Vol. 38, No. 6809 Biol. Pharm. Bull. 38, 809–816 (2015)

Review Role of Sulfatide in Influenza A Virus Replication Tadanobu Takahashi and Takashi Suzuki* Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka; Shizuoka 422–8526, Japan. Received February 6, 2015; accepted March 22, 2015 Sulfatide is a 3-O-sulfated galactosylceramide that is abundantly expressed in the gastrointestinal tract, kidney, trachea, and particularly the central nervous system. Cellular sulfatide is mainly localized in the Golgi apparatus, cellular membrane, and lysosomes in cytosol. Since our earlier report showed that the influenza A virus specifically binds to sulfatide, we have investigated the roles of sulfatide in the influenza A virus lifecycle. The viral binding is independent of sialic acids, which function as virus receptors in virus attachment to the host cell surface. Sulfatide is recognized by the ectodomain of the viral envelope glycoprotein hemagglutinin (HA). Nascent HA is transported on the surface membrane of infected cells. The binding of HA with sulfatide on the cell surface induces apoptosis through potential loss of the mitochondrial membrane and nuclear translocation of apoptosis-inducing factor in mitochondria, where PB1-F2 peptide from the viral gene is accumulated. In the nucleus of infected cells, viral ribonucleoprotein (vRNP) complexes are formed from viral RNA genomes, viral nucleoprotein, and viral RNA polymerase subunits, and these complexes are selectively exported into cytosol through the nuclear membrane. The apoptosis significantly enhances the nuclear export of vRNP complexes, resulting in efficient formation of progeny viruses and facilitation of virus replication. At that time, activation of the Raf/mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK) pathway through sulfatide is associated with virus replication. Our studies have demonstrated that sulfatide is not a viral receptor for virus infection, and that the binding of HA with sulfatide functions as an initiation switch for the formation of progeny viruses. Key words  influenza virus; sulfatide; hemagglutinin; virus replication; apoptosis

1.

INTRODUCTION

Influenza  A  virus  infects  various  hosts  such  as  humans,  birds  (aquatic  birds  and  ground  birds),  pigs,  and  equines.  Influenza  A  virus  possesses  two  major  envelope  glycoproteins,  hemagglutinin  (HA)  and  neuraminidase  (NA).  As  of  2014,  antigenicities  of  the  viral  envelope  glycoproteins  have  been  classified  into  18  for  HA  and  11  for  NA.  Influenza  A  virus  initiates cell entry after attachment to the host cellular surface through  HA  binding  to  sialic  acid  on  the  terminals  of  glycoconjugates. Influenza A virus recognizes a difference of sialyl  linkages  to  galactose,  α2,3 and α2,6.  HA  of  human  virus  shows preferential binding to α2,6-linked sialic acids, whereas  HA  of  avian  virus  shows  preferential  binding  to  α2,3-linked sialic  acids.  The  difference  in  such  HA  binding  specificities  is believed to determine the viral host such as humans and birds.1)  Under  an  endocytic  pathway  of  cell  entry,  influenza  A  virus  is  exposed  to  an  acidic  condition  in  endosomes.  HA  exerts  membrane  fusion  activity  by  conformation  change  and  invokes  fusion  between  the  viral  membrane  and  host  cell  membrane.  Complexes  including  viral  RNA  genes  and  some  viral  internal  proteins  are  injected  into  the  cytosol  and  transported to the nucleus.2)  In  the  nucleus,  viral  RNA  genes  are  replicated  and  viral  ribonucleoprotein  (vRNP)  complexes  are  formed  from  viral  RNA  genes,  viral  nucleoprotein  (NP),  and  three  viral  RNA  polymerase  subunits  (PB1,  PB2,  and  PA).  vRNP  complexes  are  selectively  transported  into  the  cytosol  from the nucleus. At that time, two nascent viral internal proteins,  M1  and  NS2,  are  likely  to  help  nuclear  export  of  vRNP  complexes. In the cytosol, vRNP complexes are transported to  the  cellular  membrane.  Nascent  viral  envelope  glycoproteins, 

