression through reverse signaling. CrmD does not affect virus replication; however, it provides the host advantage by enabling survival. Host survival would facilitate virus spread, which would also provide an advantage to the virus.
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http://dx.doi.org/10.1073/pnas.2004688117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604498PMC
October 2020

SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

Nat Commun 2020 10 2;11(1):4938. Epub 2020 Oct 2.

Department of Biomedicine, Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus, Denmark.

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.
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http://dx.doi.org/10.1038/s41467-020-18764-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532469PMC
October 2020

Viral cGAMP nuclease reveals the essential role of DNA sensing in protection against acute lethal virus infection.

Sci Adv 2020 Sep 18;6(38). Epub 2020 Sep 18.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid, Spain.

Cells contain numerous immune sensors to detect virus infection. The cyclic GMP-AMP (cGAMP) synthase (cGAS) recognizes cytosolic DNA and activates innate immune responses via stimulator of interferon genes (STING), but the impact of DNA sensing pathways on host protective responses has not been fully defined. We demonstrate that cGAS/STING activation is required to resist lethal poxvirus infection. We identified viral Schlafen (vSlfn) as the main STING inhibitor, and ectromelia virus was severely attenuated in the absence of vSlfn. Both vSlfn-mediated virulence and STING inhibitory activity were mapped to the recently discovered poxin cGAMP nuclease domain. Animals were protected from subcutaneous, respiratory, and intravenous infection in the absence of vSlfn, and interferon was the main antiviral protective mechanism controlled by the DNA sensing pathway. Our findings support the idea that manipulation of DNA sensing is an efficient therapeutic strategy in diseases triggered by viral infection or tissue damage-mediated release of self-DNA.
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http://dx.doi.org/10.1126/sciadv.abb4565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500930PMC
September 2020

Was smallpox a widespread mild disease?

Authors:
Antonio Alcamí

Science 2020 07;369(6502):376-377

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM), Nicolás Cabrera 1, 28049 Madrid, Spain.

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http://dx.doi.org/10.1126/science.abd1214DOI Listing
July 2020

Herpes Simplex Virus 2 Counteracts Neurite Outgrowth Repulsion during Infection in a Nerve Growth Factor-Dependent Manner.

J Virol 2020 09 29;94(20). Epub 2020 Sep 29.

Institute of Virology, Hannover Medical School, Hannover, Germany

During primary infection, herpes simplex virus 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors and attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesized that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis, we exposed sensory neurons to nerve growth factor (NGF) and mock- or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secrete factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduces this repelling phenotype, increasing neurite numbers. The HSV-2-mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that ecombinant, oluble lycoprotein of HSV- (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection has not been investigated. Therefore, we addressed whether gG2 contributes to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of nonneuronal cells reduces their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype, but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2-mediated pathogenesis. Herpes simplex virus 2 (HSV-2) is a prevalent human pathogen that establishes lifelong latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that nonneuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition and, rather, stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.
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http://dx.doi.org/10.1128/JVI.01370-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527038PMC
September 2020

Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands.

Vaccines (Basel) 2020 Jun 22;8(2). Epub 2020 Jun 22.

Centro de Investigación en Sanidad Animal (CISA-INIA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, 28130 Madrid, Spain.

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, OX40L and CD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of OX40L and CD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-κB signalling pathway through their cognate receptor. These adenovirus-secreted OX40L and CD70 forms could also activate ovine T cell proliferation and enhance IFN-γ production in CD4 and CD8 T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants.
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http://dx.doi.org/10.3390/vaccines8020333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350415PMC
June 2020

T cells with dysfunctional mitochondria induce multimorbidity and premature senescence.

Science 2020 06 21;368(6497):1371-1376. Epub 2020 May 21.

Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Madrid, Spain.

