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Surface localization of the nuclear receptor CAR in influenza A virus-infected cells
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Biochemical and Biophysical Research Communications 368 (2008) 550–555 www.elsevier.com/locate/ybbrc
Surface localization of the nuclear receptor CAR in influenza A virus-infected cells Tadanobu Takahashi a,b, Yusuke Moriyama a, Akira Ikari a, Junko Sugatani a, Takashi Suzuki b, Masao Miwa a,* a
Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and COE Program in the 21st Century, 52-1 Yada, Shizuoka 422-8526, Japan b Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526, Japan Received 8 January 2008 Available online 7 February 2008
Abstract Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Nuclear receptor; CAR; Cell surface; Hemagglutinin; Influenza virus; Neuraminidase
CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes, cytochrome P450 genes [1]. It is retained in the cytosol under normal conditions and is translocated into the nucleus upon stimuli such as xenobiotic exposure [2]. Recently, interactions between CAR and apoptosis signaling, in addition to the established effects of CAR on metabolic regulation in vivo, have been discussed [3]. Studies of biological functions of CAR other than metabolic regulation have not included studies of the involvement of CAR in virus infection. To address this issue, we examined the localization of CAR in tissue culture cells infected with influenza A virus (IAV). IAV is a zoonotic virus that can infect a wide spectrum of hosts, including human and avian hosts. IAV possesses two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and the most abundant internal protein, nucleoprotein (NP). Based on the antigenicity of viral envelope proteins, HA is classified *
Corresponding author. Fax: +81 054 264 5779. E-mail address: [email protected] (M. Miwa).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.145
into 16 subtypes and NA into 9 subtypes. In infected cells, newly synthesized HA and NA are preferentially localized in the apical cell surface membrane, whereas NP is localized in the cell nucleus [4,5]. Here, we found that CAR was strongly localized at the cell surface in IAV-infected cells. Although Koike et al. have reported that CAR is localized at the cell surface in the mouse liver in vivo [6], our finding is the first report to demonstrate the apparent localization of CAR in a patch-like pattern at the cell surface in vitro, and to suggest an interaction between CAR and IAV infection. Additionally, we demonstrated that the expression of either HA or NA, but not NP, was responsible for this localization. At the cell surface, CAR may have a novel function associated with the IAV infection mechanism, distinct from its established function in metabolic enzyme regulation. Materials and methods Materials. Human IAV A/Memphis/1/71 (H3N2) (M71) and avian IAV A/duck/HK/313/4/78 (H5N3) (D313) were propagated in 10-day-old
T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555 embryonated hens’ eggs for 2 days at 34 °C and used at biosafety level 2. 293T cells, A549 cells, and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS). Virus infection. Cells were washed with phosphate-buffered saline (PBS) (pH 7.2, 131 mM NaCl, 14 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl) and infected with multiplicity of infection (moi) of 68–272 plaque forming units (pfu)/cell (for confocal microscopy) or 17 pfu/cell (for FACS analysis) of M71, or with moi of 4.4 103 pfu/cell of D313 in serum-free DMEM at 37 °C for 30 min. After washing with PBS, infected cells were maintained in medium supplemented with 1% FCS at 37 °C for the indicated times. Infectious virus titers (pfu) were measured by using Madin-Darby canine kidney MDCK cells as previously described [7]. Plasmids and transfection. pCAGGS-Mem71HA was generated by insertion of IAV M71 HA cDNA (GenBank Accession No. AB298687) into the multicloning site between the KpnI and XmaI sites of expression vector pCAGGS/MCS. pCAGGS-Mem71NA and pCAGGS-WSN-NP0/ 4 contain of M71 NA and A/WSN/33 (H1N1) NP cDNA, respectively [7– 10]. pCR3-hCAR contains of human CAR cDNA [11]. These vectors (0.5 or 0.2 lg) were transfected into 293T cells in 24-well tissue culture plates or six-well glass coverslips (Teflon printed glass slides, Erie Scientific Company, Portsmouth, NH), respectively, by using TransIT-293 (Panvera, Madison, WI) according to the manufacturer’s instructions. After 6 h, cells were washed with PBS and placed in fresh medium supplemented with 1% FCS.