HA and NA, are selectively transported to the cellular surface  membrane  through  N-glycosylation  in  the  Golgi  apparatus.  In  the  cellular  surface  membrane,  vRNP  complexes,  the  viral  envelope and internal proteins, and the host cellular membrane,  are  selectively  packaged  into  progeny  viruses.3)  NA  shows  sialidase  activity  that  removes  the  terminal  sialic  acid  from  sugar  chains  of  glycoconjugates.  The  sialidase  activity  facilitates  release  of  progeny  virus  from  the  cellular  surface  by removal of sialic acids on the host cellular surface. The sialidase  activity  also  prevents  aggregation  among  progeny  viruses by removal of sialic acids on the viral envelope proteins. The sialidase activity has been suggested to be involved  in enhancement of virus infection and replication at the early stage  of  the  virus  lifecycle  such  as  virus  attachment  to  the  host cellular surface and cell entry process, but the mechanism remains unknown.4–7) We  have  found  that  influenza  A  virus  also  showed  strong  binding to a non-sialo glycoconjugate, sulfatide8) (Fig. 1). Sulfatide is a 3-O-sulfated  galactosylceramide  (GalCer),  which  is  the  first  sulfoglycolipid  isolated  from  the  human  brain  in  1884.9) Sulfatide is abundantly expressed in the gastrointestinal  tract, islet of Langerhans, kidney, trachea, and particularly the  central  nervous  system.  Abundant  expression  of  sulfatide  is  detected in many human cancer tissues, such as human serous papillary ovarian carcinoma tissues,10)  human  ovarian  malignant  and  benign  cancer  tissues,11) primary human colorectal cancer tissues,12) human renal carcinoma tissues,13) human gastric cancer tissues,14) and primary human lung adenocarcinoma  tissues.15)  Cellular  sulfatide  is  mainly  localized  in  the  Golgi  apparatus,16) cellular membrane, and lysosomes in the cytosol.17) Sulfatide is synthesized by sulfation of GalCer, which is 

 * To whom correspondence should be addressed.  e-mail: [email protected] © 2015 The Pharmaceutical Society of Japan

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Fig.  1.  Binding of Influenza A Virus to Sulfatide (A)  Thin-layer  chromatography  virus  binding  assay.  Each  lipid  (lanes  1  to  2)  was  developed  on  a  silica  gel  thin-layer  plate.  After  blocking,  the  plate  was  incubated  with  human  influenza  A  virus  (A/Memphis/1/1971-A/Bellamy/42  H3N1).  Virus  bound  to  glycolipids  was  immunostained  with  rabbit  anti-influenza  A  virus  antibody  and  horseradish peroxidase-labeled Protein A. Location of each lipid was confirmed by orcinol-H 2SO4 staining. A band in lane 2 indicates binding of the virus to sulfatide. (B)  Solid-phase virus binding assay. Each lipid (red closed circle, sulfatide; empty circle, sialoglycolipid GD1a) was immobilized on a 96-well plate. After blocking, the plate  was incubated with each influenza A virus (human virus A/Memphis/1/1971 H3N2; equine virus A/equine/TN/5/1986 H3N8; avian virus A/duck/Ukr/1/1963 H3N8; swine  virus  A/swine/Colorado/1977  H3N2).  Viruses  bound  to  glycolipids  were  immunostained  with  rabbit  anti-influenza  A  virus  antibody  and  horseradish  peroxidase-labeled  Protein A.

catalyzed by 3′-phosphoadenosine-5′-phosphosulfate : cerebroside sulfotransferase (CST) (EC 2.8.2.11).18,19) CST has been reported to be localized as a homodimeric protein in the late Golgi  apparatus.20)  The  sulfatide  precursor,  GalCer,  is  synthesized by addition of galactose to ceramide, which is catalyzed  by uridine 5′-diphosphate  (UDP)-galactose : ceramide  galactosyltransferase  (CGT)  (EC  2.4.1.45).21)  The  sulfate  group  of  sulfatide  is  specifically  removed  by  arylsulfatase  A  (ASA)  (EC 3.1.6.8) in lysosomes. Enzymatic activity of lysosomal ASA  requires  saposin  B  (SapB),  which  extracts  sulfatide  from lipid membranes and thereby enables accessibility of ASA  to  sulfatide22)  (Fig.  2).  An  ASA-independent  pathway  of  sulfatide  has  been  suggested  in  a  neuroblastoma  cell  line,  which directly generates ceramide from sulfatide without prior  desulfation in lysosomes.16) Deficiency of ASA activity or mutations  in  the  gene  coding  for  SapB  lead  to  the  accumulation  of sulfatide in lysosomes, resulting in a demyelinating disease  called metachromatic leukodystrophy (MLD).23) Sulfatide has been  reported  to  be  involved  in  many  biological  activities  including  the  nervous  system,  cancer,  insulin  secretion  in  the  islet  of  Langerhans,  hemostasis/thrombosis,  immune  system,  and microbial infection.24)  For  viruses  other  than  influenza  A  virus,  roles  of  sulfatide  have  been  shown  or  suggested  in  infection  and  replication  of  human  immunodeficiency  virus-1  (HIV-1),25–31) Vaccinia virus,32) hepatitis C virus,33) and human parainfluenza  virus  type  3.34) The function of sulfatide as a  coreceptor  of  HIV-1  has  been  discussed  in  many  reports.  However,  the  function  has  remained  unclear.  Sulfatide  and  compounds based on the sulfatide structure have been proposed as inhibitors of HIV-1.29,30,35)

