Virology 464-465 (2014) 424–431

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Virology

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Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3

Keijo Fukushima a,1, Tadanobu Takahashi a,1, Seigo Ito a, Masahiro Takaguchi a, Maiko Takano a, Yuuki Kurebayashi a, Kenta Oishi a, Akira Minami a, Tatsuya Kato b,f, Enoch Y Park b,e,f, Hidekazu Nishimura c, Toru Takimoto d, Takashi Suzuki a,n a Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 4228526, Japan b Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 4228529, Japan c Virus Research Center, Sendai Medical Center, 2-8-8 Miyagino, Miyagino-ku, Sendai, Miyagi 9838520, Japan d Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA e Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 4228529, Japan f Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 4228529, Japan article info abstract

Article history: Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Received 22 May 2014 Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by Returned to author for revisions using sialic acid linkage-modified cells treated with sialidases or . The hPIV1 is bound to 8 July 2014 only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in Accepted 11 July 2014 addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase Available online 21 August 2014 treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Keywords: Treatment of A549 cells with α2,3 linkage-specific increased infectivities of both hPIV1 fl Human parain uenza virus and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Sialic acid Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes Receptor specificity only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages hPIV1 sialic acid linkages as viral receptors. hPIV3 & 2014 Published by Elsevier Inc.

Introduction the host cell surface through HN (Scheid et al., 1972; Tozawa et al., 1973). Human parainfluenza virus type 1 (hPIV1) is a respiratory Sialic acids are typically found at the terminals of oligosacchar- pathogen that most frequently causes laryngotracheobronchitis ides on and , and they are mainly in children, and human parainfluenza virus type 3 (hPIV3), which linked to the galactose residue by α2,3- or α2,6-linkage or are is generally more virulent than hPIV1, is the second most fre- polymerized by α2,8- or α2,9-linkage (Kirsch et al., 2009; Suzuki, quently isolated virus from patients with pneumonia and bronch- 2005). Influenza A viruses isolated from several animal species iolitis, mainly in infants younger than 5 years old (Chanock, 2001; recognized different terminal sialic acid linkages. Avian influenza Sinaniotis, 2004). These human parainfluenza viruses (hPIVs), A viruses preferentially bind to α2,3-linked sialic acid residues. In which belong to the genus Respirovirus and the family Paramyx- contrast, human influenza A viruses show predominant binding oviridae, have two spike glycoproteins, hemagglutinin-neura- specificity to α2,6-linked sialic acid residues (Matrosovich et al., minidase (HN) glycoprotein and fusion glycoprotein, on the viral 1997). Such a virus binding specificity for sialic acid linkages is surfaces. hPIVs bind to terminal sialic acids of on believed to be one of the major determinants of viral host range and tissue tropism. We previously reported that hPIV1 and hPIV3 show different

n binding specificities for the terminal sialic acid linkages of Corresponding author. Tel./fax: þ81 54 264 5725. α E-mail address: [email protected] (T. Suzuki). some gangliosides: hPIV1 preferentially binds to 2,3-linked 1 They contributed equally as first authors. sialic acids, whereas hPIV3 strongly binds both to α2,3- and http://dx.doi.org/10.1016/j.virol.2014.07.033 0042-6822/& 2014 Published by Elsevier Inc. K. Fukushima et al. / Virology 464-465 (2014) 424–431 425

