Antonio Alcami

Centro De Biología Molecular Severo Ochoa, Madrid, Madrid, Spain

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Publications (91)612.81 Total impact

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    ABSTRACT: Most people in the world (~90%) are infected by the Epstein-Barr virus (EBV), which establishes itself permanently in B-cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. To perform a detailed comparative genomics analysis of EBV including, for the first time, EBV strains derived from healthy individuals we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. Since strain B95-8 was used to transform B-cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605) we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. We found that latency genes harbour more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host-parasite co-evolution.
    Genome Biology and Evolution 03/2014; · 4.76 Impact Factor
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    ABSTRACT: T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.
    The EMBO Journal 02/2014; · 9.82 Impact Factor
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    ABSTRACT: The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain.
    Genome announcements. 01/2014; 2(1).
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    ABSTRACT: Nuclear factor kappa B (NF-κB) and type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-κB and its interplay with T1-IFN in antiviral immunity is poorly understood. We show that NF-κB is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-κB inhibitor activates NF-κB more effectively in vivo, resulting in increased proinflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-κB activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-κB pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease.
    Cell host & microbe 06/2013; 13(6):701-10. · 13.02 Impact Factor
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    ABSTRACT: BACKGROUND: Lymphocystis disease virus (LCDV) is a large icosahedral dsDNA-containing virus of the Lymphocystivirus genus within the Iridoviridae family that can cause disease in more than 140 marine and freshwater fish species. While several isolates have been charcaterized and classified into distinct genotypes the complete genomic sequence is currently only available from two species, the LCDV-1, isolated from flounder (Platichtys flesus) in Europe and the LCDV-C, isolated from Japanese cultured flounder (Paralichthys olivaceus) in China. Analysis of the genome of LCDV-C showed it to encode a protein named LDVICp016 with similarities to the Tumour necrosis factor receptor (TNFR) superfamily with immunomodulatory potential. FINDINGS: We have expressed and purified the recombinant protein LDVICp016 and screened for potential interaction partners using surface plasmon resonance. Commercially available human and mouse members of the TNF superfamily (TNFSF), along with a representative set of fish-derived TNFSF were tested.We have found the LDVICp016 protein to be secreted and we have identified a second viral TNFR encoded by ORF 095 of the same virus. None of the 42 tested proteins were found to interact with LDVICp016. CONCLUSIONS: We show that LDVICp016 is a secreted protein belonging to the TNF receptor family that may be part of a larger gene family in Lymphocystiviruses. While the ligand of this protein remains unknown, possibly due to the species specific nature of this interaction, further investigations into the potential role of this protein in the blockade of immune responses in its fish host are required.
    Virology Journal 06/2013; 10(1):188. · 2.09 Impact Factor
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    ABSTRACT: Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health.
    Journal of Virology 06/2012; 86(11):6365-6. · 5.08 Impact Factor
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    ABSTRACT: Worldwide amphibian population declines have been ascribed to global warming, increasing pollution levels, and other factors directly related to human activities. These factors may additionally be favoring the emergence of novel pathogens. In this report, we have determined the complete genome sequence of the emerging common midwife toad ranavirus (CMTV), which has caused fatal disease in several amphibian species across Europe. Phylogenetic and gene content analyses of the first complete genomic sequence from a ranavirus isolated in Europe show that CMTV is an amphibian-like ranavirus (ALRV). However, the CMTV genome structure is novel and represents an intermediate evolutionary stage between the two previously described ALRV groups. We find that CMTV clusters with several other ranaviruses isolated from different hosts and locations which might also be included in this novel ranavirus group. This work sheds light on the phylogenetic relationships within this complex group of emerging, disease-causing viruses.
    Journal of Virology 02/2012; 86(7):3617-25. · 5.08 Impact Factor
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    ABSTRACT: Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses.
    PLoS Pathogens 02/2012; 8(2):e1002497. · 8.14 Impact Factor
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    ABSTRACT: Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.
    PLoS Pathogens 01/2012; 8(1):e1002475. · 8.14 Impact Factor
  • Imma Montanuy, Ali Alejo, Antonio Alcami
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    ABSTRACT: Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.
