Host sphingomyelin increases West Nile virus infection in vivo
2016; Elsevier BV; Volume: 57; Issue: 3 Linguagem: Inglês
10.1194/jlr.m064212
ISSN1539-7262
AutoresMiguel A. Martín-Acebes, Enrique Gabandé‐Rodríguez, Ana M. García-Cabrero, Marina P. Sánchez, María Dolores Ledesma, Francisco Sobrino, Juan‐Carlos Sáiz,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoFlaviviruses, such as the dengue virus and the West Nile virus (WNV), are arthropod-borne viruses that represent a global health problem. The flavivirus lifecycle is intimately connected to cellular lipids. Among the lipids co-opted by flaviviruses, we have focused on SM, an important component of cellular membranes particularly enriched in the nervous system. After infection with the neurotropic WNV, mice deficient in acid sphingomyelinase (ASM), which accumulate high levels of SM in their tissues, displayed exacerbated infection. In addition, WNV multiplication was enhanced in cells from human patients with Niemann-Pick type A, a disease caused by a deficiency of ASM activity resulting in SM accumulation. Furthermore, the addition of SM to cultured cells also increased WNV infection, whereas treatment with pharmacological inhibitors of SM synthesis reduced WNV infection. Confocal microscopy analyses confirmed the association of SM with viral replication sites within infected cells. Our results unveil that SM metabolism regulates flavivirus infection in vivo and propose SM as a suitable target for antiviral design against WNV. Flaviviruses, such as the dengue virus and the West Nile virus (WNV), are arthropod-borne viruses that represent a global health problem. The flavivirus lifecycle is intimately connected to cellular lipids. Among the lipids co-opted by flaviviruses, we have focused on SM, an important component of cellular membranes particularly enriched in the nervous system. After infection with the neurotropic WNV, mice deficient in acid sphingomyelinase (ASM), which accumulate high levels of SM in their tissues, displayed exacerbated infection. In addition, WNV multiplication was enhanced in cells from human patients with Niemann-Pick type A, a disease caused by a deficiency of ASM activity resulting in SM accumulation. Furthermore, the addition of SM to cultured cells also increased WNV infection, whereas treatment with pharmacological inhibitors of SM synthesis reduced WNV infection. Confocal microscopy analyses confirmed the association of SM with viral replication sites within infected cells. Our results unveil that SM metabolism regulates flavivirus infection in vivo and propose SM as a suitable target for antiviral design against WNV. The flaviviruses comprise a group of arthropod-borne viruses that include important human and animal pathogens responsible for life-threatening diseases, such as dengue, yellow fever, Japanese encephalitis, or West Nile fever (1Gould E.A. Solomon T. Pathogenic flaviviruses.Lancet. 2008; 371: 500-509Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). The flavivirus, West Nile virus (WNV), is a neurotropic enveloped plus-strand RNA virus transmitted through the bite of mosquitoes. This virus is responsible for recurrent outbreaks of febrile illness and meningoencephalitis worldwide, accounting for hundreds of human deaths every year (2Martín-Acebes M.A. Saiz J.C. West Nile virus: a re-emerging pathogen revisited.World J. Virol. 2012; 1: 51-70Crossref PubMed Google Scholar). The WNV lifecycle is intimately associated to cellular lipids, and RNA replication and virion biogenesis are coupled into highly remodeled intracellular membranes (3Gillespie L.K. Hoenen A. Morgan G. Mackenzie J.M. The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex.J. Virol. 2010; 84: 10438-10447Crossref PubMed Scopus (277) Google Scholar, 4Apte-Sengupta S. Sirohi D. Kuhn R.J. Coupling of replication and assembly in flaviviruses.Curr. Opin. Virol. 2014; 9: 134-142Crossref PubMed Scopus (124) Google Scholar). 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Dengue virus-induced autophagy regulates lipid metabolism.Cell Host Microbe. 2010; 8: 422-432Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 12Perera R. Riley C. Isaac G. Hopf-Jannasch A.S. Moore R.J. Weitz K.W. Pasa-Tolic L. Metz T.O. Adamec J. Kuhn R.J. Dengue virus infection perturbs lipid homeostasis in infected mosquito cells.PLoS Pathog. 