α2,6-linked sialic acid residues at the terminals of oligosacchar- Resialylation hardly induced lysis of AUSD-treated hRBC. We ides containing branched N-acetyllactosaminoglycans (Suzuki measured viral hemagglutination activity by using these resialy- et al., 2001b). In the present study, we compared cell infectiv- lated hRBC (ST3-hRBC and ST6-hRBC). ST3 treatment resulted in ities of hPIV1 and hPIV3 with their binding specificities for recovery of both hemagglutination activities of hPIV1 and hPIV3; overall sialic acid linkages on cell surfaces containing ganglio- however, ST6 treatment resulted in the recovery of the activity of sides and glycoproteins. We evaluated the binding activities and hPIV3 but not that of hPIV1 (Table 1). We also measured viral infectivities of hPIV1 and hPIV3 for sialic acid linkage-modified hemagglutination activity by using resialylated hRBC treated with cells by treatment with sialidase or/and sialyltransferase. We mammalian sialyltransferase, rat α2,3-(N)-sialyltransferase found that hPIV1 uses terminal α2,3-linked sialic acids only and ST3Gal-III (ST3N) or α2,6-(N)-sialyltransferase ST6Gal-I (ST6N), that hPIV3 uses terminal α2,6-linked sialic acid residues in which transfers α2,3- or α2,6-linked sialic acids to the terminal addition to α2,3-sialic acid linkages as the viral cell receptor. galactosides of N-glycoproteins, respectively (Kaludov et al., 2001; Lee et al., 1994; Sánchez-Felipe et al., 2012; Williams et al., 1995). Viral hemagglutination with ST3N-treated hRBC (ST3N-hRBC) or Results ST6N-treated hRBC (ST6N-hRBC) was similar to that with ST3- or ST6-treated hRBC (Table 1). We concluded that hPIV1 recognized α Hemagglutination of sialic acid linkage-modified hRBC by hPIV1 and 2,3-linked sialic acid residues only and that hPIV3 recognized α α hPIV3 2,6-linked sialic acid residues in addition to 2,3-linked sialic acid residues on the surface of hRBC. To investigate the difference in virus binding to the sialic acid linkages on the cell surface, we modified the terminal sialic acid Hemadsorption activities of cell-expressed HN1 and HN3 linkages on the surface of human red blood cells (hRBC), on which glycoproteins with sialidase- or sialyltransferase-treated hRBC various sialic acid linkages for hRBC including α2,3 and α2,6 linkages existed (Aminoff et al., 1980; Bratosin et al., 1995; Bulai We checked the binding activities of HN glycoprotein from et al., 2003), by treatment with sialidases. hRBC were treated with hPIV1 (HN1 glycoprotein) and HN glycoprotein from hPIV3 (HN3 α α sialidase from Arthrobacter ureafaciens (AUSD) to remove all glycoprotein) to 2,3-linked or 2,6-linked sialic acids by hemad- terminal sialic acids or with cloned α2,3 from sorption assays using sialidase- or sialyltransferase-treated hRBC. Salmonella typhimurium LT2 (23SD) to remove preferentially Monolayers of HN1 or HN3 glycoprotein-expressing African green α2,3-linked sialic acid residues (Hoyer et al., 1991; Rogerieux et monkey kidney (COS-7) cells were incubated with sialidase- al., 1993). These sialidases hydrolyze both the terminal sialic acids treated hRBC (AUSD-hRBC and 23SD-hRBC) or sialyltransferase- at gangliosides and glycoproteins (Hoyer et al., 1991; Uchida et al., treated hRBC (ST3-hRBC, ST3N-hRBC, ST6-hRBC and ST6N-hRBC). fi 1979). hPIV1 and hPIV3 binding specificities for α2,3 and α2,6 Bound hRBC were quanti ed by the amount of hemoglobin. HN1 sialic acid linkages were evaluated by hemagglutination of these glycoprotein-expressing cells absorbed to ST3-hRBC, whereas HN3 sialidases-treated hRBC (AUSD-hRBC and 23SD-hRBC). AUSD treat- glycoprotein-expressing cells absorbed to 23SD-hRBC, ST3-hRBC ment resulted in the loss of both hemagglutination activities of and ST6-hRBC (Fig. 1A and B). Neither HN1 nor HN3 glycoprotein- hPIV1 and hPIV3, indicating that hPIV1 and hPIV3 bind to hRBC via expressing cells absorbed to AUSD-hRBC (Fig. 1A). The results for terminal sialic acids only (Table 1). 23SD treatment resulted in the mammalian sialyltransferase-treated hRBC (ST3N-hRBC and ST6N- loss of hemagglutination activity of hPIV1, whereas hemagglutina- hRBC) were similar to those for ST3- and ST6-treated hRBC tion activity of hPIV3 remained, although the activity of hPIV3 was (Fig. 1C). Fusion glycoproteins did not show any hemadsorption decreased (Table 1). hPIV3 probably recognized α2,6-linked sialic activity (data not shown) (Chu et al., 2013). Each HN glycoprotein fi acids which remained on the surface of hRBC after 23SD showed the same sialic acid linkage speci city as the correspond- fi treatment. ing virus particle, indicating that the hPIV binding speci cities for To confirm that the remaining binding of hPIV3 after 23SD sialic acid linkages were dependent on the HN glycoproteins only. treatment is not due to the recognition of α2,3-linked sialic acid residues, we evaluated viral hemagglutination activity using α2,3- Infectivities of hPIV1 and hPIV3 to sialidase-treated cells or α2,6-linked resialylated hRBC. AUSD-treated hRBC were incu- fi bated with a reaction solution containing cytidine-5'-monopho- To determine whether binding speci cities of hPIV1 and hPIV3 spho-N-acetylneuraminic acid sodium salt (CMP-Sia) as a sialic for sialic acid linkages affect infectivities of host cells, we inves- acid donor and α2,3-sialyltransferase from Photobacterium phos- tigated the infectivities of hPIV1 and hPIV3 to sialidase-treated fl phoreum (ST3) or α2,6-sialyltransferase from Photobacterium dam- cells. Con uent monolayers of Lewis lung carcinoma-monkey selae (ST6), which transfers α2,3- or α2,6-linked sialic acids to the kidney (LLC-MK2) cells were treated with serially diluted 23SD α fi terminal galactosides, respectively, with a wide substrate range for 2,3 linkage-speci c desialylation or AUSD for all linkage (Tsukamoto et al., 2007; Yamamoto et al., 1998, 1996). desialylation. Infectivities of hPIV1 and hPIV3 were measured in these sialidase-treated cells. 23SD treatment decreased the infec- tivity of hPIV1 in 23SD-dependent manner. In contrast, infectivity Table 1 of hPIV3 was hardly affected by 23SD treatment (Fig. 2A). Both Hemagglutination of hRBC modified with different sialic acid linkages by hPIV1 and hPIV3. hPIV1 and hPIV3 scarcely infected the AUSD-treated monolayer (Fig. 2A). We also measured amounts of α2,3- and α2,6-linked Hemagglutination titers by: sialic acid residues on the surfaces of sialidase-treated cells by flowcytometric analysis using Maackia amurensis agglutinin (MAA) hRBC hPIV1 hPIV3 or Sambucus nigra agglutinin (SNA), which recognize α2,3-linked Normal-hRBC 128 128 α fi AUSD-hRBC o2 o2 or 2,6-linked sialic acid residues, respectively. We con rmed 23SD-hRBC o216that 23SD treatment specifically removed α2,3-linked sialic acids ST3-hRBC 256 256 and that AUSD treatment removed both α2,3-linked and α2,6- ST6-hRBC o2128linked sialic acids (Fig. 2B). We also measured viral infectivities in ST3N-hRBC 128 256 23SD-treated human lung adenocarcinoma epithelial cells (A549 ST6N-hRBC o264 cells) cells. Similar to the results for LLC-MK2 cells, 23SD treatment 426 K. Fukushima et al. / Virology 464-465 (2014) 424–431

Fig. 1. Hemadsorption activities of cell-expressed HN1 and HN3 glycoproteins by using hRBC modified with different sialic acid linkages. (A) Hemadsorption activities (%) of HN1 and HN3 glycoproteins for sialidase-treated hRBC. HN1 or HN3 glycoproteins were expressed on the surfaces of COS-7 cells and the cells were incubated with the AUSD- (not detected) or 23SD-treated hRBC (filled column) for 30 min on ice. hRBC that bound to the cell surfaces were quantified and calculated as a percentage relative to the enzyme-untreated hRBC (B) Hemadsorption activities (%) of HN1 and HN3 glycoproteins for sialyltransferase-treated hRBC. The AUSD-treated hRBC were additionally incubated with ST3 (gray column) or ST6 (hatched column) derived from Photobacterium for α2,3-linked or α2,6-linked specific resialylation. Hemadsorption activities were calculated as described in (A). (C) Hemadsorption activities (%) of HN1 and HN3 glycoproteins for mammalian sialyltransferase-treated hRBC. The AUSD-treated hRBC were incubated with ST3N (gray column) or ST6N (hatched column) derived from mammals. The hemadsorption activities were calculated as described in (A). The values are means and standard deviations of triplicate experiments. N. D. means "not detected".