Fig.  2.  Metabolism of Sulfatide Cer is converted to GalCer by galactose addition of CGT. GalCer is converted to  sulfatide by 3-O-sulfation of CST. The sulfate group of sulfatide was degraded and  converted to GalCer by ASA in the presence of SapB.

We have investigated roles of sulfatide in influenza A virus  infection  and  replication.  Influenza  A  viruses  bind  to  sulfatide,  regardless  of  various  viral  hosts  and  viral  antigenicities  (subtypes)  (Fig.  1B).  Sulfatide  is  necessary  for  the  efficient  formation  of  progeny  virus  in  infected  cells.  The  mechanism  begins  with  binding  of  HA  with  sulfatide  on  the  host  cellular  surface,  which  enhances  nuclear  export  of  vRNP  complexes  through  apoptosis  signaling.  Here,  we  review  the  roles  of  sul-

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fatide in influenza A virus replication from our studies.

2.  SULFATIDE  ENHANCES  REPLICATION  OF  INFLUENZA A VIRUS To evaluate the contribution of sulfatide to infection and replication  of  influenza  A  virus,  we  generated  sulfatideenriched  cells  from  sulfatide-defective  African  green  monkey  kidney  COS-7  cells  because  of  no  GalCer  expression,36) by stable  expression  of  dog  CGT  and  CST  genes  from  Madin– Darby  canine  kidney  (MDCK)  cells,  which  are  usually  used  for  replication  of  and  experiments  on  influenza  A  virus  and  express  abundant  sulfatide.  Replication  of  influenza  A  virus  (A/WSN/1933  H1N1)  was  strikingly  enhanced  in  sulfatideenriched cells compared to that in parent cells (Fig. 3). Transfection  of  the  human  ASA  gene  and  RNA  interfering  against 

CST  mRNA  in  MDCK  cells  decreased  sulfatide  expression,  resulting  in  reduction  of  virus  replication.  ASA  activity  requires  SapB.  Since  transfection  of  the  ASA  gene  produced  high  sulfatase  activity,  it  is  thought  that  SapB  expression  in  MDCK  cells  is  sufficient  for  showing  ASA  activity  in  ASAexpressed  cells.  The  remarkable  virus  replication,  which  was  induced  by  sulfatide  expression,  resulted  from  enhanced  production of progeny virus in the cells. Mouse  anti-sulfatide  monoclonal  antibody  (GS-5)  suppressed  virus  replication  and  production  of  progeny  virus  in  virus-infected cells but did not suppress virus infection. The inhibitory  effect  of  the  antibody  suggested  that  binding  between  the  virus  and  sulfatide  on  the  host  cellular  surface  was  important for production of progeny virus. The enhanced virus  replication in sulfatide-enriched cells and the suppressed virus replication by GS-5 were also confirmed by using human influ-

Fig.  3.  Sulfatide Expression Enhances Influenza A Virus Replication Sulfatide-enriched cells were generated by transfection of the parent COS-7 cells with both dog CST and CGT genes and cloning of cells stably expressing both genes.  The  cells  were  infected  with  influenza  A  virus  and  incubated  in  the  presence  of  acetylated  trypsin  (required  for  virus  replication  through  activation  of  HA  fusion  activity) at 37°C for 24 h. The infected cells were stained with mouse anti-viral NP monoclonal antibody. The infected cells (blue) were observed under an optical microscope  at a magnification of 40 (upper panel) and in a well (middle panel). Sulfatide in the cells was stained with mouse anti-sulfatide monoclonal antibody and observed with a  confocal microscope (lower panel). Sulfatide and nuclei are colored in green and blue, respectively. Scale bar=50 µm.