2012; 8: e1002584Crossref PubMed Scopus (226) Google Scholar). Among the cellular lipids co-opted by WNV, sphingolipids merit special attention because they are particularly enriched in the nervous system, a major target tissue during WNV infection (13Holthuis J.C. Pomorski T. Raggers R.J. Sprong H. Van Meer G. The organizing potential of sphingolipids in intracellular membrane transport.Physiol. Rev. 2001; 81: 1689-1723Crossref PubMed Scopus (254) Google Scholar, 14Ceccaldi P.E. Lucas M. Despres P. New insights on the neuropathology of West Nile virus.FEMS Microbiol. Lett. 2004; 233: 1-6Crossref PubMed Scopus (45) Google Scholar). Sphingolipids derive from sphingosine, a long-chain amino alcohol that is acylated with a long-chain fatty acid to give ceramide, which is in turn the central core of SM (15Gault C.R. Obeid L.M. Hannun Y.A. An overview of sphingolipid metabolism: from synthesis to breakdown.Adv. Exp. Med. Biol. 2010; 688: 1-23Crossref PubMed Scopus (665) Google Scholar). The sphingolipid content of biological membranes defines important physical and biological properties (16Lorizate M. Krausslich H.G. Role of lipids in virus replication.Cold Spring Harb. Perspect. Biol. 2011; 3: a004820Crossref PubMed Scopus (172) Google Scholar, 17Schneider-Schaulies J. Schneider-Schaulies S. Sphingolipids in viral infection.Biol. Chem. 2015; 396: 585-595Crossref PubMed Scopus (57) Google Scholar). Viruses can take advantage of these properties to develop specialized membrane sites for RNA replication and particle biogenesis (18Chukkapalli V. Heaton N.S. Randall G. Lipids at the interface of virus-host interactions.Curr. Opin. Microbiol. 2012; 15: 512-518Crossref PubMed Scopus (80) Google Scholar). Lipidomic analyses have shown an increase in the content of both ceramide and SM in flavivirus-infected cells (8Martín-Acebes M.A. Merino-Ramos T. Blázquez A.B. Casas J. Escribano-Romero E. Sobrino F. Saiz J.C. The composition of West Nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis.J. Virol. 2014; 88: 12041-12054Crossref PubMed Scopus (90) Google Scholar, 12Perera R. Riley C. Isaac G. Hopf-Jannasch A.S. Moore R.J. Weitz K.W. Pasa-Tolic L. Metz T.O. Adamec J. Kuhn R.J. Dengue virus infection perturbs lipid homeostasis in infected mosquito cells.PLoS Pathog. 2012; 8: e1002584Crossref PubMed Scopus (226) Google Scholar), and ceramide has specifically been associated with WNV replication and viral particle biogenesis (8Martín-Acebes M.A. Merino-Ramos T. Blázquez A.B. Casas J. Escribano-Romero E. Sobrino F. Saiz J.C. The composition of West Nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis.J. Virol. 2014; 88: 12041-12054Crossref PubMed Scopus (90) Google Scholar, 9Aktepe T.E. Pham H. Mackenzie J.M. Differential utilisation of ceramide during replication of the flaviviruses West Nile and dengue virus.Virology. 2015; 484: 241-250Crossref PubMed Scopus (41) Google Scholar). Regarding SM, it is enriched in membranes from the replication complex of viruses phylogenetically related to WNV, such as dengue virus and hepatitis C virus (12Perera R. Riley C. Isaac G. Hopf-Jannasch A.S. Moore R.J. Weitz K.W. Pasa-Tolic L. Metz T.O. Adamec J. Kuhn R.J. Dengue virus infection perturbs lipid homeostasis in infected mosquito cells.PLoS Pathog. 2012; 8: e1002584Crossref PubMed Scopus (226) Google Scholar, 19Hirata Y. Ikeda K. Sudoh M. Tokunaga Y. Suzuki A. Weng L. Ohta M. Tobita Y. Okano K. Ozeki K. et al.Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.PLoS Pathog. 2012; 8: e1002860Crossref PubMed Scopus (64) Google Scholar). For WNV, an enrichment of SM in the viral envelope relative to total cellular membranes has been described (8Martín-Acebes M.A. Merino-Ramos T. Blázquez A.B. Casas J. Escribano-Romero E. Sobrino F. Saiz J.C. The composition of West Nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis.J. Virol. 