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Confocal fluorescence microscopy. To visualize the distribution of viral proteins and CAR, infected or transfected cells (5 103 cells/well) grown on six-well glass coverslips were fixed with cold methanol for 30 s or 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. The fixed cells were incubated with rabbit anti-CAR polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-NP (4E6), -HA (2E10 or 2G3), or -NA (SI-4) monoclonal antibodies (IgG), or mouse anti-CAR monoclonal antibody (Perceus Proteomics Inc., Japan) and anti-IAV (H3N2) polyclonal antibody from immunized rabbit at 4 °C for 1 h. These anti-IAV monoclonal antibodies against M71 or D313 were obtained by a procedure described previously [12]. Cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) and tetramethyl rhodamine (TRITC)-conjugated goat anti-mouse IgG (Sigma–Aldrich Corp., St. Louis, MO) at 4 °C for 1 h. Nuclei were visualized with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Dojindo Laboratories, Kumamoto, Japan). Stained cells were observed under a confocal microscope (LSM 510 meta; Carl Zeiss Inc., Thornwood, NY). Fluorescence-activated cell sorting (FACS) analysis. 293T cells (5 104 cells/well) in the supernatant and on 24-well plates were collected by suspension and centrifugation. Cells were stained as described above. The fluorescence of cells was measured using a flow cytometer BD FACSCanto II (BD Biosciences, San Jose, CA). At least 7000 cells were analyzed for each sample.
Fig. 1. The cell surface localization of CAR in IAV-infected cells. 293T cells were infected with human IAV M71 (A,B) or avian IAV D313 (A,C) for 24 h or 12 h, respectively. Cells were fixed with methanol (A,C) or PFA (B) and stained with mouse anti-HA or NP (red) and rabbit anti-CAR (green). Nuclei were stained with DAPI (blue). Scale bar is 20 lm. The arrows show the cell surface localization of CAR in infected cells. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
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T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555
Results The cell surface localization of CAR in IAV-infected cells We investigated the localization of CAR in tissue culture cells infected with human or avian IAV. In non-infected cells, CAR was mostly localized in the cytosol and nucleus, as previously reported. However, in infected cells, CAR was localized at the cell surface, where HA was also distributed at the apical cell surface membrane (Fig. 1). This localization of CAR was shown in infected A549 cells derived from lung tissues, which were effectively infected by clinical IAV strains [13] (data not shown), and in infected 293T cells, which were utilized for an IAV reverse genetics system to produce infectious virus [8] (Fig. 1A). These cells showed a much lower CAR mRNA expression
level than cells derived from liver tissues [14,15], and CAR exhibited an apparent cell surface localization with a patchlike pattern, unlike HA, which was distributed in a uniform pattern. This pattern of CAR is thought to indicate its clustering at the cell surface. Similar localization of CAR was also detected in infected HepG2 cells, which have often been utilized in studies of CAR (data not shown). Staining of the cell surface only by PFA fixation showed that CAR was localized at the cell surface (Fig. 1B). CAR and viral NP staining in the infected cells indicate that anti-CAR antibody used here have no cross-reactivity and non-specificity with anti-HA antibody (Fig. 1C). Additionally, the lack of difference of CAR localization between human IAV M71 (H3N2) infection and avian IAV D313 (H5N3) infection indicates that the cell surface localization of CAR was independent of the viral host and subtype.
Fig. 2. The cell surface localization of CAR during expression of newly synthesized HA during virus infection. 293T cells were infected with M71. (A) Cells were fixed with PFA at 2, 4, and 6 h p.i. and stained with mouse anti-HA (red) and rabbit anti-CAR (green). Nuclei were stained with DAPI (blue). Scale bar is 20 lm. The arrows show the cell surface localization of CAR in infected cells. (B) Infected cells were fixed with PFA at 2, 4, and 6 h p.i. and stained with mouse anti-HA (vertical axis) and rabbit anti-CAR (horizontal axis). HA and CAR at the cell surface were measured by using FACS analysis. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555
The cell surface localization of CAR occurs during expression of newly synthesized HA during virus infection We investigated at which stage of the IAV infection cycle the cell surface localization of CAR occurs. At 2 h postinfection (p.i.), no newly synthesized HA was expressed. At 4 h p.i., CAR was weakly detected at the cell
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surface, where HA was also localized. At 6 h p.i., HA and CAR were both clearly found at the cell surface (Fig. 2A). Quantitative FACS analysis of proteins at the cell surface revealed that CAR was strongly detected in all HA-positive cells (infected cells) at 4 and 6 h p.i. (Fig. 2B). These results indicate that the cell surface localization of CAR occurs when HA is expressed in virus-infected cells.