Fig.  4.  Sulfatide Knockdown Suppresses Nuclear Export of vRNP Complexes Sulfatide-enriched  COS-7  (SulCOS1)  cells  were  transfected  with  an  RNA  interfering  plasmid  vector  against  CST  mRNA  and  maintained  for  3 d.  At  7 h  after  infection  with influenza A virus (A/Memphis/1/1971 H3N2), the cells were fixed with cold methanol. The nucleus (blue), sulfatide (green), and vRNP complexes (red) were stained  with  4′,6′-diamidino-2-phenylindole  dihydrochloride  (DAPI),  mouse  anti-sulfatide  monoclonal  antibody  (GS-5),  and  mouse  anti-NP  monoclonal  antibody,  respectively.  Since  vRNP  complexes  contain  viral  NP  in  addition  to  viral  genome  RNA  and  viral  RNA  polymerase  subunits,  they  are  detected  by  anti-NP  antibody.  Arrows  indicate  suppression of nuclear export of vRNP complexes in sulfatide-knockdown cells. Scale bar=20 µm.

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enza  A  virus  (A/Memphis/1/1971  H3N2)  and  avian  influenza  A virus (A/dcuk/Hong Kong/313/4/1978 H5N3), indicating that  the effect of sulfatide was not dependent on antigenicities (subtypes)  of  the  viral  envelope  glycoproteins  and  the  viral  hosts.  When  mice  were  intranasally  infected  with  a  lethal  amount  of  mouse-adapted  influenza  A  virus  (A/WSN/1933  H1N1),  GS-5  was  administered  intranasally  once  daily  for  5 d  beginning  1 d  preinfection  (the  protocols  were  pre-approved  by  the  Animal  Ethical  Committee  of  the  University  of  Shizuoka).  GS-5  administration improved body weight reduction and survival rate  in the infected mice.37)  Taken  together,  these  results  demonstrated that sulfatide plays a significant role in virus replication  and progeny virus formation of influenza A virus.

3.  VIRAL  HA  IS  A  BINDING  MOLECULE  OF  SULFATIDE We  tried  to  identify  molecules  of  influenza  A  virus  showing  sulfatide  binding  ability.  Since  the  viral  envelope  glycoproteins,  HA  and  NA,  were  predicted  to  be  sulfatidebinding  molecules,  we  investigated  the  inhibitory  effects  of  anti-influenza  A  virus  (A/Memphis/1/1971  H3N2)  HA  and  NA  monoclonal  antibodies  on  sulfatide  binding.  Anti-H3  HA  monoclonal  antibody  (2E10)  inhibited  sulfatide  binding  of  the  virus, but anti-N2 NA monoclonal antibody (SI-4) and anti-H3  HA  monoclonal  antibody  (1F8)  did  not.  2E10  also  inhibited  sialic  acid  binding  of  the  virus,  but  1F8  did  not.  The  results  suggested  that  the  sulfatide  binding  site  was  near  the  sialic  acid  binding  site  in  HA.  To  prove  that  HA  was  a  sulfatidebinding  molecule,  we  generated  recombinant  whole  HA  of  avian  influenza  A  virus  (A/Hong  Kong/313/4/1978  H5N3)  by  using  a  baculovirus  protein  expression  system.  The  purified  whole  HA  with  a  his-tag  in  the  C-terminal  region  maintained  antigenicity  (binding  of  a  specific  monoclonal  antibody)  and  sialic  acid  binding  ability  similar  to  those  of  the  parent  virus.  The  purified  whole  HA  also  showed  sulfatide  binding  ability.  Anti-H5  HA  monoclonal  antibody  (1H10)  inhibited  sulfatide  binding of the whole HA, but anti-H3 HA 2E10 did not. 1H10  also inhibited sialic acid binding of the whole HA, supporting  a  suggestion  that  the  sulfatide  binding  site  is  near  the  sialic  acid  binding  site  in  HA.  These  results  demonstrated  that  HA  is a sulfatide-binding molecule of influenza A virus.38) The  whole  HA  contains  a  transmembrane  region  because  of  the  membrane  glycoprotein.  Since  the  whole  HA  was  purified  from  HA-expressing  insect  cells,  the  purified  HA  might  contain  a  small  amount  of  cellular  proteins.  A  detergent,  3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)  was  continuously  needed  because  of  solubilization  of  the  whole  HA.  The  presence  of  CHAPS  may  have  an  inappropriate  effect  on  some  experimental  assays.  Therefore,  we  generated  recombinant  secreted  HA  of  avian  influenza  A  virus (A/Hong Kong/313/4/1978 H5N3) by using a baculovirus  protein expression system. The secreted HA was generated by  deletion of the cytoplasmic tail and transmembrane region and  replacement  of  the  N-terminal  signal  peptide  to  a  secretion  signal and a his-tag including a linker peptide. High solubility  of  the  secreted  HA  did  not  need  a  detergent  at  all.  Furthermore, since the secreted HA was purified from serum-free supernatant of HA-secreted insect cells, we were able to obtain a  high yield of the purified HA containing few cellular proteins.  The  purified  secreted  HA  also  maintained  antigenicity,  sialic 