The effect of immunometabolism on age-associated diseases remains uncertain. In this work, we show that T cells with dysfunctional mitochondria owing to mitochondrial transcription factor A (TFAM) deficiency act as accelerators of senescence. In mice, these cells instigate multiple aging-related features, including metabolic, cognitive, physical, and cardiovascular alterations, which together result in premature death. T cell metabolic failure induces the accumulation of circulating cytokines, which resembles the chronic inflammation that is characteristic of aging ("inflammaging"). This cytokine storm itself acts as a systemic inducer of senescence. Blocking tumor necrosis factor-α signaling or preventing senescence with nicotinamide adenine dinucleotide precursors partially rescues premature aging in mice with -deficient T cells. Thus, T cells can regulate organismal fitness and life span, which highlights the importance of tight immunometabolic control in both aging and the onset of age-associated diseases.
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http://dx.doi.org/10.1126/science.aax0860DOI Listing
June 2020

Virus-encoded cytokine and chemokine decoy receptors.

Curr Opin Immunol 2020 10 8;66:50-56. Epub 2020 May 8.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), 28049, Madrid, Spain. Electronic address:

Poxviruses and herpesviruses encode secreted versions of cytokine receptors as a unique strategy to evade the host immune response. Recent advances in the field have shown the great impact of some of these proteins in immune modulation and viral pathogenesis, and have uncovered unique properties of these viral proteins not found in the cellular counterparts. These modifications inspired by viruses lead to improved immune modulatory activity of the soluble cytokine receptors, information that has been used to develop more efficient therapeutics to treat inflammatory conditions.
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http://dx.doi.org/10.1016/j.coi.2020.04.008DOI Listing
October 2020

Deriving Immune Modulating Drugs from Viruses-A New Class of Biologics.

J Clin Med 2020 Mar 31;9(4). Epub 2020 Mar 31.

Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.
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http://dx.doi.org/10.3390/jcm9040972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230489PMC
March 2020

Antiviral, Immunomodulatory and Antiproliferative Activities of Recombinant Soluble IFNAR2 without IFN-ß Mediation.

J Clin Med 2020 Mar 31;9(4). Epub 2020 Mar 31.

Instituto de Investigación Biomédica de Málaga-IBIMA, 29009 Málaga, Spain.

Soluble receptors of cytokines are able to modify cytokine activities and therefore the immune system, and some have intrinsic biological activities without mediation from their cytokines. The soluble interferon beta (IFN-ß) receptor is generated through alternative splicing of IFNAR2 and has both agonist and antagonist properties for IFN-ß, but its role is unknown. We previously demonstrated that a recombinant human soluble IFN-ß receptor showed intrinsic therapeutic efficacy in a mouse model of multiple sclerosis. Here we evaluate the potential biological activities of recombinant sIFNAR2 without the mediation of IFN-ß in human cells. Recombinant sIFNAR2 down-regulated the production of IL-17 and IFN-ɣ and reduced the cell proliferation rate. Moreover, it showed a strong antiviral activity, fully protecting the cell monolayer after being infected by the virus. Specific inhibitors completely abrogated the antiviral activity of IFN-ß, but not that of the recombinant sIFNAR2, and there was no activation of the JAK-STAT signaling pathway. Consequently, r-sIFNAR2 exerts immunomodulatory, antiproliferative and antiviral activities without IFN-ß mediation, and could be a promising treatment against viral infections and immune-mediated diseases.
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http://dx.doi.org/10.3390/jcm9040959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230527PMC
March 2020

Addition of a Viral Immunomodulatory Domain to Etanercept Generates a Bifunctional Chemokine and TNF Inhibitor.

J Clin Med 2019 Dec 20;9(1). Epub 2019 Dec 20.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain.