    The FASEB Journal 03/2011; 25(6):1960-71. · 5.70 Impact Factor
  • Alí Alejo, Sergio M Pontejo, Antonio Alcami
    Advances in experimental medicine and biology 01/2011; 691:203-10. · 1.83 Impact Factor
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    ABSTRACT: Tumor necrosis factor α (TNFα) is a key pathogenic factor in Crohn's disease and rheumatoid arthritis. TNF(ΔARE) mice express high levels of TNFα and present Crohn's-like ileitis and arthritis. Alterations in the chemokine network could underline the TNF-driven ileitis. The aim of this study was to evaluate the role of TNF and chemokines in ileitis using ectromelia virus cytokine response modifier D (CrmD), a protein that binds TNFα and a limited number of chemokines. We generated transgenic mice expressing CrmD in intestinal epithelial cells (vCrmD mice) and crossed them with the TNF(ΔARE) mice to test whether CrmD could affect TNF-driven inflammatory processes. During homeostasis, only the number of B cells in the lamina propria was reduced by CrmD expression. Interestingly, CrmD expression in the intestine markedly attenuated the inflammatory infiltrates in the ileum of TNF(ΔARE) mice, but did not affect development of arthritis. Our results suggest that CrmD affects development of ileitis by locally affecting both TNF and chemokine function in the ileum.
    Mucosal Immunology 11/2010; 3(6):633-44. · 7.00 Impact Factor
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    Antonio Alcami, Sergio A Lira
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    ABSTRACT: Viruses encode a variety of mechanisms to evade host immune pathways. Large DNA viruses (herpesviruses and poxviruses) encode proteins that mimic chemokines and chemokine receptors. Also, some viruses encode secreted proteins that bind chemokines and have structure unrelated to host proteins. Recent research in this area has led to the identification of new viral proteins that modulate the chemokine system, has provided information on the molecular mechanisms leading to interference of chemokine signaling, and has shed light into the function of these proteins in the context of infection. The therapeutic value of these viral proteins to inhibit immune responses that cause pathology has been explored further. Finally, a new family of chemokine binding proteins identified in ticks expands this strategy of immune modulation beyond the virus world.
    Current opinion in immunology 08/2010; 22(4):482-7. · 10.88 Impact Factor
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    Antonio Alcami
    Current opinion in microbiology 08/2010; 13(4):501-2. · 7.87 Impact Factor
  • Nazario Rubio, Marcos Palomo, Antonio Alcami
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    ABSTRACT: This article reports the production of interferon alpha/beta (IFN-alpha/beta) by SJL/J mouse brain astrocyte cultures infected with Theiler's murine encephalomyelitis virus (TMEV). cRNA from mock- and TMEV-infected SJL/J astrocytes was hybridized to the Affymetrix whole murine genome DNA microarray. Analysis revealed the up-regulation of 3 sequences coding for the IFN-alpha/beta domain. Increased expression of mRNA coding for IFN-alpha was shown by conventional RT-PCR and quantitative real-time RT-PCR. According to ELISA, the concentration of IFN-alpha in the supernatants of infected astrocyte cultures varied with the multiplicity of infection and post-infection time. The IFN-alpha/beta secreted was biologically active, as shown by a virus-based IFN bioassay involving Cocal virus and TMEV infection. The contribution to total interferon activity was 29% +/- 3.0% for IFN-alpha and 52% +/- 3.6% for IFN-beta. IFN-alpha/beta was induced by whole TMEV virions; induction was not achieved with either purified isolated virion capsid proteins or UV-inactivated virus. Further, induction was inhibited by specific anti-TMEV antibodies. The receptor for IFN-alpha/beta, which is absent in uninfected astrocytes, was up-regulated after infection, as suggested by DNA hybridization analysis. The brains of infected mice contained IFN-alpha/beta mRNA during the acute encephalitis phase, peaking at day 5 post-infection. Our findings could have significance for human diseases such as viral encephalitis and multiple sclerosis.
    Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 04/2010; 30(4):253-62. · 1.63 Impact Factor
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    ABSTRACT: Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.
    The FASEB Journal 12/2009; 24(5):1479-88. · 5.70 Impact Factor
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    ABSTRACT: Viruses are the most abundant biological entities and can control microbial communities, but their identity in terrestrial and freshwater Antarctic ecosystems is unknown. The genetic structure of an Antarctic lake viral community revealed unexpected genetic richness distributed across the highest number of viral families that have been found to date in aquatic viral metagenomes. In contrast to other known aquatic viromes, which are dominated by bacteriophage sequences, this Antarctic virus assemblage had a large proportion of sequences related to eukaryotic viruses, including phycodnaviruses and single-stranded DNA (ssDNA) viruses not previously identified in aquatic environments. We also observed that the transition from an ice-covered lake in spring to an open-water lake in summer led to a change from a ssDNA- to a double-stranded DNA-virus-dominated assemblage, possibly reflecting a seasonal shift in host organisms.