Fig. 2. Infectivities of hPIV1 and hPIV3 in sialidase-treated cells. (A) Infectivities (%) of hPIV1 and hPIV3 in 23SD-treated LLC-MK2 cells. Confluent LLC-MK2 cells were treated with serially diluted 23SD or AUSD for 2 h and were infected with hPIV1 (filled column) or hPIV3 (empty column). Virus-infected cells were detected by anti-virus antibodies, and the infectivities were calculated as a percentage relative to the sialidase-untreated cells. (B) Flowcytometric analysis of the terminal sialic acid linkages on the surface of sialidase-treated LLC-MK2 cells. Sialidase-treated LLC-MK2 cells were incubated with digoxigenin-conjugated MAA (filled column) or SNA (empty column) and then with the FITC-conjugated anti-digoxin antibody. Lectin bindings were quantified as fluorescence and calculated as a mean fluorescence intensity (MFI) ratio relative to the sialidase- untreated LLC-MK2 cells. (C) Infectivities (%) of hPIV1 and hPIV3 in 23SD-treated A549 cells. Confluent A549 cells were treated with 23SD and were infected with hPIV1 (filled column) or hPIV3 (empty column). Infectivities were calculated as described in (A). The values are means and standard deviations of triplicate experiments. nnPo0.01 versus the results for hPIV1. decreased the infectivity of hPIV1 but not that of hPIV3 (Fig. 2C). To confirm whether hPIV3 bound α2,6-linked sialic acids on the The results suggested that hPIV1 can use α2,3-linked sialic acids Lec2 cell surface or not, we measured amounts of hPIV3 binding and that hPIV3 can use α2,6-linked sialic acids in addition to α2,3- on the sialylated Lec2 cell surface by flowcytometric analysis. The linked sialic acids as virus receptors of host cell infection. analysis revealed that hPIV3 could hardly bind to the surface of ST6-treated Lec2 cells (Fig. 3B). Existence of α2,6-linked sialic acid residues on the surface of ST6-treated Lec2 cells was confirmed by Infectivities of hPIV1 and hPIV3 in sialyltransferase-treated cells fluorescent observation using SNA (Fig. 3C). In addition, influenza A virus A/Memphis/1/1971 (H3N2) strain, which preferentially To determine whether sialic acid linkages on the cell surface binds to α2,6-linked sialic acids (Suzuki et al., 2000), infected regulate infectivity of hPIV1 and hPIV3, we investigated the ST6-treated Lec2 cells but scarcely infected non-sialylated Lec2 infectivities of hPIV1 and hPIV3 in sialyltransferase-treated Lec2 cells (data not shown). In contrast to the results for hRBC, the cells. Lec2 cells, surface sialic acid-deficient mutant cells derived results suggested that hPIV3 could not bind to the main α2,6- from Chinese hamster ovary (CHO) Pro-5 cells, were sialylated linked sialylated glycochains on Lec2 cells that SNA could recog- with ST3 or ST6. We measured viral infectivities in these sialylated nize and that hPIV binding specificity to sialic acid linkages Lec2 cells. Both hPIV1 and hPIV3 infected ST3-treated cells but not determines the viral infectivity. non-sialylated Lec2 cells (Fig. 3A). Unexpectedly, not only hPIV1 Furthermore, we demonstrated infectivities of hPIV1 and hPIV3 but also hPIV3 scarcely infected ST6-treated Lec2 cells, indicating in resialylated A549 cells. To prevent endogenous cellular resialyla- that hPIV3 cannot recognize the main α2,6-linked sialic acid tion, A549 cells were pretreated with 3Fax-Peracetyl Neu5Ac (ST-I), residues on the surface of Lec2 cells. a sialic acid analog and intracellular silalyltransferase inhibitor K. Fukushima et al. / Virology 464-465 (2014) 424–431 427

Fig. 3. Infectivities of hPIV1 and hPIV3 in sialyltransferase-treated Lec2 cells and A549 cells. (A) Infectivities (%) of hPIV1 and hPIV3 in sialyltransferase-treated Lec2 cells. Lec2 cell monolayers were incubated with a reaction solution containing CMP-Sia and ST3 or ST6 (ST3, gray column; ST6, hatched column). As a control, a Lec2 cell monolayer was incubated with CMP-Sia only (No ST, filled column). The sialylated cells were infected with hPIV1 or hPIV3 and their infectivities were calculated as a percentage relative to parental Pro-5 cell monolayers. The values are means and standard deviations of triplicate experiments. N.D. means “not detected”. (B) hPIV3 binding activities to sialylated Lec2 cells. The ST3-treated (ST3, solid line), ST6-treated (ST6, dotted line), or enzyme-untreated Lec2 cells (No ST, gray shading) were incubated with hPIV3 for 1 h on ice. The cells were incubated with anti-virus antibodies on ice for 30 min and then incubated with PE-conjugated anti-rabbit IgG on ice for 30 min. The fluorescence intensities of the cells were analyzed using a flowcytometer. (C) Lectin staining of sialyltransferase-treated Lec2 cells. The ST3- or ST6-treated Lec2 cells were stained with FITC-conjugated SNA (green) or biotin-conjugated MAL-II, followed by FITC-conjugated streptavidin (green). Nuclei were stained with DAPI (blue). Scale bar¼40 μm. (D) Infectivities (%) of hPIV1 and hPIV3 in sialyltransferase-treated A549 cells. A549 cell monolayers were pretreated with ST-I for 4 days and were treated with AUSD for 3 h. The desialylated cells were incubated with a reaction solution containing CMP-Sia and ST3 or ST6 for 2 h to resialylate α2,3-linked or α2,6-linked sialic acids (ST3, gray column; ST6, hatched column). As a control, the cells were incubated with CMP-Sia only (No ST, filled column). The resialylated cells were infected with hPIV1 or hPIV3 and their infectivities were calculated as a percentage relative to ST-I-treated and AUSD-untreated monolayers. The values are means and standard deviations of triplicate experiments. nPo0.05, nnPo0.01 versus the results for each no ST.