The inhibition of tumor necrosis factor (TNF) through the use of either antibodies or soluble receptors is a highly effective strategy for the clinical control of chronic inflammatory conditions such as rheumatoid arthritis. Different viruses have similarly exploited this concept by expressing a set of specifically tailored secreted TNF decoy receptors to block host inflammatory responses. Poxviruses have been shown to encode at least two distinct molecules, termed Cytokine response modifier D (CrmD) and CrmB, in which a TNF inhibitor is combined with a chemokine inhibitor on the same molecule. The ectromelia virus CrmD protein was found to be a critical determinant of virulence in vivo, being able to control local inflammation to allow further viral spread and the establishment of a lethal infection. Strikingly, both the TNF and the chemokine inhibitory domains are required for the full activity of CrmD, suggesting a model in which inhibition of TNF is supported by the concomitant blockade of a reduced set of chemokines. Inspired by this model, we reasoned that a similar strategy could be applied to modify the clinically used human TNF receptor (etanercept), producing a generation of novel, more effective therapeutic agents. Here we show the analysis of a set of fusion proteins derived from etanercept by addition of a viral chemokine-binding protein. A bifunctional inhibitor capable of binding to and blocking the activity of TNF as well as a set of chemokines is generated that is active in the prevention of arthritis in a murine disease model.
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http://dx.doi.org/10.3390/jcm9010025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020083PMC
December 2019

Subversion of natural killer cell responses by a cytomegalovirus-encoded soluble CD48 decoy receptor.

PLoS Pathog 2019 04 4;15(4):e1007658. Epub 2019 Apr 4.

Immunology Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain.

Throughout evolution, cytomegaloviruses (CMVs) have been capturing genes from their hosts, employing the derived proteins to evade host immune defenses. We have recently reported the presence of a number of CD48 homologs (vCD48s) encoded by different pathogenic viruses, including several CMVs. However, their properties and biological relevance remain as yet unexplored. CD48, a cosignaling molecule expressed on the surface of most hematopoietic cells, modulates the function of natural killer (NK) and other cytotoxic cells by binding to its natural ligand 2B4 (CD244). Here, we have characterized A43, the vCD48 exhibiting the highest amino acid sequence identity with host CD48. A43, which is encoded by owl monkey CMV, is a soluble molecule released from the cell after being proteolytically processed through its membrane proximal region. A43 is expressed with immediate-early kinetics, yielding a protein that is rapidly detected in the supernatant of infected cells. Remarkably, surface plasmon resonance assays revealed that this viral protein binds to host 2B4 with high affinity and slow dissociation rates. We demonstrate that soluble A43 is capable to abrogate host CD48:2B4 interactions. Moreover, A43 strongly binds to human 2B4 and prevents 2B4-mediated NK-cell adhesion to target cells, therefore reducing the formation of conjugates and the establishment of immunological synapses between human NK cells and CD48-expressing target cells. Furthermore, in the presence of this viral protein, 2B4-mediated cytotoxicity and IFN-γ production by NK cells are severely impaired. In summary, we propose that A43 may serve as a functional soluble CD48 decoy receptor by binding and masking 2B4, thereby impeding effective NK cell immune control during viral infections. Thus, our findings provide a novel example of the immune evasion strategies developed by viruses.
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http://dx.doi.org/10.1371/journal.ppat.1007658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448830PMC
April 2019

Insights into ligand binding by a viral tumor necrosis factor (TNF) decoy receptor yield a selective soluble human type 2 TNF receptor.

J Biol Chem 2019 03 5;294(13):5214-5227. Epub 2019 Feb 5.

From the Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, 28049 Madrid, Spain

Etanercept is a soluble form of the tumor necrosis factor receptor 2 (TNFR2) that inhibits pathological tumor necrosis factor (TNF) responses in rheumatoid arthritis and other inflammatory diseases. However, besides TNF, etanercept also blocks lymphotoxin-α (LTα), which has no clear therapeutic value and might aggravate some of the adverse effects associated with etanercept. Poxviruses encode soluble TNFR2 homologs, termed viral TNF decoy receptors (vTNFRs), that display unique specificity properties. For instance, cytokine response modifier D (CrmD) inhibits mouse and human TNF and mouse LTα, but it is inactive against human LTα. Here, we analyzed the molecular basis of these immunomodulatory activities in the ectromelia virus-encoded CrmD. We found that the overall molecular mechanism to bind TNF and LTα from mouse and human origin is fairly conserved in CrmD and dominated by a groove under its 50s loop. However, other ligand-specific binding determinants optimize CrmD for the inhibition of mouse ligands, especially mouse TNF. Moreover, we show that the inability of CrmD to inhibit human LTα is caused by a Glu-Phe-Glu motif in its 90s loop. Importantly, transfer of this motif to etanercept diminished its anti-LTα activity in >60-fold while weakening its TNF-inhibitory capacity in 3-fold. This new etanercept variant could potentially be used in the clinic as a safer alternative to conventional etanercept. This work is the most detailed study of the vTNFR-ligand interactions to date and illustrates that a better knowledge of vTNFRs can provide valuable information to improve current anti-TNF therapies.
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http://dx.doi.org/10.1074/jbc.RA118.005828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442024PMC
March 2019

