Role of Sulfatide in Influenza A Virus Replication

Abstract

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.

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 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. 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-H₂SO₄ 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.

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 sulfatide 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 sulfatide-enriched 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 sulfatide-enriched 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 ASA-expressed cells. The remarkable virus replication, which was induced by sulfatide expression, resulted from enhanced production of progeny virus in the cells.

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.

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 influenza 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 sulfatide-binding 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 sulfatide-binding 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 acid binding ability, and sulfatide binding ability similar to those of the whole HA and the virus.

We measured dissociation constant (K_d), binding rate constant (K_on), and dissociation rate constant (K_off) for sialic acid binding and sulfatide binding of the secreted HA by using a quartz crystal microbalance (QCM). The K_d value, K_on value, and K_off 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⁾

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.

5. APOPTOSIS INDUCED BY BINDING BETWEEN NASCENT HA AND SULFATIDE ENHANCES NUCLEAR 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 caspase-independent apoptosis of AIF by inhibiting the mitochondrial permeability transition pore)⁴⁴⁾ had an inhibitory effect on the apoptosis through suppression of potential loss 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-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).

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.

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.

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.

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 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

The authors declare no conflict of interest.

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