(Rillahan et al., 2012), for 4 days. For ST-I-treated A549 cells, both COS-7 cells infected with an hPIV1 clinical isolate hPIV1/Sendai-H/ infectivities of hPIV1 and hPIV3 were decreased but still remained 1381/2007 (CL-1381) and an hPIV3 clinical isolate hPIV3/Sendai- (Supplementary Fig. 1). The decrease of hPIV3 infectivity was more H/918/2009 (CL-918), which were isolated from infected patients moderate than that of hPIV1, possibly because ST-I was a somewhat in 2007 and 2009, and we compared reduction of viral infectivity weak inhibitor of endogenous cellular α2,6-sialylation (Rillahan et by sialidase treatment (23SD and AUSD). COS-7 cells were infected al., 2012). ST-I-treated A549 cells were treated with AUSD for with CL-1381 or CL-918, and the sialic acid linkage recognition complete desialylation and were then incubated with a reaction specificities of the isolates were evaluated by hemadsorption solution containing CMP-Sia and ST3 or ST6 to resialylate α2,3- activities of HN glycoproteins expressed on the virus-infected cell linked or α2,6-linked sialic acids, respectively. We measured infec- surfaces. CL-1381-infected cells bound to ST3-treated hRBC. In tivities of hPIV1 and hPIV3 in these resialylated A549 cells. ST3 contrast, CL-918-infected cells adsorbed both ST3- and ST6-treated treatment increased both infectivities of hPIV1 and hPIV3 compared hRBC (Fig. 4A). The cell infected with the clinical strains hardly to incubation with CMP-Sia only (No ST) (Fig. 3D). In contrast to the bound to AUSD-hRBC. We also measured the infectivities of CL- results for sialylated Lec2 cells, ST6 treatment increased the 1381 and CL-918 in 23SD-treated LLC-MK2 cells and confirmed infectivity of hPIV3 but not that of hPIV1 (Fig. 3D). hPIV3 recognized that CL-918 infection shows more resistance to 23SD treatment α2,6-linked sialic acids on the A549 cell surfaces as viral receptors. than does CL-1381 infection (Fig. 4B). The hPIV clinical isolates The results indicated that hPIV1 and hPIV3 can use various showed sialic acid linkage recognition specificities similar to those terminal α2,3-linked sialic acids as viral receptors and that hPIV3 of hPIV laboratory strains. can also use terminal α2,6-linked sialic acid residues that exist in specific glycochains on the surface of A549 cells but not on Lec2 cells. Discussion and conclusion

Sialic acid linkage recognition specificities of hPIV clinical isolates In the present study, we confirmed binding specificities of hPIV1 and hPIV3 for sialic acid linkages at all glycoconjugates on

To confirm that clinical isolates of hPIV1 and hPIV3 show the the surface of hRBC, LLC-MK2 cells, A549 cells and Chinese hamster same silayl linkage recognition specificities as those of hPIV ovary cell mutants (Lec2 cells and Pro-5 cells). The cell surfaces laboratory strains, we examined the hemadsorption activity of were treated with several sialidases and sialyltransferases. A 428 K. Fukushima et al. / Virology 464-465 (2014) 424–431

Fig. 4. Sialic acid linkage recognition specificities of hPIV clinical isolates. (A) Hemadsorption activities (%) of hPIV1 clinical isolate (CL-1381)-infected cells and hPIV3 clinical isolate (CL-918)-infected cells for sialyltransferase-treated hRBC. COS-7 cells were infected with CL-1381 or CL-918 and incubated for 2 days. The cells were incubated with AUSD- (empty column), ST3- (gray column), or ST6-treated hRBC (hatched column) for 30 min on ice. hRBC that bound to the cell surfaces were quantified and calculated as a percentage relative to enzyme-untreated hRBC. Non-infected cells were used as a negative control. (B) Infectivities (%) of CL-1381 and CL-918 in 23SD- or AUSD-treated LLC-

MK2 cells. Confluent LLC-MK2 cells were treated with 23SD or AUSD for 2 h and infected with CL-1381 (filled column) or CL-918 (empty column). Virus-infected cells were detected by anti-virus antibodies. The infectivities were calculated as a percentage relative to those of sialidase-untreated cells. The values are means and standard deviations of triplicate experiments. nPo0.05, nnPo0.01 versus the results for each CL-1381. N. D. means "not detected". hemagglutination assay of the virus particles and a hemadsorption blood group I antigens that contain internal branching polylacto- assay of HN glycoproteins-expressing COS-7 cells revealed that samine motifs and exist on the surfaces of hRBC, A549 cells and both hPIV1 and hPIV3 are bound to α2,3-linked sialic acid residues ciliated human bronchial epithelial cells but not on CHO cells on the surface of hRBC and that hPIV3 is additionally bound to (Inaba et al., 2003; Loveless and Feizi, 1989), and hPIV1 and hPIV3 α2,6-linked sialic acid residues. Viral infectivity assays using the strongly bind (Suzuki et al., 2001b). sialidase-treated A549 cells, sialidase-treated LLC-MK2 cells and In the clinical site, hPIV3 causes more severe bronchiolitis and sialyltransferase-treated A549 cells showed that the different lower pneumonia. On the other hand, hPIV1 causes only mild binding specificities of hPIV1 and hPIV3 for sialic acid linkages is infection that is limited to the upper respiratory tract (Chanock, a critical factor for viral infectivity. In addition, the hPIV clinical 2001). Human trachea cells predominantly express α2,6-linked isolates showed sialic acid linkage recognition specificities similar sialic acids (Baum and Paulson, 1990). The difference between to those of hPIV laboratory strains in a hemadsorption assay for hPIV1 and hPIV3 in tropism and pathogenesis in vivo may result ST3- or ST6-treated hRBC and infectivity assay for 23SD-treated from the ability of hPIV3 to recognize α2,6-linked sialic acid.

LLC-MK2 cells. Taken together, we concluded that both hPIV1 and Further characterization of sialic acid linkages and the internal hPIV3 mainly recognize terminal α2,3-linked sialic acid residues as glycochain structure motif distributions in the human respiratory virus receptors and that hPIV3 also recognizes terminal α2,6-linked tract would reveal the biological significance of tropism and sialic acid residues. pathogenesis of hPIV1 and hPIV3. Some previous reports have indicated that hPIVs recognize not only terminal sialic acids but also internal glycochain structures, especially the polylactosamine motif. hPIV1 and hPIV3 preferen- Materials and Methods tially bind to sialic acids at the terminals of branching polylacto- samine structures (Suzuki et al., 2001b). In the primary human Cells, viruses and antibodies airway epithelium, hPIV3 exclusively infects ciliated epithelial cells