Fig. 3. The cell surface localization of CAR in HA- or NA-expressing cells. Cells were transfected with the HA, NA, or NP gene, together with the CAR gene. After 24 h, cells were fixed with methanol (A,C) or PFA (B). 293T cells in A and B were stained with mouse anti-HA, NA, or NP (red) and rabbit anti-CAR (green). HepG2 cells in C were stained with mouse anti-CAR monoclonal antibody (red) and rabbit anti-HA polyclonal antibody (green). Nuclei were stained with DAPI (blue). Scale bar is 20 lm. The arrows show the cell surface localization of CAR in HA- or NA-expressing cells. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
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T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555
Expression of either HA or NA is responsible for the cell surface localization of CAR To identify the viral component(s) responsible for the cell surface localization of CAR, we investigated the localization of CAR in cells transfected with the HA, NA, or NP gene, together with the human CAR gene. Staining of permeabilized cells revealed that CAR was mostly localized at the cell surface in a patch-like pattern in HA- or NA-expressing cells, but not in NP-expressing cells (Fig. 3A). In non-transfected and NP-expressing cells, CAR was mostly localized in the cytosol and nucleus. When only the cell surface was stained, CAR was detected together with either HA or NA (Fig. 3B). These results indicate that expression of either HA or NA is responsible for the cell surface localization of CAR. The same result was obtained when using mouse anti-CAR monoclonal antibody and rabbit anti-IAV polyclonal antibody in HA-expressed HepG2 cells, supporting the strict specificity of each antibody (Fig. 3C). Weak apparent localization of CAR was shown in 293T cells transfected with HA gene only. This suggests that the virus infection may increase CAR expression. We confirmed the cell surface localization of CAR in HA- or NA-expressing cells quantitatively by using FACS analysis. At the surface of cells transfected with the HA or NA gene together with the CAR gene, almost all cells positive for CAR exhibited expression of HA (Fig. 4A and B) or NA (Fig. 4C and D). These results
further support the conclusion that expression of either HA or NA is responsible for the cell surface localization of CAR. Discussion We found that CAR was localized on the cell surface in IAV-infected cells. Expression of either HA or NA during virus infection was responsible for this localization. Koike et al. published the only report showing that CAR exists at the cell surface membrane in the mouse liver in vivo [10]. Our present study supports their finding and is the first study to show this novel localization of CAR in tissue culture cells in vitro and to suggest an interaction between CAR and IAV infection. Furthermore, it also suggests a novel function of CAR at the cell surface, distinct from its established function in xenobiotic metabolism regulation. Its biological significance of the localization of CAR at the cell surface remains unknown, and is of great interest. Some nuclear receptors for steroid hormones are transferred to the plasma membrane [16]. Plasma membraneassociated estrogen receptor contributes to transcription signaling as well as to the functional modulation of lipid raft-associated enzymes and intracellular signaling (a ‘‘non-genomic” effect). CAR at the cell surface may have non-genomic effects, such as contributing to efficient IAV replication. For example, CAR may help HA and NA to translocate to the cell surface membrane.
Fig. 4. Measurement of the cell surface localization of CAR in HA- or NA-expressing cells by using FACS analysis. 293T cells were transfected with the HA (A,B) or NA gene (C,D), together with the CAR gene. After 24 h, cells were fixed with PFA and stained with mouse anti-HA (vertical axis) and rabbit anti-CAR (horizontal axis). HA, NA, and CAR at the cell surface were measured by using FACS analysis. Representative data are shown in (A) and (C). Mean and standard deviation of cell counts (% of total cells) within each quadrant region in three independent experiments are shown in (B) and (D). 1, upper left; 2, upper right; 3, lower left; 4, lower right.