A virus-encoded type I interferon decoy receptor enables evasion of host immunity through cell-surface binding.

Nat Commun 2018 12 21;9(1):5440. Epub 2018 Dec 21.

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28049, Madrid, Spain.

Soluble cytokine decoy receptors are potent immune modulatory reagents with therapeutic applications. Some virus-encoded secreted cytokine receptors interact with glycosaminoglycans expressed at the cell surface, but the biological significance of this activity in vivo is poorly understood. Here, we show the type I interferon binding protein (IFNα/βBP) encoded by vaccinia and ectromelia viruses requires of this cell binding activity to confer full virulence to these viruses and to retain immunomodulatory activity. Expression of a variant form of the IFNα/βBP that inhibits IFN activity, but does not interact with cell surface glycosaminoglycans, results in highly attenuated viruses with a virulence similar to that of the IFNα/βBP deletion mutant viruses. Transcriptomics analysis and infection of IFN receptor-deficient mice confirmed that the control of IFN activity is the main function of the IFNα/βBP in vivo. We propose that retention of secreted cytokine receptors at the cell surface may largely enhance their immunomodulatory activity.
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http://dx.doi.org/10.1038/s41467-018-07772-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303335PMC
December 2018

Complete Genome Sequence of Herpes Simplex Virus 2 Strain 333.

Microbiol Resour Announc 2018 Sep 6;7(9). Epub 2018 Sep 6.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Madrid, Spain.

Herpes simplex virus 2, or human herpesvirus 2, is a ubiquitous human pathogen that causes genital ulcerations and establishes latency in sacral root ganglia. We fully sequenced and manually curated the viral genome sequence of herpes simplex virus 2, strain 333 using Pacific Biosciences and Illumina sequencing technologies.
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http://dx.doi.org/10.1128/MRA.00870-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256523PMC
September 2018

A New Putative Caulimoviridae Genus Discovered through Air Metagenomics.

Microbiol Resour Announc 2018 Oct 11;7(14). Epub 2018 Oct 11.

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

Members of the Caulimoviridae family are important plant pathogens. These circular double-stranded DNA viruses may integrate into the host genome, although this integration is not required for the viral replication cycle. Here, we describe three complete genomes belonging to a new putative Caulimoviridae genus discovered through air metagenomics.
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http://dx.doi.org/10.1128/MRA.00955-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256638PMC
October 2018

Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.

Virulence 2018 ;9(1):1669-1684

a Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School , Hannover , Germany.

Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-β. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.
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http://dx.doi.org/10.1080/21505594.2018.1535785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000197PMC
April 2019

Mechanism of action of the viral chemokine-binding protein E163 from ectromelia virus.

J Biol Chem 2018 11 26;293(45):17418-17429. Epub 2018 Sep 26.