(Zhang et al., 2005), which have lactosamine motifs that are LLC-MK2 cells were maintained in Eagle's minimum essential recognized by erythrina crista lectin or sequence- medium (MEM) supplemented with 5% heat-inactivated fetal specific antibodies (Loveless and Feizi, 1989; Vierbuchen et al., bovine serum (FBS). COS-7 cells and A549 cells were maintained 1988). Different from other respiratory paramyxoviruses, such as in Dulbecco's modified Eagle's medium (DMEM) supplemented Sendai virus, that mainly recognize terminal sialic acids (Markwell with 10% FBS. Lec2 cells and Pro-5 cells, derivatives of a CHO cell and Paulson, 1980; Markwell et al., 1981; Suzuki et al., 2001b, line, were maintained in alpha minimal essential medium supple- 1985), the internal polylactosamine motifs as well as terminal mented with 10% FBS and 40 μg/ml of L-proline (Sigma-Aldrich, St. sialic acids may be important factors for cell surface binding and Louis, MO). The hPIV1 C35 strain and the hPIV3 C243 strain were infection of hPIV1 and hPIV3. In studies on glycochain recognition kindly provided by Allen Portner (St. Jude Children's Research of hPIV1 and hPIV3 by a glycoarray, it was shown that hPIV3 has Hospital). These hPIVs were propagated in LLC-MK2 cells. An hPIV1 no binding to glycochains containing α2,6-linked sialic acid clinical isolate CL-1381 and an hPIV3 clinical isolate CL-918 were (Amonsen et al., 2007; Song et al., 2011). However, the glycoarray provided by the Virus Research Center, Clinical Research Division, probably contains a few kinds of internal glycochain structures in Sendai Medical Center, Sendai, Japan. CL-1381 and CL-918 were length and branching, compared with natural glycochains on cell isolated from infected patients in 2007 and 2009, respectively, and surfaces. hPIV3 recognized α2,6-linked sialic acids on human A549 were passaged two times in human melanoma HMV-II cells and cells and hRBC but not those on Lec2 cells derived from the CHO then four times in LLC-MK2 cells. Rabbit anti-hPIV1 antibody and cell line. hPIV3 may need a specific internal glycochain when it rabbit anti-hPIV3 antibody were prepared by immunization with binds to terminal α2,6-linked sialic acids, for example, human each hPIV as described previously (Suzuki et al., 2001a) and K. Fukushima et al. / Virology 464-465 (2014) 424–431 429 purified on a Protein G HiTrapTM Protein G HP (CE Healthcare Invitrogen, Corp., Carlsbad, CA). After incubation at 37 1C for 48 h, Biosciences Corp., Priscataway, NJ). the cells were washed with PBS and incubated with 0.5% of each sialic acid linkage-modified hRBC in SFM for 30 min on ice. The Preparation of sialic acid linkage-modified red blood cells cells were washed 3 times with SFM and incubated with 100 μlof ultrapure water for 10 min on ice. Fifty microliters of the super- hRBC were collected from a healthy adult and washed 3 times natant was applied to a 96-well plate, and the amount of with phosphate-buffered saline (PBS; pH 7.2, 131 mM NaCl, 14 mM hemoglobin was determined by measuring the absorbance at

Na2HPO4 and 2.7 mM KCl) (Normal-hRBC). For complete desialyla- 415 nm with a reference wavelength of 630 nm (Takaguchi et al., tion, 10% of Normal-hRBC were incubated with PBS containing 2011). The amount of hemoglobin in non-transfected cells was 25 mU/ml of AUSD (Nacalai Tesque, Kyoto, Japan) at 37 1C for 2 h tested as a negative control. and washed with PBS 5 times (AUSD-hRBC). For desialylation of To determine the hemadsorption activity of hPIV clinical α2,3-linked sialic acids, 10% of Normal-hRBC were incubated with isolate-infected cells, a confluent monolayer of COS-7 cells in the PBS containing 100 U/ml of 23SD (New England Biolabs Inc., 48-well plate was infected with CL-1381 or CL-918 diluted by SFM

Ipswich, MA) and 5 mM CaCl2 at 37 1C for 1 h and washed with at a multiplicity of infection (MOI) of 1 for 30 min at room PBS 5 times (23SD-hRBC). For resialylation of α2,3-linked sialic temperature. The wells were washed with PBS and then the cells acids, 10% of AUSD-hRBC were incubated with PBS containing 1% were cultured in 300 μl of SFM. After incubation at 37 1C for albumin from bovine serum (BSA; Sigma-Aldrich, St. Louis, MO), 2 days, the cells were washed with SFM and incubated with 0.5% 1.5 mg/ml of CMP-Sia (Nacalai Tesque, Kyoto, Japan) and 20 mU/ml each sialic acid linkage-modified hRBC in SFM for 30 min on ice. of ST3N, which was prepared from BmNPV bacmid-injected silk- The amount of hemoglobin was determined as described above. worm larvae as described previously (Ogata et al., 2009), at 37 1C Non-infected cells were tested as a negative control. for 16 h and washed with PBS 5 times (ST3N-hRBC) or were incubated with PBS containing 1.5 mg/ml of CMP-Sia and Virus infectivity assay for sialidase-treated cells 100 mU/ml of ST3 (Cosmo Bio Co., Ltd., Tokyo, Japan) at 37 1C for