T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555
Lipid rafts are sphingolipid- and cholesterol-enriched microdomains on the plasma membrane. Some nuclear receptors are known to interact with lipid rafts [16]. In fact, the localization of CAR at the cell surface was consistent with the localization of caveolin, a marker of lipid rafts [6]. In this study, the localization of CAR in a patch-like pattern may have indicated its clustering at the cell surface, further suggesting the association of CAR with lipid rafts. As the virus buds from lipid rafts-enriched domain of the cell surface membrane, CAR may provide the efficient virus budding associated with lipid rafts. These further investigations will reveal a novel mechanism induced by IAV infection, and may contribute to the development of novel therapy against IAV, including avian H5 strain, as well as revealing a novel function of CAR. Acknowledgment
[6]
[7]
[8]
[9]
[10]
[11]
This work was supported by a grant from the University of Shizuoka (No. 3). References [1] P. Honkakoski, I. Zelko, Y. Sueyoshi, M. Negishi, The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the Cyp 2b10 gene, Mol. Cell. Biol. 18 (1998) 5652–5658. [2] T. Kawamoto, T. Sueyoshi, I. Zelko, R. Moore, K. Washburn, M. Negishi, Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene, Mol. Cell. Biol. 19 (1999) 6318–6322. [3] E.S. Baskin-Bey, W. Huang, N. Ishimura, H. Isomoto, S.F. Bronk, K. Braley, R.W. Craig, D.D. Moore, G.J. Gores, Constitutive androstane receptor (CAR) ligand, TCPOBOP, attenuates Fasinduced murine liver injury by altering Bcl-2 proteins, Hepatology 44 (2006) 252–262. [4] Y. Suzuki, Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses, Biol. Pharm. Bull. 28 (2005) 399– 408. [5] H. Marjuki, M.I. Alam, C. Ehrhardt, R. Wagner, O. Planz, H.D. Klenk, S. Ludwig, S. Pleschka, Membrane accumulation of influenza A virus hemagglutinin triggers nuclear export of the viral genome via
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protein kinase Calpha-mediated activation of ERK signaling, J. Biol. Chem. 281 (2006) 16707–16715. C. Koike, R. Moore, M. Negishi, Localization of the nuclear receptor CAR at the cell membrane of mouse liver, FEBS Lett. 579 (2005) 6733–6736. T. Suzuki, T. Takahashi, C.T. Guo, K.I. Hidari, D. Miyamoto, H. Goto, Y. Kawaoka, Y. Suzuki, Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication, J. Virol. 79 (2005) 11705–11715. G. Neumann, T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, M. Hughes, D.R. Perez, R. Donis, E. Hoffmann, G. Hobom, Y. Kawaoka, Generation of influenza A viruses entirely from cloned cDNAs, Proc. Natl. Acad. Sci. USA 96 (1999) 9345–9350. T. Takahashi, T. Suzuki, K.I. Hidari, D. Miyamoto, Y. Suzuki, A molecular mechanism for the low-pH stability of sialidase activity of influenza A virus N2 neuraminidases, FEBS Lett. 543 (2003) 71–75. T. Suzuki, T. Takahashi, T. Saito, C.T. Guo, K.I. Hidari, D. Miyamoto, Y. Suzuki, Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity, FEBS Lett. 557 (2004) 228–232. J. Sugatani, S. Nishitani, K. Yamakawa, K. Yoshinari, T. Sueyoshi, M. Negishi, M. Miwa, Transcriptional regulation of human UGT1A1 gene expression: Activated glucocorticoid receptor enhances constitutive androstane receptor/pregnane X receptor-mediated UDPglucuronosyltransferase 1A1 regulation with glucocorticoid receptor-interacting protein 1, Mol. Pharmacol. 67 (2005) 845–855. D. Miyamoto, T. Ueno, S. Takashima, K. Ohta, T. Miyawaki, T. Suzuki, Y. Suzuki, Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt’s lymphoma and germinal centre B cells, Glycoconj. J. 14 (1997) 379–388. Y.T. Huang, B.M. Turchek, Mink lung cells and mixed mink lung and A549 cells for rapid detection of influenza virus and other respiratory viruses, J. Clin. Microbiol. 38 (2000) 422–423. Z. Dovorak, M. Modriansky, L. Pichard-Garcia, P. Balaguer, M.J. Vilarem, J. Ulrichova, P. Maurel, J.M. Pascussi, Colchicine downregulates cytochrome P450 2B6, 2C8, 2C9, and 3A4 in human hepatocytes by affecting their glucocorticoid receptor-mediated regulation, Mol. Pharmacol. 64 (2003) 160–169. R. Huang, D.J. Murry, D. Kolwankar, S.D. Hall, D.R. Foster, Vincristine transcriptional regulation of efflux drug transporters in carcinoma cell lines, Biochem. Pharmacol. 71 (2006) 1695–1704. D.C. Marquez, H.W. Chen, E.M. Curran, W.V. Welshons, R.J. Pietras, Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer, Mol. Cell. Endocrinol. 246 (2006) 91– 100.