From the Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid 28049, Spain

Chemokines interact with glycosaminoglycans (GAGs) at the cellular surface and to specific cell-surface receptors to activate signaling pathways. The GAG interaction allows the formation of a chemotactic gradient of chemokine required for cell haptotaxis and chemokine oligomerization. Poxviruses encode secreted chemokine-binding proteins with no sequence similarity to their cellular counterparts to modulate the host immune system. The E163 protein from ectromelia virus, the causative agent of mousepox, binds chemokines through their GAG-binding domain. In addition, E163 interacts with GAGs to be anchored at the cell surface, but its ability to interfere with chemokine-GAG interactions has not been demonstrated. We report the identification of the GAG-binding regions in E163 and the generation of mutant forms deficient of GAG binding. Chemokine binding assays show that some of the E163 GAG-binding sites are also involved in the interaction with chemokines. By using recombinant GAG-binding mutant forms we demonstrate that E163 prevents the interaction of chemokines with cell-surface GAGs, providing mechanisms for the immunomodulatory activity of the viral chemokine-binding protein E163.
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http://dx.doi.org/10.1074/jbc.RA118.004432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231125PMC
November 2018

New insights into the immunomodulatory properties of poxvirus cytokine decoy receptors at the cell surface.

F1000Res 2018 11;7. Epub 2018 Jun 11.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Nicolás Cabrera 1, Cantoblanco, 28049 , Madrid, Spain.

Poxviruses encode a set of secreted proteins that bind cytokines and chemokines as a strategy to modulate host defense mechanisms. These viral proteins mimic the activity of host cytokine decoy receptors but have unique properties that may enhance their activity. Here, we describe the ability of poxvirus cytokine receptors to attach to the cell surface after secretion from infected cells, and we discuss the advantages that this property may confer to these viral immunomodulatory proteins.
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http://dx.doi.org/10.12688/f1000research.14238.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998005PMC
June 2018

Viruses in Polar Lake and Soil Ecosystems.

Adv Virus Res 2018 26;101:39-54. Epub 2018 Apr 26.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Madrid, Spain. Electronic address:

Viruses play an important role in the control of microbial communities, and it has been suggested that the influence of viruses in polar ecosystems, with low nutrients and under extreme environmental conditions, may be greater. Viral metagenomics allows the genetic characterization of complex viral communities without the need to isolate and grow viruses. Recent investigations in Antarctica and the Arctic are uncovering a great diversity of DNA viruses, including bacteriophages, circular single-stranded DNA viruses, algal-infecting phycodnaviruses, and virophages, adapted to these extreme environments. The limited sequence similarity between viruses in Antarctica and the Arctic suggests that viral communities in the two polar regions have evolved independently since the formation of the Antarctic continent, estimated to occur 25 million years ago. The community of RNA viruses in Antarctica is dominated by the order Picornavirales and their quasispecies composition suggests that higher genetic variability may correlate with viral adaptation to new environmental conditions.
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http://dx.doi.org/10.1016/bs.aivir.2018.02.002DOI Listing
November 2018

Chemokines cooperate with TNF to provide protective anti-viral immunity and to enhance inflammation.

Nat Commun 2018 05 3;9(1):1790. Epub 2018 May 3.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Cantoblanco, Madrid, 28049, Spain.

The role of cytokines and chemokines in anti-viral defense has been demonstrated, but their relative contribution to protective anti-viral responses in vivo is not fully understood. Cytokine response modifier D (CrmD) is a secreted receptor for TNF and lymphotoxin containing the smallpox virus-encoded chemokine receptor (SECRET) domain and is expressed by ectromelia virus, the causative agent of the smallpox-like disease mousepox. Here we show that CrmD is an essential virulence factor that controls natural killer cell activation and allows progression of fatal mousepox, and demonstrate that both SECRET and TNF binding domains are required for full CrmD activity. Vaccination with recombinant CrmD protects animals from lethal mousepox. These results indicate that a specific set of chemokines enhance the inflammatory and protective anti-viral responses mediated by TNF and lymphotoxin, and illustrate how viruses optimize anti-TNF strategies with the addition of a chemokine binding domain as soluble decoy receptors.
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http://dx.doi.org/10.1038/s41467-018-04098-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934441PMC
May 2018

Composition of the Schistosoma mansoni worm secretome: Identification of immune modulatory Cyclophilin A.

PLoS Negl Trop Dis 2017 Oct 26;11(10):e0006012. Epub 2017 Oct 26.