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acid  binding  ability,  and  sulfatide  binding  ability  similar  to  those of the whole HA and the virus. We measured dissociation constant (Kd),  binding  rate  constant (Kon), and dissociation rate constant (Koff ) for sialic acid binding  and  sulfatide  binding  of  the  secreted  HA  by  using  a quartz crystal microbalance (QCM). The Kd value, Kon value, and Koff  value  were  1.44×10−8 (M),  1.41×104 (M−1 s−1), and 2.03×10−4 (s−1) for sulfatide and 6.10×10−9 (M), 3.61×104 (M−1 s−1), and 2.20×10−4 (s−1)  for  GD1a  (sialoglycolipid),  respectively. No values could be obtained for GA1 (non-sialoglycolipid).39)  These  binding  parameters  mean  that  there  is  little  difference  in  the  dissociation  rate  for  the  secreted  HA  binding  between  sulfatide  and  GD1a  and  that  the  association  rate  for  the  secreted  HA  binding  to  sulfatide  is  2.56-times  slower  than  that  to  GD1a.  No  inhibition  of  GS-5  in  infection  demonstrated  that  sulfatide  was  not  a  virus  receptor  of  infection.37) Moreover,  sulfatide  is  a  much  smaller  sphingoglycolipid  than  various  glycoconjugates  containing  sialic  acids,  which  are  believed to be virus receptors of infection. Sulfatide is thought  to be a molecule with low accessibility in virus attachment to  the  host  cellular  surface.  The  binding  parameters  also  suggest  that  the  HA  preferentially  binds  to  sialic  acids  on  glycoconjugates  compared  to  sulfatide  for  receptor  attachment  of  the  virus, further supporting the notion that sulfatide has no function as a virus receptor.

4.  SULFATIDE  BINDING  OF  NASCENT  HA  ENHANCES NUCLEAR EXPORT OF vRNP COMPLEXES Viral  RNA  genomes  of  influenza  A  virus  are  replicated  in  the  nucleus  of  the  infected  cell.  Viral  genomes  form  vRNP  complexes  together  with  viral  RNA  polymerase  subunits  (PB1,  PB2,  and  PA)  and  viral  NP,  which  are  transported  into the nucleus after protein synthesis in the cytosol. vRNP complexes  are  selectively  exported  from  the  nucleus  to  the  cytosol  through  the  cellular  chromosome  region  maintenance  1  (CRM1)-mediated  nuclear  export  pathway.40) vRNP complexes  in  the  cytosol  are  absolutely  required  for  progeny  virus  formation  as  internal  components  of  progeny  viruses.  Sulfatide expression by transfection of CGT and CST genes in  COS-7  cells  enhanced  nuclear  export  of  vRNP  complexes  in  the  cells  infected  with  influenza  A  virus  (A/Memphis/1/1971  H3N2),  resulting  in  an  increase  of  progeny  virus  production.  Conversely,  sulfatide  knockdown  by  RNA  interfering  against  CST  mRNA  in  MDCK  cells  and  sulfatide-enriched  COS-7  cells  suppressed  the  nuclear  export  (Fig.  4).  The  nuclear  export  of  vRNP  complexes  was  also  inhibited  by  treatment  of  virus  (A/Memphis/1/1971  H3N2)-infected  cells  with  GS-5  or  anti-HA  monoclonal  antibody  that  could  inhibit  the  sulfatide  binding  of  HA.  Addition  of  GS-5  after  4 h  postinfection  had  an inhibitory effect on virus replication, but it had no effect before  4 h  postinfection.  HA  is  abundantly  expressed  on  the  cellular  surface  after  5  or  6 h  postinfection.  It  is  thought  that  GS-5  masked  sulfatide  on  the  celllular  surface  and  prevented  binding  with  HA.  These  results  indicated  that  sulfatide  bound  with nascent HA was transported on the surface membrane of  infected  cells  and  that  the  binding  induced  nuclear  export  of  vRNP complexes.37)