Biochemical and Biophysical Research Communications 368 (2008) 550–555 www.elsevier.com/locate/ybbrc
Surface localization of the nuclear receptor CAR in influenza A virus-infected cells Tadanobu Takahashi a,b, Yusuke Moriyama a, Akira Ikari a, Junko Sugatani a, Takashi Suzuki b, Masao Miwa a,* a
Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and COE Program in the 21st Century, 52-1 Yada, Shizuoka 422-8526, Japan b Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526, Japan Received 8 January 2008 Available online 7 February 2008
Abstract Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Nuclear receptor; CAR; Cell surface; Hemagglutinin; Influenza virus; Neuraminidase
CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes, cytochrome P450 genes [1]. It is retained in the cytosol under normal conditions and is translocated into the nucleus upon stimuli such as xenobiotic exposure [2]. Recently, interactions between CAR and apoptosis signaling, in addition to the established effects of CAR on metabolic regulation in vivo, have been discussed [3]. Studies of biological functions of CAR other than metabolic regulation have not included studies of the involvement of CAR in virus infection. To address this issue, we examined the localization of CAR in tissue culture cells infected with influenza A virus (IAV). IAV is a zoonotic virus that can infect a wide spectrum of hosts, including human and avian hosts. IAV possesses two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and the most abundant internal protein, nucleoprotein (NP). Based on the antigenicity of viral envelope proteins, HA is classified *
Corresponding author. Fax: +81 054 264 5779. E-mail address: [email protected] (M. Miwa).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.145
into 16 subtypes and NA into 9 subtypes. In infected cells, newly synthesized HA and NA are preferentially localized in the apical cell surface membrane, whereas NP is localized in the cell nucleus [4,5]. Here, we found that CAR was strongly localized at the cell surface in IAV-infected cells. Although Koike et al. have reported that CAR is localized at the cell surface in the mouse liver in vivo [6], our finding is the first report to demonstrate the apparent localization of CAR in a patch-like pattern at the cell surface in vitro, and to suggest an interaction between CAR and IAV infection. Additionally, we demonstrated that the expression of either HA or NA, but not NP, was responsible for this localization. At the cell surface, CAR may have a novel function associated with the IAV infection mechanism, distinct from its established function in metabolic enzyme regulation. Materials and methods Materials. Human IAV A/Memphis/1/71 (H3N2) (M71) and avian IAV A/duck/HK/313/4/78 (H5N3) (D313) were propagated in 10-day-old
T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555 embryonated hens’ eggs for 2 days at 34 °C and used at biosafety level 2. 293T cells, A549 cells, and HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS). Virus infection. Cells were washed with phosphate-buffered saline (PBS) (pH 7.2, 131 mM NaCl, 14 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl) and infected with multiplicity of infection (moi) of 68–272 plaque forming units (pfu)/cell (for confocal microscopy) or 17 pfu/cell (for FACS analysis) of M71, or with moi of 4.4 103 pfu/cell of D313 in serum-free DMEM at 37 °C for 30 min. After washing with PBS, infected cells were maintained in medium supplemented with 1% FCS at 37 °C for the indicated times. Infectious virus titers (pfu) were measured by using Madin-Darby canine kidney MDCK cells as previously described [7]. Plasmids and transfection. pCAGGS-Mem71HA was generated by insertion of IAV M71 HA cDNA (GenBank Accession No. AB298687) into the multicloning site between the KpnI and XmaI sites of expression vector pCAGGS/MCS. pCAGGS-Mem71NA and pCAGGS-WSN-NP0/ 4 contain of M71 NA and A/WSN/33 (H1N1) NP cDNA, respectively [7– 10]. pCR3-hCAR contains of human CAR cDNA [11]. These vectors (0.5 or 0.2 lg) were transfected into 293T cells in 24-well tissue culture plates or six-well glass coverslips (Teflon printed glass slides, Erie Scientific Company, Portsmouth, NH), respectively, by using TransIT-293 (Panvera, Madison, WI) according to the manufacturer’s instructions. After 6 h, cells were washed with PBS and placed in fresh medium supplemented with 1% FCS.