Trinity Biomedical Sciences Institute, School of Medicine, Trinity College Dublin, Dublin 2, Ireland.

The helminth Schistosoma mansoni modulates the infected host's immune system to facilitate its own survival, by producing excretory/secretory molecules that interact with a variety of the host's cell types including those of the immune system. Herein, we characterise the S. mansoni adult male worm secretome and identify 111 proteins, including 7 vaccine candidates and several molecules with potential immunomodulatory activity. Amongst the molecules present in the secretome, a 17-19kDa protein analogous to human cyclophilin A was identified. Given the ability of cyclophilin A to modulate the immune system by regulating antigen presenting cell activity, we sought to determine whether recombinant S. mansoni Cyclophilin A (rSmCypA) is capable of modulating bone-marrow derived dendritic cell (BMDC) and T cell responses under in vitro conditions. rSmCypA was enzymatically active and able to alter the pro-inflammatory cytokine profile of LPS-activated dendritic cells. rSmCypA also modulated DC function in the induction of CD4+ T cell proliferation with a preferential expansion of Treg cells. This work demonstrates the unique protein composition of the S. mansoni male worm secretome and immunomodulatory activity of S. mansoni Cyclophilin A.
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http://dx.doi.org/10.1371/journal.pntd.0006012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681295PMC
October 2017

Validation of the Hirst-Type Spore Trap for Simultaneous Monitoring of Prokaryotic and Eukaryotic Biodiversities in Urban Air Samples by Next-Generation Sequencing.

Appl Environ Microbiol 2017 07 16;83(13). Epub 2017 Jun 16.

Escuela Técnica Superior de Ingenieros Industriales, Universidad Politécnica de Madrid (ETSII-UPM), Madrid, Spain

Pollen, fungi, and bacteria are the main microscopic biological entities present in outdoor air, causing allergy symptoms and disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not yet been developed. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the 16S rRNA gene and internal transcribed spacer for the taxonomic assignment. We showed that aerobiological communities are well represented by this approach. The operational taxonomic units (OTUs) of two traps working synchronically compiled >87% of the total relative abundance for bacterial diversity collected in each sampler, >89% for fungi, and >97% for pollen. We found a good correspondence between traditional characterization by microscopy and genetic identification, obtaining more-accurate taxonomic assignments and detecting a greater diversity using the latter. We also demonstrated that DNA sequencing accurately detects differences in biodiversity between samples. We concluded that high-throughput DNA sequencing applied to aerobiological samples obtained with Hirst spore traps provides reliable results and can be easily implemented for monitoring prokaryotic and eukaryotic entities present in the air of urban areas. Detection, monitoring, and characterization of the wide diversity of biological entities present in the air are difficult tasks that require time and expertise in different disciplines. We have evaluated the use of the Hirst spore trap (an instrument broadly employed in aerobiological studies) to detect and identify these organisms by DNA-based analyses. Our results showed a consistent collection of DNA and a good concordance with traditional methods for identification, suggesting that these devices can be used as a tool for continuous monitoring of the airborne biodiversity, improving taxonomic resolution and characterization together. They are also suitable for acquiring novel DNA amplicon-based information in order to gain a better understanding of the biological particles present in a scarcely known environment such as the air.
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http://dx.doi.org/10.1128/AEM.00472-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478992PMC
July 2017

Infection with diverse immune-modulating poxviruses elicits different compositional shifts in the mouse gut microbiome.

PLoS One 2017 10;12(3):e0173697. Epub 2017 Mar 10.

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Madrid, Spain.