    Science 11/2009; 326(5954):858-61. · 31.20 Impact Factor
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    ABSTRACT: Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes severe respiratory disease in poultry. Glycoprotein G (gG) is a virulence factor in ILTV. Recent studies have shown that gG-deficient ILTV is an effective attenuated vaccine however the function of ILTV gG is unknown. This study examined the function and in vivo relevance of ILTV gG. The results showed that ILTV gG binds to chemokines with high affinity and inhibits leukocyte chemotaxis. Specific-pathogen-free (SPF) chickens infected with gG-deficient virus had altered tracheal leukocyte populations and lower serum antibody levels compared with those infected with the parent virus. The findings suggest that the absence of chemokine-binding activity during infection with gG-deficient ILTV results in altered host immune responses.
    Vaccine 11/2009; 28(5):1325-32. · 3.77 Impact Factor
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    ABSTRACT: Pseudorabies virus (PRV), also known as suid herpesvirus, is the aetiological agent of Aujeszky's disease in swine. In other animals, except higher-order primates, PRV infection is often fatal. The mechanisms of PRV pathogenesis and immune modulation are largely unknown. PRV codes for 11 glycoproteins. Among them, glycoprotein G (gG) is the most abundant PRV protein found in the supernatant of PRV-infected cell cultures. PRV-gG has low amino acid sequence similarity with gG from other animal alphaherpesviruses and its function is unknown. gG from other animal alphaherpesviruses, with the exception of at least equine herpesvirus 4, binds to chemokines. We show here that PRV-gG binds to the human chemokine CL1 and several CC and CXC human chemokines with high affinity. Chemokine-binding activity can be detected in the supernatants of PRV-infected cell cultures, and insertional inactivation of the gene encoding gG from the PRV genome results in loss of chemokine-binding activity. Binding of PRV-gG to chemokines inhibits chemokine-mediated cell migration, suggesting a role for PRV-gG in immune evasion.
    Journal of General Virology 09/2009; 91(Pt 1):23-31. · 3.13 Impact Factor
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    ABSTRACT: Chemokines are small proteins that direct leukocyte trafficking under homeostatic and inflammatory conditions. We analyzed the differential expression of chemokines in distinct segments of the intestine and investigated the importance of chemokines for the distribution of leukocytes in the intestine during homeostatic and inflammatory conditions. We analyzed messenger RNA for all known chemokines in different segments of the gut by quantitative polymerase chain reaction. To study the effect of multiple-chemokine blockade in the gut, we generated transgenic mice that expressed the chemokine binding protein M3 in the intestine (V-M3 mice). We used flow cytometry to evaluate the changes in the numbers of leukocytes. We observed distinct chemokine expression profiles in the 6 segments of the gut. Some chemokines were expressed throughout the intestine (CCL28, CCL6, CXCL16, and CX3CL1), whereas others were expressed preferentially in the small (CCL25 and CCL5) or large intestine (CCL19, CCL21, and CXCL5). Expression of the chemokine blocker M3 in intestinal epithelial cells resulted in reduced numbers of B and T cells in Peyer's patches, reduced numbers of intraepithelial CD8alphabeta(+)/TCRalphabeta(+) and CD8alphaalpha(+)/TCRalphabeta(+) T cells, and reduced numbers of lamina propria CD8(+) T cells. Strikingly, M3 expression markedly reduced the number of eosinophils and macrophages in the small and large intestines. Dextran sulfate sodium treatment of control mice led to marked changes in the expression of chemokines and in the number of myeloid cells in the colon. These cellular changes were significantly attenuated in the presence of M3. Our study reveals a complex pattern of chemokine expression in the intestine and indicates that chemokines are critical for leukocyte accumulation in the intestine during homeostasis and inflammation.
    Gastroenterology 06/2009; 137(3):1006-18, 1018.e1-3. · 12.82 Impact Factor

Publication Stats

4k Citations
612.81 Total Impact Points

Institutions

  • 2007–2014
    • Centro De Biología Molecular Severo Ochoa
      Madrid, Madrid, Spain
  • 2008–2012
    • Spanish National Research Council
      • • Department of Virology and Microbiology
      • • Centro de Biología Molecular "Severo Ochoa"
      Madrid, Madrid, Spain
  • 1989–2012
    • Universidad Autónoma de Madrid
      • • Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM)
      • • Facultad de Ciencias
      Madrid, Madrid, Spain
  • 2009–2011
    • Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria
      • Centre for Research in Animal Health
      Madrid, Madrid, Spain
  • 2010
    • Cajal Institute
      Madrid, Madrid, Spain
  • 2000–2010
    • University of Cambridge
      • • Department of Medicine
      • • Department of Pathology
      Cambridge, England, United Kingdom
  • 2004–2009
    • Mount Sinai School of Medicine
      • • Immunology Institute
      • • Department of Medicine
      Manhattan, NY, United States
  • 2006
    • Trinity College Dublin
      Dublin, Leinster, Ireland
  • 1992–2002
    • University of Oxford
      • Sir William Dunn School of Pathology
      Oxford, ENG, United Kingdom