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5.  APOPTOSIS  INDUCED  BY  BINDING  BETWEEN  caspase-independent  apoptosis  of  AIF  by  inhibiting  the  miNASCENT  HA  AND  SULFATIDE  ENHANCES  NU- tochondrial permeability transition pore)44) had an inhibitory effect  on  the  apoptosis  through  suppression  of  potential  loss  CLEAR EXPORT OF vRNP COMPLEXES Caspase-3-mediated  apoptosis  and  Raf/mitogen-activated  protein  extracellular  kinase  (MEK)/extracellular  signal-regulated  kinase  (ERK)  activation  of  mitogen-activated  protein  kinase (MAPK) increase influenza A virus replication through  enhancement  of  nuclear  export  of  vRNP  complexes.41) Moreover,  HA  accumulation  on  the  cellular  surface  membrane  increases nuclear export of vRNP complexes by Raf/MEK/ERK  activation via protein kinase Cα (PKCα).42) U0126, an inhibitor of  the  Raf/MEK/ERK  pathway,  strongly  inhibited  virus  replication  in  sulfatide-enriched  COS-7  cells.  On  the  other  hand,  12-O-tetradecanoyl phorbol-13-acetate (TPA), a PKC activator,  significantly  increased  virus  replication  in  sulfatide-enriched  COS-7  cells.  Activation  of  the  Raf/MEK/ERK  pathway  via PKC,  which  probably  occurred  from  sulfatide  binding  of  HA  transported to the cellular surface, was necessary for enhancement  of  virus  replication  by  sulfatide  expression.  A  high  degree  of  virus-inducing  apoptosis  (Annexin  V  and  propydium  iodide  detection)  was  observed  in  sulfatide-enriched  COS-7  cells compared to that in parent cells. Nuclear translocation of  apoptosis-inducing  factor  (AIF)  in  mitochondria  is  known  as a representative caspase-3-independent apoptosis signal and  follows  potential  loss  of  the  mitochondrial  membrane.43)  Apoptosis was invoked through potential loss of the mitochondrial  membrane  and  nuclear  translocation  of  AIF  in  mitochondria  without  caspase-3  activation  in  sulfatide-enriched  cells.  On  the  other  hand,  activation  of  caspase-8  and  -9  was  detected  by  sulfatide  expression.  Both  GS-5  (inhibitor  of  binding  between  HA  and  sulfatide)  and  cyclosporin  A  (inhibitor  of 

of the mitochondrial membrane and nuclear translocation of AIF  in  mitochondria,  resulting  in  reduction  of  virus  replication  by  suppression  of  nuclear  export  of  vRNP  complexes.  However,  Z-VAD-FMK,  a  pan-caspase  inhibitor,  showed  no  effect  on  these  apoptosis  signals,  also  suggesting  that  activation of caspase-8 and -9 is not associated with enhancement of  virus  replication  by  sulfatide  expression.  AIF  knockdown  had  inhibitory  effects  on  virus-inducing  apoptosis,  nuclear  export  of  vRNP  complexes,  and  virus  replication.  The  AIF-mediated  virus-inducing  apoptosis  enhanced  nuclear  export  of  vRNP  complexes  and  virus  replication.  These  results  demonstrated  that  the  binding  between  sulfatide  and  HA  on  the  cellular  surface  invoked  apoptosis  through  nuclear  translocation  of  AIF  following  potential  loss  of  the  mitochondrial  membrane  and that the apoptosis enhanced nuclear export of vRNP complexes linking to progeny virus production.45) Apoptosis through potential loss of the mitochondrial membrane  is  induced  by  mitochondrial  accumulation  of  PB1-F2,  a  frame-shift  peptide  of  the  viral  PB1  gene  and  mitochondrial localization peptide, in some cell lines such as human lymphoma  U937  cells.46)  Two  PB1-F2-knockout  influenza  A  viruses  were  generated  by  a  reverse  genetics  system  based  on  a  backbone  of  A/WSN/1933  H1N1  strain.  One  virus  (PB1  T120C)  carried  the  PB1  gene  with  a  T-to-C  substitution  at  nucleotide  120,  introducing  an  alteration  in  a  Met  start  codon  to  Thr  without  affecting  the  PB1  standard  reading  frame.  The  other  (PB1  G144A)  carried  the  PB1  gene  with  a  G-to-A  substitution  at  nucleotide  144,  introducing  a  stop  codon  after  translation  of  only  eight  residues  of  PB1-F2,  and  a  Met-to-

Fig.  5.  Mechanism of Influenza A Virus Formation Initiated from Binding of Nascent HA with Sulfatide Viral nascent HA is transported on the surface membrane of the infected cells. Viral PB1-F2 is localized in mitochondria. Viral NP, viral genome RNA, and viral RNA  polymerase subunits form vRNP complexes in the nucleus. Binding of nascent HA with sulfatide on the cell surface induces potential loss of the PB1-F2-accumulating mitochondrial membrane and then nuclear translocation of AIF in mitochondria that invokes apoptosis. The apoptosis enhances nuclear export of vRNP complexes, resulting  in efficient formation of progeny viruses. Dotted-lined arrows are putative pathways.