It is often not possible to demonstrate causality within the context of gut microbiota dysbiosis-linked diseases. Thus, we need a better understanding of the mechanisms whereby an altered host immunophysiology shapes its resident microbiota. In this regard, immune-modulating poxvirus strains and mutants could differentially alter gut mucosal immunity in the context of a natural immune response, providing a controlled natural in vivo setting to deepen our understanding of the immune determinants of microbiome composition. This study represents a proof-of-concept that the use of an existing collection of different immune-modulating poxviruses may represent an innovative tool in gut microbiome research. To this end, 16S rRNA amplicon sequencing and RNAseq transcriptome profiling were employed as proxies for microbiota composition and gut immunophysiological status in the analysis of caecal samples from control mice and mice infected with various poxvirus types. Our results show that different poxvirus species and mutants elicit different shifts in the mice mucosa-associated microbiota and, in some instances, significant concomitant shifts in gut transcriptome profiles, thus providing an initial validation to the proposed model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173697PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345840PMC
August 2017

RNA-Seq Based Transcriptome Analysis of the Type I Interferon Host Response upon Vaccinia Virus Infection of Mouse Cells.

J Immunol Res 2017 9;2017:5157626. Epub 2017 Feb 9.

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), 28049 Madrid, Spain.

Vaccinia virus (VACV) encodes the soluble type I interferon (IFN) binding protein B18 that is secreted from infected cells and also attaches to the cell surface, as an immunomodulatory strategy to inhibit the host IFN response. By using next generation sequencing technologies, we performed a detailed RNA-seq study to dissect at the transcriptional level the modulation of the IFN based host response by VACV and B18. Transcriptome profiling of L929 cells after incubation with purified recombinant B18 protein showed that attachment of B18 to the cell surface does not trigger cell signalling leading to transcriptional activation. Consistent with its ability to bind type I IFN, B18 completely inhibited the IFN-mediated modulation of host gene expression. Addition of UV-inactivated virus particles to cell cultures altered the expression of a set of 53 cellular genes, including genes involved in innate immunity. Differential gene expression analyses of cells infected with replication competent VACV identified the activation of a broad range of host genes involved in multiple cellular pathways. Interestingly, we did not detect an IFN-mediated response among the transcriptional changes induced by VACV, even after the addition of IFN to cells infected with a mutant VACV lacking B18. This is consistent with additional viral mechanisms acting at different levels to block IFN responses during VACV infection.
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http://dx.doi.org/10.1155/2017/5157626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322442PMC
March 2017

Genome Sequence of Herpes Simplex Virus 1 Strain SC16.

Genome Announc 2017 Jan 26;5(4). Epub 2017 Jan 26.

Department of Virology and Microbiology, Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Cantoblanco, Madrid, Spain

Herpes simplex virus 1 (HSV-1), also known as Human herpesvirus 1, is a highly prevalent human neurotropic pathogen that causes a variety of diseases, including lethal encephalitis. Here, we report the genome sequence of the HSV-1 strain SC16.
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http://dx.doi.org/10.1128/genomeA.01392-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270689PMC
January 2017

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Sci Transl Med 2016 12;8(370):370ra184

Centro de Biología Molecular Severo Ochoa, Spanish National Research Council-Autonomous University of Madrid (CSIC-UAM), Madrid, Spain.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.
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http://dx.doi.org/10.1126/scitranslmed.aaf2140DOI Listing
December 2016

Aquatic viral metagenomics: Lights and shadows.

Virus Res 2017 07 24;239:87-96. Epub 2016 Nov 24.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas y Universidad Autónoma de Madrid), Madrid, Spain. Electronic address:

Viruses are the most abundant biological entities on Earth, exceeding bacteria in most of the ecosystems. Specially in oceans, viruses are thought to be the major planktonic predators shaping microorganism communities and controlling ocean biological capacity. Plankton lysis by viruses plays an important role in ocean nutrient and energy cycles. Viral metagenomics has emerged as a powerful tool to uncover viral diversity in aquatic ecosystems through the use of Next Generation Sequencing. However, many of the commonly used viral sample preparation steps have several important biases that must be considered to avoid a misinterpretation of the results. In addition to biases caused by the purification of virus particles, viral DNA/RNA amplification and the preparation of genomic libraries could also introduce biases, and a detailed knowledge about such protocols is required. In this review, the main steps in the viral metagenomic workflow are described paying special attention to the potential biases introduced by each one.
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http://dx.doi.org/10.1016/j.virusres.2016.11.021DOI Listing
July 2017