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Confocal fluorescence microscopy. To visualize the distribution of viral proteins and CAR, infected or transfected cells (5 103 cells/well) grown on six-well glass coverslips were fixed with cold methanol for 30 s or 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. The fixed cells were incubated with rabbit anti-CAR polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-NP (4E6), -HA (2E10 or 2G3), or -NA (SI-4) monoclonal antibodies (IgG), or mouse anti-CAR monoclonal antibody (Perceus Proteomics Inc., Japan) and anti-IAV (H3N2) polyclonal antibody from immunized rabbit at 4 °C for 1 h. These anti-IAV monoclonal antibodies against M71 or D313 were obtained by a procedure described previously [12]. Cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) and tetramethyl rhodamine (TRITC)-conjugated goat anti-mouse IgG (Sigma–Aldrich Corp., St. Louis, MO) at 4 °C for 1 h. Nuclei were visualized with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Dojindo Laboratories, Kumamoto, Japan). Stained cells were observed under a confocal microscope (LSM 510 meta; Carl Zeiss Inc., Thornwood, NY). Fluorescence-activated cell sorting (FACS) analysis. 293T cells (5 104 cells/well) in the supernatant and on 24-well plates were collected by suspension and centrifugation. Cells were stained as described above. The fluorescence of cells was measured using a flow cytometer BD FACSCanto II (BD Biosciences, San Jose, CA). At least 7000 cells were analyzed for each sample.
Fig. 1. The cell surface localization of CAR in IAV-infected cells. 293T cells were infected with human IAV M71 (A,B) or avian IAV D313 (A,C) for 24 h or 12 h, respectively. Cells were fixed with methanol (A,C) or PFA (B) and stained with mouse anti-HA or NP (red) and rabbit anti-CAR (green). Nuclei were stained with DAPI (blue). Scale bar is 20 lm. The arrows show the cell surface localization of CAR in infected cells. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
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Results The cell surface localization of CAR in IAV-infected cells We investigated the localization of CAR in tissue culture cells infected with human or avian IAV. In non-infected cells, CAR was mostly localized in the cytosol and nucleus, as previously reported. However, in infected cells, CAR was localized at the cell surface, where HA was also distributed at the apical cell surface membrane (Fig. 1). This localization of CAR was shown in infected A549 cells derived from lung tissues, which were effectively infected by clinical IAV strains [13] (data not shown), and in infected 293T cells, which were utilized for an IAV reverse genetics system to produce infectious virus [8] (Fig. 1A). These cells showed a much lower CAR mRNA expression
level than cells derived from liver tissues [14,15], and CAR exhibited an apparent cell surface localization with a patchlike pattern, unlike HA, which was distributed in a uniform pattern. This pattern of CAR is thought to indicate its clustering at the cell surface. Similar localization of CAR was also detected in infected HepG2 cells, which have often been utilized in studies of CAR (data not shown). Staining of the cell surface only by PFA fixation showed that CAR was localized at the cell surface (Fig. 1B). CAR and viral NP staining in the infected cells indicate that anti-CAR antibody used here have no cross-reactivity and non-specificity with anti-HA antibody (Fig. 1C). Additionally, the lack of difference of CAR localization between human IAV M71 (H3N2) infection and avian IAV D313 (H5N3) infection indicates that the cell surface localization of CAR was independent of the viral host and subtype.
Fig. 2. The cell surface localization of CAR during expression of newly synthesized HA during virus infection. 293T cells were infected with M71. (A) Cells were fixed with PFA at 2, 4, and 6 h p.i. and stained with mouse anti-HA (red) and rabbit anti-CAR (green). Nuclei were stained with DAPI (blue). Scale bar is 20 lm. The arrows show the cell surface localization of CAR in infected cells. (B) Infected cells were fixed with PFA at 2, 4, and 6 h p.i. and stained with mouse anti-HA (vertical axis) and rabbit anti-CAR (horizontal axis). HA and CAR at the cell surface were measured by using FACS analysis. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
T. Takahashi et al. / Biochemical and Biophysical Research Communications 368 (2008) 550–555
The cell surface localization of CAR occurs during expression of newly synthesized HA during virus infection We investigated at which stage of the IAV infection cycle the cell surface localization of CAR occurs. At 2 h postinfection (p.i.), no newly synthesized HA was expressed. At 4 h p.i., CAR was weakly detected at the cell
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surface, where HA was also localized. At 6 h p.i., HA and CAR were both clearly found at the cell surface (Fig. 2A). Quantitative FACS analysis of proteins at the cell surface revealed that CAR was strongly detected in all HA-positive cells (infected cells) at 4 and 6 h p.i. (Fig. 2B). These results indicate that the cell surface localization of CAR occurs when HA is expressed in virus-infected cells.