2014; 88: 12041-12054Crossref PubMed Scopus (90) Google Scholar). Despite this observation, the contribution of SM, the most abundant complex sphingolipid in mammalian cells (15Gault C.R. Obeid L.M. Hannun Y.A. An overview of sphingolipid metabolism: from synthesis to breakdown.Adv. Exp. Med. Biol. 2010; 688: 1-23Crossref PubMed Scopus (665) Google Scholar), has not been addressed in detail in WNV infection. The levels of SM are tightly controlled by cellular sphingomyelinases (15Gault C.R. Obeid L.M. Hannun Y.A. An overview of sphingolipid metabolism: from synthesis to breakdown.Adv. Exp. Med. Biol. 2010; 688: 1-23Crossref PubMed Scopus (665) Google Scholar). Consistently, defects in the activity of acid sphingomyelinase (ASM) result in the accumulation of SM, causing a sphingolipidosis termed Niemann-Pick disease type A (NPA) (20Brady R.O. Kanfer J.N. Mock M.B. Fredrickson D.S. The metabolism of sphingomyelin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease.Proc. Natl. Acad. Sci. USA. 1966; 55: 366-369Crossref PubMed Scopus (333) Google Scholar). Because ASM is a ubiquitous enzyme, SM accumulates in all kinds of cells in NPA patients leading to peripheral symptoms, especially evident in enlarged liver and spleen. However, the most affected organ is the brain, resulting in severe cognitive deficits, neurodegeneration, and early death. Mice lacking ASM [ASM knockout mice (ASMko)] reproduce NPA and accumulate SM, but not ceramide, in their tissues (21Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar, 22Ledesma M.D. Prinetti A. Sonnino S. Schuchman E.H. Brain pathology in Niemann-Pick disease type A: insights from the acid sphingomyelinase knockout mice.J. Neurochem. 2011; 116: 779-788Crossref PubMed Scopus (56) Google Scholar, 23Galvan C. Camoletto P.G. Cristofani F. Van Veldhoven P.P. Ledesma M.D. Anomalous surface distribution of glycosyl phosphatidyl inositol-anchored proteins in neurons lacking acid sphingomyelinase.Mol. Biol. Cell. 2008; 19: 509-522Crossref PubMed Scopus (43) Google Scholar). Accumulation of SM in the ASMko mouse brain cells is progressive, reaching a 5-fold increase compared with wild-type (wt) mice (20Brady R.O. Kanfer J.N. Mock M.B. Fredrickson D.S. The metabolism of sphingomyelin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease.Proc. Natl. Acad. Sci. USA. 1966; 55: 366-369Crossref PubMed Scopus (333) Google Scholar, 21Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar, 22Ledesma M.D. Prinetti A. Sonnino S. Schuchman E.H. Brain pathology in Niemann-Pick disease type A: insights from the acid sphingomyelinase knockout mice.J. Neurochem. 2011; 116: 779-788Crossref PubMed Scopus (56) Google Scholar). This increase is similar to that reported in the cerebral cortex of NPA patients (24Rodriguez-Lafrasse C. Vanier M.T. Sphingosylphosphorylcholine in Niemann-Pick disease brain: accumulation in type A but not in type B.Neurochem. Res. 1999; 24: 199-205Crossref PubMed Scopus (85) Google Scholar). ASMko mice and primary cell cultures from NPA patients have provided useful models to study the role of SM in viral infection (25Ng C.G. Griffin D.E. Acid sphingomyelinase deficiency increases susceptibility to fatal alphavirus encephalomyelitis.J. Virol. 2006; 80: 10989-10999Crossref PubMed Scopus (28) Google Scholar, 26Ng C.G. Coppens I. Govindarajan D. Pisciotta J. Shulaev V. Griffin D.E. Effect of host cell lipid metabolism on alphavirus replication, virion morphogenesis, and infectivity.Proc. Natl. Acad. Sci. USA. 2008; 105: 16326-16331Crossref PubMed Scopus (32) Google Scholar). Using these models, we here show, for the first time, that SM levels modulate WNV infection in vitro and in vivo, thus identifying this sphingolipid as a key cellular factor for WNV replication. All virus manipulations were performed in our biosafety level 3 (BSL-3) facilities. The origin and passage history of WNV strain NY99 has been described (27Merino-Ramos T. Blazquez A.B. Escribano-Romero E. Canas-Arranz R. Sobrino F. Saiz J.C. Martin-Acebes M.A. Protection of a single dose West Nile virus recombinant subviral particle vaccine against lineage 1 or 2 strains and analysis of the cross-reactivity with Usutu virus.PLoS One. 2014; 9: e108056Crossref PubMed Scopus (33) Google Scholar, 28Martín-Acebes M.A. Saiz J.C. A West Nile virus mutant with increased resistance to acid-induced inactivation.J. Gen. Virol. 