17 h and washed with PBS 5 times (ST3-hRBC). For resialylation of Confluent monolayers of LLC-MK2 cells in a 12-well plate were α2,6-linked sialic acids, 10% of AUSD-hRBC were incubated with washed with PBS and treated with pH 5.5 SFM containing 40 mU/ PBS containing 1% BSA, 1.5 mg/ml of CMP-Sia and 20 mU/ml of ml of AUSD or 0, 40, 80 and 100 mU/ml of 23SD (TaKaRa Bio Inc., ST6N (Calbiochem, San Diego, CA) at 37 1C for 16 h and washed Shiga, Japan) at 37 1C for 2 h. The wells were washed with PBS and with PBS 5 times (ST6N-hRBC) or were incubated with PBS con- infected with each virus diluted by SFM at an MOI of 0.01 for 1 h taining 1.5 mg/ml of CMP-Sia and 75 mU/ml of ST6 (Cosmo Bio Co., on ice. The wells were washed with PBS and then the cells were Ltd., Tokyo, Japan) at 37 1C for 17 h and washed with PBS 5 times cultured in 1 ml of MEM containing 5% FBS. The plate was (ST6-hRBC). incubated at 37 1C for 12 h. The monolayers were fixed with 1 ml of cold methanol for 5 min. Virus-infected cells were detected by Hemagglutination assays probing with a rabbit anti-hPIV1 antibody or rabbit anti-hPIV3 antibody at room temperature for 30 min, followed by horseradish Each virus was serially diluted with 50 μl of PBS on a microtiter peroxidase (HRP)-conjugated Protein A (Sigma-Aldrich, St. Louis, plate. Fifty microliters of 0.8% (vol/vol) of each type of hRBC MO) at room temperature for 30 min as described previously (Normal-hRBC, AUSD-hRBC, 23SD-hRBC, ST3N-hRBC, ST3-hRBC, (Takahashi et al., 2012). Each step was preceded by washing the ST6N-hRBC and ST6-hRBC) in PBS was added to each well. The wells three times with PBS. Finally, the virus-infected cells were hemagglutination unit (HAU) was defined as the maximum dilu- stained by addition of an immunostaining reagent containing tion of virus that showed hemagglutination after 2 h. The plates H2O2, N,N-diethyl-p-phenylenediamine sulfate and 4-chloro-1- were kept on ice during the assays to prevent the viral sialidase naphthol in 100 mM citrate buffer (pH 6.0) as described previously and fusion activities (Conyers and Kidwell, 1991; Takahashi et al., 2008). Infectivity (%) was calculated as a percentage of the number of stained cells Plasmid construction of HN genes relative to the sum total of that in the sialidase-untreated well under 3 optical observations at a magnification of 100. HN genes were subcloned into the pCAGGS/MCS expression For the A549 cell line, confluent monolayers in a 24-well plate vector as described previously (Takaguchi et al., 2011). Briefly, each were washed with PBS and treated with pH 5.5 SFM containing viral RNA was isolated from virus particles with TRIzol reagent 50 U/ml of 23SD (New England BioLabs, Ipswich, MA) at 37 1C for (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's 2 h. The wells were washed with PBS and infected with each virus instructions. Full-length cDNAs of the HN gene from hPIV1 (HN1 diluted by SFM at an MOI of 0.01 for 30 min at room temperature. gene) and the HN gene from hPIV3 (HN3 gene) were amplified by The wells were washed with PBS and then the cells were cultured using a TaKaRa RNA PCR™ kit Ver. 3.0 (TaKaRa Bio Inc., Shiga, in 500 μl of SFM. The plate was incubated at 37 1C for 14 h. The Japan). The HN1 and HN3 genes were subcloned into the site monolayers were fixed with 500 μl of cold methanol for 5 min. between EcoR I and Xho I of the pCAGGS/MCS vector (pCAGGS- Virus-infected cells were detected by anti-hPIV antibodies and HN1 and pCAGGS-HN3, respectively). infectivity (%) was calculated as a percentage relative to the sialidase-untreated cells as described above. Hemadsorption assays To determine virus infectivity of hPIV clinical isolates, confluent

monolayers of LLC-MK2 cells in the 48-well plate were washed To determine the hemadsorption activity of HN glycoproteins, a with PBS and treated with pH 5.5 SFM containing 50 U/ml of 23SD confluent monolayer of COS-7 cells in a 48-well plate was (New England BioLabs, Ipswich, MA) or 40 mU/ml of AUSD at 37 1C transfected with 300 ng of pCAGGS-HN1 or pCAGGS-HN3 using for 2 h. The wells were washed with PBS and infected with hPIV1 the transfection reagent TransIT-LT1 (Mirus, Madison, WI) accord- and hPIV3 clinical isolates, CL-1381 or CL-918, diluted by SFM at an ing to the manufacturer's instructions. The cells were mock- MOI of 0.01 for 30 min at room temperature. The wells were treated without plasmids as a control. After incubation at 37 1C washed with PBS and then the cells were cultured in 300 μlof for 6 h, the transfected cells were washed with PBS and then SFM. The plate was incubated at 37 1C for 14 h. The monolayers cultured in 300 μl of a serum-free medium (Hybridoma-SFM; SFM, were fixed with 300 μl of cold methanol for 5 min. Virus-infected 430 K. Fukushima et al. / Virology 464-465 (2014) 424–431 cells were detected by anti-hPIV antibodies. Infectivity (%) was excitation of 488-nm line of an argon laser on a FACSCanto II flow calculated as a percentage relative to the sialidase-untreated cells cytometer. At least 1 104 cells were analyzed for each sample. as described above. Lectin staining Lectin binding assay Seventy percent confluent monolayers of Lec2 cells in the