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Fig.  6.  Sulfatide Expression of Human Tracheal NHBE Cells Differentiated  NHBE  cells  were  fixed  with  4%  paraformaldehyde  for  15 min  and  then  permeabilized  with  TritonX-100  for  15 min.  The  cells  were  reacted  with  mouse  anti-β-tubulin IgG and mouse anti-sulfatide IgM (GS-5) monoclonal antibodies for 1 h. Anti-β-tubulin (red) and sulfatide (green) were stained with goat tetramethylrhodamine  (TRITC)-labeled anti-mouse IgG and goat fluorescein isothiocyanate (FITC)-labeled anti-mouse IgM secondary antibodies for 1 h, respectively. Scale bar=50 µm.

Ile  substitution  in  PB1  at  position  40.  Both  PB1-F2-knockout  viruses  showed  reduction  of  apoptosis  through  suppression  of potential loss of the mitochondrial membrane and nuclear translocation  of  AIF  in  mitochondria,  resulting  in  reduction  of  virus  replication,  compared  to  the  parent  virus.  PB1-F2  was  required  for  AIF-mediated  virus-inducing  apoptosis  in  sulfatide-enriched COS-7 cells.45) Taken together, the results indicate that the binding between  sulfatide  and  nascent  HA  on  the  cellular  surface  invokes  potential  loss  of  the  mitochondrial  membrane  when  PB1-F2  from  the  viral  PB1  gene  is  accumulated  in  mitochondria.  The  potential  loss  promotes  nuclear  translocation  of  AIF  in  mitochondria,  which  induces  apoptosis.  The  apoptosis  enhances  nuclear export of vRNP complexes, resulting in progeny virus  formation.  The  binding  between  sulfatide  and  nascent  HA  is  thought to be an initiation switch for the progeny virus formation process. Activation of the Raf/MEK/ERK pathway, which  probably  begins  with  binding  between  sulfatide  and  nascent  HA  on  the  cellular  surface,42)  is  likely  to  be  associated  with  these  processes.  Activation  of  caspase-8  and  -9  by  sulfatide  expression  is  not  directly  associated  with  enhancement  of  virus  replication  through  AIF-mediated  virus-inducing  apoptosis,  because  of  no  activation  of  caspase-3  at  the  final  stage  of caspase cascades and no effect of a pan-caspase inhibitor in virus replication. Since activation of caspase-8 is initiated by stimulation of death receptors on the cellular surface,47) sulfatide  expression  on  the  cellular  surface  may  interact  with  such death receptors and may increase receptor sensitivity. Caspase-9 is activated by cytochrome c (Cyt c),  which  is  released to the cytosol from the mitochondria in apoptosis.47) Activation  of  caspase-9  possibly  results  from  the  release  of  Cyt c because of potential loss of the mitochondrial membrane in sulfatide-enriched COS-7 cells (Fig. 5). There  is  a  concern  about  whether  sulfatide  is  actually  expressed  on  human  tracheal  epithelial  cells.  The  existence  of  sulfatide in human tracheal epithelial cells remains unclear, but  we  have  data  showing  a  high  level  of  sulfatide  expression  in  ciliated  cells  of  human  bronchial  epithelial  (NHBE)  cells  (Fig.  6).  Sulfatide  is  a  highly  multifunctional  glycolipid  that  is involved in cancer, the nervous system, insulin secretion in the  islet  of  Langerhans,  hemostasis/thrombosis,  immune  system, and microbial infection. Moreover, abnormal metabolism of  sulfatide  is  associated  with  the  development  of  many  diseases,  including  MLD,  diabetes,  and  autoimmune  diseases.24) Myelin  and  lymphocyte  protein  (MAL)  is  expressed  on  the  apical  surface  of  the  kidney  and  forms  complexes  with  glycosphingolipids  including  sulfatide.  Sulfatide  is  abundant  on  the 