2011; 92: 831-840Crossref PubMed Scopus (36) Google Scholar). Vero 76 cells, clone E6 (ATTC CRL-1586), were used for amplification and titration of viruses. Primary human skin fibroblasts from unaffected individuals (AG07310 and AG07471) and NPA patients (GM13205 and GM16195) were from Coriell Institute for Medical Research (Camden, NJ). GM13205 carried a deletion of a single C in exon 2 at codon 330 of the SMPD1 gene (which encodes human ASM) resulting in a frameshift leading to the formation of a premature stop at codon 382. GM16195 is homozygous for a T to C transition at nucleotide 905 of the SPMD1 gene, resulting in a Leu to Pro substitution at codon 302. Cells were cultured (37°C, 5% CO2) in DMEM supplemented with 2 mM glutamine, penicillin-streptomycin, and 5% FBS for Vero cells or 10% FBS for fibroblasts. For infections in liquid medium, the viral inoculum was incubated with cell monolayers for 1 h, and then the inoculum was removed and fresh medium containing 1% FBS was added. This time point was considered 1 h postinfection (p.i.). Virus titrations were performed by standard plaque assay on Vero cells (28Martín-Acebes M.A. Saiz J.C. A West Nile virus mutant with increased resistance to acid-induced inactivation.J. Gen. Virol. 2011; 92: 831-840Crossref PubMed Scopus (36) Google Scholar). The multiplicity of infection (MOI) used in each experiment was expressed as the number of plaque-forming units (PFUs) per cell and is indicated in the corresponding figure legend. Age- and sex-matched 5-month-old wt (C57BL/6) or ASMko (21Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar) mice were used. A breeding colony was established from a couple of ASM heterozygous C57BL/6 mice kindly donated by Prof. E. H. Schuchman (Mount Sinai School of Medicine, New York) and genotyped as described (29Gabandé-Rodríguez E. Boya P. Labrador V. Dotti C.G. Ledesma M.D. High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann-Pick disease type A.Cell Death Differ. 2014; 21: 864-875Crossref PubMed Scopus (111) Google Scholar). Mice were intraperitoneally inoculated with 104 PFU per mouse of WNV and monitored daily for signs of infection up to 20 days p.i. Animals were kept with ad libitum access to food and water and those exhibiting clear signs of disease were anesthetized and euthanized, as were all surviving mice, at the end of the experiment. All animals were handled in strict accordance with the guidelines of the European Community 86/609/CEE at the BSL-3 animal facilities of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the CBMSO. The protocols were approved by the Committee on Ethics of Animal Experimentation of INIA (permit number 2015-004E and PROEX 05/14) and the Animal Welfare Committee of Centro de Biología Molecular "Severo Ochoa" (CBMSO) (permit numbers CEEA-CBMSO-22I149 and PROEX 034/15). Organotypic slice cultures of the hippocampus were prepared from 5-month-old wt or ASMko mice, as previously described (30Jurado-Arjona J. Goni-Oliver P. Rodriguez-Prada L. Engel T. Henshall D.C. Avila J. Hernandez F. Excitotoxicity induced by kainic acid provokes glycogen synthase kinase-3 truncation in the hippocampus.Brain Res. 2015; 1611: 84-92Crossref PubMed Scopus (3) Google Scholar). Six slices (400 μm thick each) were placed per insert and cultured for 24 h at 37°C in a 5% CO2 atmosphere prior to infection. Each slice was individually infected with 105 PFU of WNV. Slices were harvested 24 h p.i. and homogenized in PBS for quantitative RT-PCR analysis. SM (Santa Cruz Biotechnology, Dallas, TX) was dissolved in ethanol and added to the cells 24 h before infection (29Gabandé-Rodríguez E. Boya P. Labrador V. Dotti C.G. Ledesma M.D. High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann-Pick disease type A.Cell Death Differ. 2014; 21: 864-875Crossref PubMed Scopus (111) Google Scholar). D609 (Sigma, St. Louis, MO), SPK-601 (LMV-601; Eurodiagnostico, Madrid, Spain), and MS-209 (dofequidar fumarate; Sigma) were dissolved in DMSO and added to infected cultures 1 h p.i. Control cells were treated in parallel with the same amount of drug vehicle. Biochemical analysis of SM in brain extracts or cultured cells containing the same amount of protein was performed using an enzymatic fluorescence assay. Briefly, lipid extracts were dried in the presence of detergent (Thesit), and SM was subsequently converted into choline by means of sphingomyelinase and alkaline phosphatase, and coupled to the production of fluorescence with choline oxidase, peroxidase, and homovanillic acid, as modified from Hojjati and Jiang (31Hojjati M.R. Jiang X.C. Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine.J. Lipid Res. 2006; 47: 673-676Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Right brain hemispheres from four randomly selected mice per condition were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Serial sagittal sections (3 μm thick) were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was inactivated by incubation in 1.5% H2O2 in methanol for 20 min. Sections were placed in blocking buffer (1% BSA, 5% FBS, 2% Triton X-100 in PBS) for 1 h and incubated overnight at 4°C with an anti-WNV E glycoprotein (3.67G) mouse monoclonal antibody (Millipore, Temecula, CA) in blocking buffer. Subsequently, sections were incubated with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) and stained using the Elite Vectastain ABC kit (Vector). Immunoreactivity was developed with diaminobenzidine (Dako, Glostrup, Denmark), which yields a brownish precipitate, and sections were counterstained with hematoxylin. Contiguous brain sections were processed in parallel without primary antibody in order to determine nonspecific staining. Images were acquired using a Leica DM LB microscope equipped with a HCX PL 40×/0.75 objective and a digital camera, Leica DC 100. Immunofluorescence was performed as described (5Martín-Acebes M.A. Blázquez A.B. Jiménez de Oya N. Escribano-Romero E. Saiz J.C. West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.PLoS One. 2011; 6: e24970Crossref PubMed Scopus (113) Google Scholar). Plasma membrane staining with lysenin, which specifically binds to SM, was accomplished as described (23Galvan C. Camoletto P.G. Cristofani F. Van Veldhoven P.P. Ledesma M.D. Anomalous surface distribution of glycosyl phosphatidyl inositol-anchored proteins in neurons lacking acid sphingomyelinase.Mol. Biol. Cell. 2008; 19: 509-522Crossref PubMed Scopus (43) Google Scholar). For intracellular SM detection, lysenin staining was performed in a similar manner, except that cells were permeabilized with 50 μg/ml digitonin after fixation (32Makino A. Abe M. Murate M. Inaba T. Yilmaz N. Hullin-Matsuda F. Kishimoto T. Schieber N.L. Taguchi T. Arai H. et al.Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis.FASEB J. 2015; 29: 477-493Crossref PubMed Scopus (64) Google Scholar). Lysenin (Peptanova, Sandhausen, Germany) was detected using a specific rabbit antiserum (Peptanova). Double-strand RNA (dsRNA) and WNV E protein were detected using mouse monoclonal antibodies J2 (English and Scientific Consulting, Hungary) and 3.67G, respectively. Actin microfilaments were visualized with TRITC-labeled phalloidin (Sigma). The ER was visualized by transfection with plasmid IgLdR1kdel (33Wölk B. Büchele B. Moradpour D. Rice C.M. A dynamic view of hepatitis C virus replication complexes.J. Virol. 2008; 82: 10519-10531Crossref PubMed Scopus (117) Google Scholar) encoding an ER-targeted red fluorescent protein (5Martín-Acebes M.A. Blázquez A.B. Jiménez de Oya N. Escribano-Romero E. Saiz J.C. West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.PLoS One. 2011; 6: e24970Crossref PubMed Scopus (113) Google Scholar). Appropriate secondary antibodies labeled with Alexa Fluor 488, 594, or 647 were from Invitrogen. For plasma membrane lysenin staining, cells were observed using an Axioskop (Zeiss, Oberkochen, Germany) epifluorescence microscope with a Plan-Neofluar Ph3 40×/1.3 oil immersion objective. Images were acquired with a monochrome Coolsnap FX camera (Roper Scientific, Tucson, AZ) and fluorescence was quantified using ImageJ software (http://imagej.nih.gov/ij/). In the case of confocal microscopy analyses, cells were observed using a Leica TCS SPE confocal laser scanning microscope using an HCX PL APO 63×/1.4 oil immersion objective. Optical slice thickness for all confocal images displayed was of 1 airy unit. For quantification of cell-associated viral RNA, infected cell monolayers were washed, supernatants were replaced by fresh medium, and the cells were subjected to three freeze-thaw cycles. In the case of animal samples or organotypic slice cultures, tissues were homogenized in PBS using TissueLyser II equipment (Quiagen, Venlo, The Netherlands). Viral RNA was extracted from samples with Speedtools RNA virus extraction kit (Biotools, Madrid, Spain). Positive-strand viral RNA was quantified by real-time RT-PCR (34Lanciotti R.S. Kerst A.J. Nasci R.S. Godsey M.S. Mitchell C.J. Savage H.M. Komar N. Panella N.A. Allen B.C. Volpe K.E. et al.Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay.J. Clin. Microbiol. 2000; 38: 4066-4071Crossref PubMed Google Scholar) as genomic equivalents to PFUs by comparison with RNA extracted from previously titrated samples (35Blázquez A.B. Sáiz J.C. West Nile virus (WNV) transmission routes in the murine model: intrauterine, by breastfeeding and after cannibal ingestion.Virus Res. 2010; 151: 240-243Crossref PubMed Scopus (32) Google Scholar). ANOVA, applying Bonferroni's correction, was performed with the statistical package, SPSS 15. Nonparametric data were analyzed using the Mann-Whitney U test (GraphPad Prism 2.01). Kaplan-Meier survival curves were analyzed by a log-rank test using GraphPad Prism 2.01. Data are presented as mean ± SD. Differences with P values of <0.05 were considered statistically significant. Asterisks in the figures indicate P values: *P < 0.05; **P < 0.005. SM accumulation is an age-related process in ASMko mice that, close to the end of their life (7 months of age), exhibits a 5-fold higher level of SM in brain, while cholesterol and ceramide content remains unchanged with respect to wt mice (22Ledesma M.D. Prinetti A. Sonnino S. Schuchman E.H. Brain pathology in Niemann-Pick disease type A: insights from the acid sphingomyelinase knockout mice.J. Neurochem. 2011; 116: 779-788Crossref PubMed Scopus (56) Google Scholar, 23Galvan C. Camoletto P.G. Cristofani F. Van Veldhoven P.P. Ledesma M.D. Anomalous surface distribution of glycosyl phosphatidyl inositol-anchored proteins in neurons lacking acid sphingomyelinase.Mol. Biol. Cell. 2008; 19: 509-522Crossref PubMed Scopus (43) Google Scholar, 36Scandroglio F. Venkata J.K. Loberto N. Prioni S. Schuchman E.H. Chigorno V. Prinetti A. Sonnino S. Lipid content of brain, brain membrane lipid domains, and neurons from acid sphingomyelinase deficient mice.J. Neurochem. 2008; 107: 329-338Crossref PubMed Scopus (44) Google Scholar). In our experiments, 5-month-old ASMko mice that exhibited a 2.7-fold increase in their brain SM content relative to wt mice were used (218 ± 19 nmol/mg and 81 ± 5 nmol/mg of protein, respectively) (Fig. 1A). To determine whether the alteration of SM levels could modulate WNV infection in vivo, we first infected ASMko mice (21Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar) with a highly neurovirulent WNV NY99 strain (28Martín-Acebes M.A. Saiz J.C. A West Nile virus mutant with increased resistance to acid-induced inactivation.J. Gen. Virol. 2011; 92: 831-840Crossref PubMed Scopus (36) Google Scholar) derived from the virus causing the outbreak of encephalitis in the Northeastern United States in 1999 (37Lanciotti R.S. Roehrig J.T. Deubel V. Smith J. Parker M. Steele K. Crise B. Volpe K.E. Crabtree M.B. Scherret J.H. et al.Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.Science. 1999; 286: 2333-2337Crossref PubMed Scopus (1277) Google Scholar). ASMko mice were significantly more susceptible to WNV-induced lethality than wt mice when challenged with 104 PFU by intraperitoneal inoculation (Fig. 1B). Quantitative RT-PCR revealed a significantly higher viral load in the brains of dead ASMko mice than in those of dead wt mice (Fig. 1C), indicating that WNV replicated to higher levels in ASMko mice. To test this hypothesis, the viral load in several neural and peripheral tissues was analyzed in randomly chosen animals at 6 days p.i. The values found were significantly higher (by several orders of magnitude) in the brain of ASMko than in wt mice (Fig. 1D). In addition, the amount of viral RNA in peripheral tissues (liver, spleen, kidney, and heart) of ASMko mice was also higher than in wt mice (Fig. 1D). These results confirm that WNV infection is exacerbated in ASMko mice. WNV-infected cells were analyzed by immunohistochemistry in brain sections from wt and ASMko mice at 6 days p.i. using a monoclonal antibody that recognizes the WNV E glycoprotein. Our analysis was focused on the cortex and hippocampus, because both areas are important targets for WNV infection (38Hunsperger E.A. Ro
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