A confluent monolayer of LLC-MK2 cells in the 12-well plate 96-well plate were resialylated with ST3 or ST6 as described was harvested by treatment with 0.125% trypsin and fixed with above. The cells were fixed with 2% paraformaldehyde for 4% paraformaldehyde for 15 min at room temperature. The cells 20 min at room temperature and were washed with PBS 3 times. were washed with PBS 3 times and treated with 100 mU/ml To determine α2,3-linked sialic acids, the cells were incubated of 23SD (TaKaRa Bio Inc., Shiga, Japan) or 40 mU/ml of AUSD at with PBS containing 100 μg/ml of biotin-conjugated Maackia 37 1C for 12 h. They were washed with PBS 3 times, incubated in amurensis lection II (MAL-II, Vector Laboratories, Inc., Burlingame, PBS containing 20 μg/ml of digoxigenin-conjugated SNA (Roche CA) at room temperature for 30 min, followed by 20 μg/ml of FITC- Applied Science, Mannheim, Germany) or 10 μg/ml of digoxigenin- conjugated streptavidin (Vector Laboratories, Inc., Burlingame, CA) conjugated MAA (Roche Applied Science, Mannheim, Germany) for at room temperature for 30 min. To determine α2,6-linked sialic 1 h at 4 1C, and then incubated with 50 μg/ml of fluorescein acids, the cells were incubated with PBS containing 20 μg/ml of isothiocyanate (FITC)-conjugated mouse anti-digoxin monoclonal FITC-conjugated SNA (Vector Laboratories, Inc., Burlingame, CA) at antibody (Sigma-Aldrich, St Louis, MO) for 1 h at 4 1C. Fluorescence room temperature for 30 min. Nuclei were stained with 2 μg/ml of for cells was measured with an excitation of the 488-nm line of an 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI; Dojindo argon laser on a FACSCanto II flow cytometer (BD, Franklin Lakes, Laboratories, Kumamoto, Japan) at room temperature for 30 min. NJ). At least 1 104 cells were analyzed for each sample. Lectin binding and nuclei staining were observed by using a fluorescent microscope (OLYMPUS IX71, Olympus, Tokyo, Japan). Virus infectivity assay for sialyltransferase-treated cells Statistical analysis To determine virus infectivity in resialylated Lec2 cells, 70% confluent monolayers of Lec2 cells in the 96-well plate were Results are shown as averages. Student's t-test was used to incubated with SFM containing 1.5 mM of CMP-Sia and 100 mU/ perform statistical analyses. ml of ST3 or 200 mU/ml of ST6 at 37 1C for 12 h. The cells were washed with PBS and infected with each virus diluted by SFM at an MOI of 0.1 for 30 min at room temperature. The wells were Acknowledgments washed with PBS and then with the cells were cultured in 50 μlof 1 SFM. The plate was incubated overnight at 37 C. The monolayers We thank Dr. Allen Portner (St. Jude Children's Research fi μ were xed with 100 l of cold methanol and virus-infected cells Hospital) for providing virus strains. Keijo Fukushima is a recipient were detected by anti-hPIV antibodies as described above. Infec- of a scholarship by Honjo International Scholarship Foundation. tivity (%) was calculated as a percentage relative to the parental This work was supported by JSPS KAKENHI, Series of single-year cell line Pro-5 cells of the Lec2 cells under 3 optical observations. grants, 2510690, in part, and by a MEXT/JSPS KAKENHI Grant To determine virus infectivity in resialylated A549 cells after (number C; 23590549). desialylation, 70% confluent monolayers of A549 cells in the 24-well plate were pretreated with 200 μM of a sialyltransferase inhibitor, ST-I (Millipore, Billerica, MA), for 4 days to deactivate Appendix A. Supporting information cellular sialyltransferase activity. The cells were washed with PBS 1 and incubated with 100 mU/ml of AUSD at 37 C for 3 h. Then the Supplementary data associated with this article can be found in cells were washed with PBS and incubated with SFM containing the online version at http://dx.doi.org/10.1016/j.virol.2014.07.033. 1.0 mM of CMP-Sia and 100 mU/ml of ST3 or 150 mU/ml of ST6 at 30 1C for 2 h. The cells were washed with PBS and infected with each virus diluted by SFM at an MOI of 0.01 for 30 min. The wells References were washed with PBS and then the cells were cultured in 500 μl of SFM. The plate was incubated overnight at 37 1C. The mono- Aminoff, D., Anderson, J., Dabich, L., Gathmann, W.D., 1980. Sialic acid content of fi μ erythrocytes in normal individuals and patients with certain hematologic layers were xed with 500 l of cold methanol and virus-infected disorders. Am. J. Hematol. 9, 381–389. cells were detected by anti-hPIV antibodies as described above. Amonsen, M., Smith, D.F., Cummings, R.D., Air, G.M., 2007. Human parainfluenza Infectivity (%) was calculated as a percentage relative to the ST-I- viruses hPIV1 and hPIV3 bind with α2-3-linked sialic acids fl treated and sialyltransferase-untreated cells under 3 optical that are distinct from those bound by H5 avian in uenza virus hemagglutinin. J. Virol. 81, 8341–8345. observations. Baum, L.G., Paulson, J.C., 1990. Sialyloligosaccharides of the respiratory epithelium in the selection of human influenza virus receptor specificity. Acta Histochem. Suppl. 40, 35–38. Virus binding assay Bratosin, D., Mazurier, J., Debray, H., Lecocq, M., Boilly, B., Alonso, C., Moisei, M., Motas, C., Montreuil, J., 1995. Flow cytofluorimetric analysis of young and Seventy percent confluent monolayers of Lec2 cells in the senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span. Glycoconj. J. 12, 258–267. 24-well plate were resialylated with ST3 or ST6 as described above Bulai, T., Bratosin, D., Pons, A., Montreuil, J., Zanetta, J.P., 2003. Diversity of the and harvested by treatment with 0.125% trypsin. The cells were human erythrocyte membrane sialic acids in relation with blood groups. FEBS 7 Lett. 534, 185–189. incubated in PBS containing 2 HAU of hPIV3 for 1 h on ice and fl – fi Chanock, R., 2001. Parain uenza viruses. Clin. Microbiol. Rev. 16, 242 264. xed with 4% paraformaldehyde for 30 min. The cells were Chu, F.-L., Wen, H.-L., Hou, G.-H., Lin, B., Zhang, W.-Q., Song, Y.-Y., Ren, G.-J., Sun, incubated in PBS containing rabbit anti-hPIV3 antibody for C.-X., Li, Z.-M., Wang, Z., 2013. Role of N-linked of the human 30 min on ice and then with phycoerythrin (PE)-conjugated goat parainfluenza virus type 3 hemagglutinin-neuraminidase protein. Virus Res. 174, 137–147. anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for Conyers, S.M., Kidwell, D.A., 1991. Chromogenic substrates for horseradish perox- 30 min on ice. Fluorescence for cells was measured with an idase. Anal. Biochem. 192, 207–211. K. Fukushima et al. / Virology 464-465 (2014) 424–431 431