apical  surface  of  distal  kidney  tubuli.  MAL  complexes  with  sulfatide  might  contribute  to  stabilization  and  apical  sorting  of lipid rafts.48,49)  Such  complexes  and  their  associated  apical  sorting  might  be  involved  in  progeny  virus  formation  at  the apical surface of tracheal epithelial cells. Further study on sulfatide function in virus replication may help to reveal mechanisms  underlying  these  biological  activities  and  these  diseases,  leading  to  the  development  of  drugs  against  sulfatide-associated diseases. Highly  pathogenic  H5N1  avian  influenza  A  virus,  which  shows  approximately  60%  fatality  in  human  infection  cases,  has often been transmitted from poultry to humans since 1997.50,51) A pandemic of swine-origin H1N1 influenza A virus  suddenly  occurred  among  humans  in  2009.52)  A  pandemic  occurs  due  to  dramatic  changes  of  antigenicities  in  viral  envelope glycoproteins, HA and NA. A vaccine has difficulty in  dealing with such antigenic changes. Most human influenza A  viruses have acquired resistance to a viral M2 ion channel inhibitor (amantadine) since 2006.53)  Currently,  influenza  A  virus-specific  NA  inhibitors  (zanamivir,  oseltamivir,  peramivir,  and laninamivir) are used for treatment of influenza. However,  there  is  a  serious  problem  of  the  emergence  of  drug-resistant  viruses.54)  Most  H1N1  human  influenza  A  viruses  have  acquired  resistance  to  oseltamivir  since  2008,  although  these  viruses  disappeared  after  the  pandemic  of  a  new  H1N1  virus  in  2009.  Luckily,  most  new  H1N1  virus  in  2009  was  sensitive to oseltamivir.54)  Our  studies  showed  that  the  signal  of  progeny  virus  formation  was  initiated  from  binding  between  sulfatide  and  nascent  HA  on  the  cellular  surface.  Inhibitors  of  the  binding  of  HA  with  sulfatide  have  potential  as  novel  and  efficient  anti-influenza  virus  drugs  that  can  be  used  for  conventional  NA  inhibitor-resistant  viruses  and  new  subtypes  of  pandemic  viruses  or  for  patients  with  serious  symptoms  from advanced infection. For treatment of influenza, inhibitors  specifically  targeting  the  sulfatide  binding  site  on  HA  would  be  suitable  for  preventing  predicted  side  effects  because  they  have  no  direct  influence  on  endogenous  sulfatide.  Moreover,  the  inhibitors  might  have  more  powerful  antiviral  efficacy  due  to  inhibition  of  progeny  virus  formation  itself,  compared  to  conventional  NA  inhibitors  that  inhibit  the  release  process  after the virus formation process. Acknowledgments We  would  like  to  acknowledge  people  in  our  laboratory  who  contributed  to  this  work.  We  would  also  like  to  acknowledge  and  appreciate  Dr.  Yasuo  Suzuki  of  Chubu  University,  Dr.  Koichi  Honke  of  Kochi  University  Medical  School,  Dr.  Kiyoshi  Ogura  and  Dr.  Tadashi  Tai  of 

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Tokyo Metropolitan Institute of Medical Science, and Dr. Hideshi  Yokoyama  and  Dr.  Satoshi  Fujii  of  the  University  of  Shizuoka.  This  work  was  supported  in  part  by  a  Grant-in-Aid  from  the  MEXT/JSPS  KAKENHI  (Scientific  Research  B,  18390142;  Scientific  Research  C,  23590549;  Young  Scientist  B,  20790357;  Challenging  Exploratory  Research  26670064),  Sasakawa  Scientific  Research  Grant  from  The  Japan  Science  Society  (20–403  and  23–439),  Grant-in-Aid  from  Tokyo  Biochemical  Research  Foundation,  Hamamatsu  Scientific  Research Foundation, Takeda Science Foundation, The Research Foundation for Pharmaceutical Sciences, Mizutani Foundation for Glycoscience, The Public Foundation of Chubu  Science  and  Technology  Center,  The  Waksman  Foundation  of  Japan  Inc.,  Showa  University  Medical  Foundation,  The  Uehara Memorial Foundation, Foundation for Promotion of Material  Science  and  Technology  of  Japan,  Adaptable  and  Seamless Technology Transfer Program through Target-driven  R&D  (A-STEP)  from  Japan  Science  and  Technology  Agency,  CREST  from  Japan  Science  and  Technology  Agency,  and  the  Gobal  COE  Program  from  the  Japan  Society  for  the  Promotion of Science. The funders had no role in study design, data  collection and analysis, decision to publish, or preparation of the manuscript. Conflict of Interest interest.

The  authors  declare  no  conflict  of 

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Role of sulfatide in influenza A virus replication.

Sulfatide is a 3-O-sulfated galactosylceramide that is abundantly expressed in the gastrointestinal tract, kidney, trachea, and particularly the centr...
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