Fig. 3. The cell surface localization of CAR in HA- or NA-expressing cells. Cells were transfected with the HA, NA, or NP gene, together with the CAR gene. After 24 h, cells were fixed with methanol (A,C) or PFA (B). 293T cells in A and B were stained with mouse anti-HA, NA, or NP (red) and rabbit anti-CAR (green). HepG2 cells in C were stained with mouse anti-CAR monoclonal antibody (red) and rabbit anti-HA polyclonal antibody (green). Nuclei were stained with DAPI (blue). Scale bar is 20 lm. The arrows show the cell surface localization of CAR in HA- or NA-expressing cells. (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)
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Expression of either HA or NA is responsible for the cell surface localization of CAR To identify the viral component(s) responsible for the cell surface localization of CAR, we investigated the localization of CAR in cells transfected with the HA, NA, or NP gene, together with the human CAR gene. Staining of permeabilized cells revealed that CAR was mostly localized at the cell surface in a patch-like pattern in HA- or NA-expressing cells, but not in NP-expressing cells (Fig. 3A). In non-transfected and NP-expressing cells, CAR was mostly localized in the cytosol and nucleus. When only the cell surface was stained, CAR was detected together with either HA or NA (Fig. 3B). These results indicate that expression of either HA or NA is responsible for the cell surface localization of CAR. The same result was obtained when using mouse anti-CAR monoclonal antibody and rabbit anti-IAV polyclonal antibody in HA-expressed HepG2 cells, supporting the strict specificity of each antibody (Fig. 3C). Weak apparent localization of CAR was shown in 293T cells transfected with HA gene only. This suggests that the virus infection may increase CAR expression. We confirmed the cell surface localization of CAR in HA- or NA-expressing cells quantitatively by using FACS analysis. At the surface of cells transfected with the HA or NA gene together with the CAR gene, almost all cells positive for CAR exhibited expression of HA (Fig. 4A and B) or NA (Fig. 4C and D). These results
further support the conclusion that expression of either HA or NA is responsible for the cell surface localization of CAR. Discussion We found that CAR was localized on the cell surface in IAV-infected cells. Expression of either HA or NA during virus infection was responsible for this localization. Koike et al. published the only report showing that CAR exists at the cell surface membrane in the mouse liver in vivo [10]. Our present study supports their finding and is the first study to show this novel localization of CAR in tissue culture cells in vitro and to suggest an interaction between CAR and IAV infection. Furthermore, it also suggests a novel function of CAR at the cell surface, distinct from its established function in xenobiotic metabolism regulation. Its biological significance of the localization of CAR at the cell surface remains unknown, and is of great interest. Some nuclear receptors for steroid hormones are transferred to the plasma membrane [16]. Plasma membraneassociated estrogen receptor contributes to transcription signaling as well as to the functional modulation of lipid raft-associated enzymes and intracellular signaling (a ‘‘non-genomic” effect). CAR at the cell surface may have non-genomic effects, such as contributing to efficient IAV replication. For example, CAR may help HA and NA to translocate to the cell surface membrane.
Fig. 4. Measurement of the cell surface localization of CAR in HA- or NA-expressing cells by using FACS analysis. 293T cells were transfected with the HA (A,B) or NA gene (C,D), together with the CAR gene. After 24 h, cells were fixed with PFA and stained with mouse anti-HA (vertical axis) and rabbit anti-CAR (horizontal axis). HA, NA, and CAR at the cell surface were measured by using FACS analysis. Representative data are shown in (A) and (C). Mean and standard deviation of cell counts (% of total cells) within each quadrant region in three independent experiments are shown in (B) and (D). 1, upper left; 2, upper right; 3, lower left; 4, lower right.
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Lipid rafts are sphingolipid- and cholesterol-enriched microdomains on the plasma membrane. Some nuclear receptors are known to interact with lipid rafts [16]. In fact, the localization of CAR at the cell surface was consistent with the localization of caveolin, a marker of lipid rafts [6]. In this study, the localization of CAR in a patch-like pattern may have indicated its clustering at the cell surface, further suggesting the association of CAR with lipid rafts. As the virus buds from lipid rafts-enriched domain of the cell surface membrane, CAR may provide the efficient virus budding associated with lipid rafts. These further investigations will reveal a novel mechanism induced by IAV infection, and may contribute to the development of novel therapy against IAV, including avian H5 strain, as well as revealing a novel function of CAR. Acknowledgment
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