Hoyer, L.L., Roggentin, P., Schauer, R., Vimr, E.R., 1991. Purification and properties of Suzuki, T., Ikeda, K., Koyama, N., Hosokawa, C., Kogure, T., Takahashi, T., Jwa Hidari, cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic pre- K.I., Miyamoto, D., Tanaka, K., Suzuki, Y., 2001a. Inhibition of human parain- ference for sialyl alpha 2→3 linkages. J. Biochem. 110, 462–467. fluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetyl- Inaba, N., Hiruma, T., Togayachi, A., Iwasaki, H., Wang, X.-H., Furukawa, Y., Sumi, R., . Glycoconj. J. 18, 331–337. Kudo, T., Fujimura, K., Iwai, T., Gotoh, M., Nakamura, M., Narimatsu, H., 2003. Suzuki, T., Portner, A., Scroggs, R.A., Uchikawa, M., Koyama, N., Matsuo, K., Suzuki, b A novel I-branching -1,6-N-acetylglucosaminyltransferase involved in human Y., Takimoto, T., 2001b. Receptor specificities of human respiroviruses. J. Virol. – blood group I antigen expression. Blood 101, 2870 2876. 75, 4604–4613. Kaludov, N., Brown, K.E., Walters, R.W., Zabner, J., Chiorini, J.A., 2001. Adeno- Suzuki, Y., 2005. Sialobiology of influenza: molecular mechanism of host range associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding variation of influenza viruses. Biol. Pharm. Bull. 28, 399–408. fi for hemagglutination and ef cient transduction but differ in sialic acid linkage Suzuki, Y., Ito, T., Suzuki, T., Holland, R.E., Chambers, T.M., Kiso, M., Ishida, H., specificity. J. Virol. 75, 6884–6893. Kawaoka, Y., 2000. Sialic acid species as a determinant of the host range of Kirsch, S., Müthing, J., Peter-Katalinić, J., Bindila, L., 2009. On-line nano-HPLC/ESI influenza A viruses. J. Virol. 74, 11825–11831. QTOF MS monitoring of α2-3 and α2-6 sialylation in granulocyte glyco- Suzuki, Y., Suzuki, T., Matsunaga, M., Matsumoto, M., 1985. Gangliosides as sphingolipidome. Biol. Chem. 390, 657–672. paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in Lee, Y.C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T., Tsuji, S., receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle 1994. Cloning and expression of cDNA for a new type of Galb1,3GalNAc alpha – 2,3-sialyltransferase. J. Biol. Chem. 269, 10028–10033. disease virus. J. Biochem. 97, 1189 1199. Loveless, R.W., Feizi, T., 1989. Sialo-oligosaccharide receptors for Mycoplasma Takaguchi, M., Takahashi, T., Hosokawa, C., Ueyama, H., Fukushima, K., Hayakawa, T., pneumoniae and related oligosaccharides of poly-N-acetyllactosamine series Itoh, K., Ikeda, K., Suzuki, T., 2011. A single amino acid mutation at position 170 fl are polarized at the cilia and apical-microvillar domains of the ciliated cells in of human parain uenza virus type 1 fusion glycoprotein induces obvious human bronchial epithelium. Infect. Immun. 57, 1285–1289. syncytium formation and caspase-3-dependent cell death. J. Biochem. 149, Markwell, M.A., Paulson, J.C., 1980. Sendai virus utilizes specific sialyloligosacchar- 191–202. ides as host cell receptor determinants. Proc. Natl. Acad. Sci. USA 77, Takahashi, T., Ito, K., Fukushima, K., Takaguchi, M., Hayakawa, T., Suzuki, Y., Suzuki, 5693–5697. T., 2012. negatively regulates the fusion process of human parain- Markwell, M.A., Svennerholm, L., Paulson, J.C., 1981. Specific gangliosides function fluenza virus type 3. J. Biochem. 152, 373–380. as host cell receptors for Sendai virus. Proc. Natl. Acad. Sci. USA 78, 5406–5410. Takahashi, T., Murakami, K., Nagakura, M., Kishita, H., Watanabe, S., Honke, K., Matrosovich, M.N., Gambaryan, A.S., Teneberg, S., Piskarev, V.E., Yamnikova, S.S., Ogura, K., Tai, T., Kawasaki, K., Miyamoto, D., Hidari, K.I.P.J., Guo, C.-T., Suzuki, Y., Lvov, D.K., Robertson, J.S., Karlsson, K.A., 1997. Avian influenza A viruses differ Suzuki, T., 2008. Sulfatide is required for efficient replication of influenza A from human viruses by recognition of sialyloligosaccharides and gangliosides virus. J. Virol. 82, 5940–5950. and by a higher conservation of the HA receptor-binding site. Virology 233, Tozawa, H., Watanabe, M., Ishida, N., 1973. Structural components of Sendai virus. 224–234. Serological and physicochemical characterization of hemagglutinin subunit Ogata, M., Nakajima, M., Kato, T., Obara, T., Yagi, H., Kato, K., Usui, T., Park, E.Y., 2009. associated with neuraminidase activity. Virology 55, 242–253. Synthesis of sialoglycopolypeptide for potentially blocking influenza virus fi α Tsukamoto, H., Takakura, Y., Yamamoto, T., 2007. Puri cation, cloning, and expres- infection using a rat 2,6-sialyltransferase expressed in BmNPV bacmid- sion of an α/b-galactoside α-2,3-sialyltransferase from a luminous marine injected silkworm larvae. BMC Biotechnol. 9, 54. bacterium, Photobacterium phosphoreum. J. Biol. Chem. 282, 29794–29802. Rillahan, C.D., Antonopoulos, A., Lefort, C.T., Sonon, R., Azadi, P., Ley, K., Dell, A., Uchida, Y., Tsukada, Y., Sugimori, T., 1979. Enzymatic properties of Haslam, S.M., Paulson, J.C., 2012. Global metabolic inhibitors of sialyl- and from Arthrobacter ureafaciens. J. Biochem. 86, 1573–1585. fucosyltransferases remodel the glycome. Nat. Chem. Biol. 8, 661–668. Vierbuchen, M., Uhlenbruck, G., Ortmann, M., Dufhues, G., Fischer, R., 1988. Rogerieux, F., Belaise, M., Terzidis-Trabelsi, H., Greffard, A., Pilatte, Y., Lambré, C.R., Occurrence and distribution of glycoconjugates in human tissues as detected 1993. Determination of the sialic acid linkage specificity of sialidases using by the Erythrina cristagalli lectin. J. Histochem. Cytochem. 36, 367–376. lectins in a solid phase assay. Anal. Biochem. 211, 200–204. Williams, M.A., Kitagawa, H., Datta, A.K., Paulson, J.C., Jamieson, J.C., 1995. Large- Sánchez-Felipe, L., Villar, E., Muñoz-Barroso, I., 2012. α2-3- and α2-6- N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target scale expression of recombinant sialyltransferases and comparison of their – cells. Glycoconj. J. 29, 539–549. kinetic properties with native enzymes. Glycoconj. J. 12, 755 761. fi Scheid, A., Caliguiri, L.A., Compans, R.W., Choppin, P.W., 1972. Isolation of para- Yamamoto, T., Nakashizuka, M., Kodama, H., Kajihara, Y., Terada, I., 1996. Puri ca- b α myxovirus glycoproteins. Association of both hemagglutinating and neurami- tion and characterization of a marine bacterial -galactoside 2,6-sialyltrans- nidase activities with the larger SV5 glycoprotein. Virology 50, 640–652. ferase from Photobacterium damsela JT0160. J. Biochem. 120, 104–110. Sinaniotis, C., 2004. Viral pneumoniae in children: incidence and aetiology. Yamamoto, T., Nakashizuka, M., Terada, I., 1998. Cloning and expression of a marine Paediatr. Respir. Rev. 5, S197–S200. bacterial b-galactoside α2,6-sialyltransferase gene from Photobacterium dam- Song, X., Yu, H., Chen, X., Lasanajak, Y., Tappert, M.M., Air, G.M., Tiwari, V.K., Cao, H., sela JT0160. J. Biochem. 123, 94–100. Chokhawala, H.a., Zheng, H., Cummings, R.D., Smith, D.F., 2011. A sialylated Zhang, L., Bukreyev, A., Thompson, C.I., Watson, B., Peeples, M.E., Collins, P.L., microarray reveals novel interactions of modified sialic acids with Pickles, R.J., 2005. Infection of ciliated cells by human parainfluenza virus type proteins and viruses. J. Biol. Chem. 286, 31610–31622. 3 in an in vitro model of human airway epithelium. Society 79, 1113–1124.