Up-regulation of epithelial Na+ channel ENaC by human parvovirus B19 capsid protein VP1

Biochemical and Biophysical Research Communications 468 (2015) 179e184

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Up-regulation of epithelial Naþ channel ENaC by human parvovirus B19 capsid protein VP1 Musaab Ahmed a, Sabina Honisch a, Lisann Pelzl a, Myriam Fezai a, Zohreh Hosseinzadeh a, C.-Thomas Bock b, 1, Reinhard Kandolf b, 1, Florian Lang a, *, 1 a b

Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076 Tuebingen, Germany Department of Molecular Pathology, University of Tuebingen, Liebermeisterstraße 8, 72076 Tuebingen, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 October 2015 Accepted 26 October 2015 Available online 29 October 2015

Background: Clinical disorders caused by parvovirus B19 (B19V) infection include endothelial dysfunction with cardiac ischemia. The virus is effective in part by lysophosphatidylcholine-producing phospholipase A2 (PLA2) activity of B19V capsid protein VP1. Mechanisms compromising endothelial function include up-regulation of amiloride sensitive epithelial Naþ-channel ENaC leading to endothelial cell stiffness. Regulators of ENaC include ubiquitin-ligase Nedd4-2. The present study explored whether VP1 modifies ENaC-activity. Methods: cRNA encoding ENaC was injected into Xenopus oocytes without or with cRNA encoding VP1. Experiments were made with or without coexpression of Nedd4-2. ENaC activity was estimated from amiloride (50 mM) sensitive current. Results: Injection of cRNA encoding ENaC into Xenopus oocytes was followed by appearance of amiloride sensitive current, which was significantly enhanced by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on ENaC was mimicked by treatment of ENaC expressing oocytes with lysophosphatidylcholine (1 mg/ml). The effect of VP1 and lysophosphatidylcholine was not additive. ENaC activity was downregulated by Nedd4-2, an effect not reversed by VP1. Conclusions: The B19V capsid protein VP1 up-regulates ENaC, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine. © 2015 Elsevier Inc. All rights reserved.

Keywords: PVB19 Lysophosphatidylcholine Viral myocarditis Phospholipase A2 Oocyte Nedd4-2

1. Introduction Infections with the erythrovirus parvovirus B19 (B19V) [1] are common [2] and are followed by several clinical entities [3,4], including erythema infectiosum (fifth disease), hydrops fetalis and transient aplastic anaemia [5,6], arthritis [7,8], hepatitis [9,10], vasculitic syndromes [11,12], neurological disorders, and myocarditis [13,14]. In pregnancy B19V-infection may cause maternal and fetal myocarditis, congenital malformations, stillbirth and abortion [15,16]. B19V preferably invades proliferating cells thus leading to particularly severe disease during antenatal infection [17]. Entry of B19V into cells is mediated by blood group P-antigen

€t Tübingen, * Corresponding author. Physiologisches Institut, der Universita Gmelinstr. 5, D-72076 Tübingen. E-mail address: fl[email protected] (F. Lang). 1 CTB, RK and FL contributed equally to this study and thus share last authorship. http://dx.doi.org/10.1016/j.bbrc.2015.10.137 0006-291X/© 2015 Elsevier Inc. All rights reserved.

[18], a5b1 integrin and Ku80 autoantigen [19] and B19V preferably invades erythroid progenitor cells with strong P antigen, a5b1 integrin and Ku80 autoantigen expression. B19V further invades fetal myocytes, follicular dendritic cells and endothelial cells [18,19]. B19V-infection of endothelial cells [20] may result in B19Veassociated myocarditis and ventricular diastolic dysfunction [21]. Along those lines endothelial rather than myocardial B19V was observed in fatal inflammatory cardiomyopathy [22,23]. Consequences of endothelial B19V expression include E-selectin expression, margination, adherence, penetration, and perivascular infiltration of T-lymphocytes and macrophages in cardiac tissue [22,23]. Proteins encoded by the B19V genome include the structural capsid proteins VP1 and VP2 [24], which are required for the viral life cycle [6,25]. The VP1 protein contains a sequence homologous to the catalytic site and Ca2þ-binding loop of secreted phospholipase A2 (sPLA2) [26,27]. Accordingly, B19V PLA2 may generate

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eicosanoids [27,28], which are required for infectivity of the virus [27]. The vPLA2 enzyme activity is disrupted by replacement of the histidine at the position 153 with alanine (H153AVP1) [27,28]. Expression of wild type VP1 but not of H153AVP1 in endothelial cells up-regulates Ca2þ entry [29], an effect mimicked by PLA2 product lysophosphatidylcholine [29]. VP1 has further been shown to inhibit Naþ/Kþ ATPase activity [30] and to down-regulate Kþ channels [31,32], effects again lacking in H153AVP1 and mimicked by lysophosphatidylcholine [30e33]. Endothelial cells may express the epithelial Naþ channel ENaC, the activation of which could result in early swelling and later stiffening of those cells [34]. Endothelial cell stiffness counteracts the endothelial formation of nitric oxide (NO) and thus compromises the endothelial stimulation of vasodilation [34]. Thus, stiffening of endothelial cells is expected to impair tissue perfusion and increase blood pressure [34,35]. Along those lines inhibition of ENaC has been shown to enhance NO formation [34]. The present study explored, whether expression of VP1 influences ENaC activity. Additional experiments tested whether the effect of VP1 could be disrupted by loss of function mutations of the PLA sequence (H153AVP1) and could be mimicked by lysophosphatidylcholine. Moreover, experiments were performed to test whether the effect of VP1 involves the ubiquitin ligase Nedd4-2, a powerful negative regulator of ENaC [36e40]. 2. Materials and methods 2.1. Ethical Statement All experiments conform with the 'European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes' (Council of Europe No 123, Strasbourg 1985) and were conducted according to the German law for the welfare of animals and the surgical procedures on the adult Xenous laevis were reviewed and approved by the respective government authority of €sidium) prior to the the state Baden-Württemberg (Regierungspra start of the study (Anzeige für Organentnahme nach x 36). 2.2. Plasmids B19V DNA was isolated from a patient with fatal B19Vassociated inflammatory cardiomyopathy as described previously [29]. Constructs encoding a,b,gENaC [41], wild-type VP1 [29] and PLA2-negative H153AVP1 mutant [29] were used for generation of cRNA as described previously [42,43]. 2.3. Voltage clamp in Xenopus oocytes Xenopus oocytes were prepared as previously described [44]. cRNA encoding a,b,g subunits of ENaC (1 ng each) and cRNA encoding wild type VP1 or PLA2-negative H153AVP1 (10 ng) were injected on the day of Xenopus oocyte preparation. All experiments were performed at room temperature (about 22 C) 3 days after the injection [45]. Amiloride (50 mM) sensitive currents were determined by two-electrode voltage-clamp at a holding potential of 80 mV. The data were filtered at 1 kHz and recorded with a Digidata 1322A A/D-D/A converter and ClampexV.9.2 software for data acquisition (Axon Instruments). The analysis of the data was performed with Clampfit 9.2 (Axon Instruments) software [30,46]. The oocytes were maintained at 17 C in ND96 solution containing: 88.5 mM NaCl, 2 mM KCl, 1 mM MgC12, 1.8 mM CaC12, 5 mM HEPES. Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Refobacin (100 mg/ l), Theophylline (90 mg/l), and Sodium Pyruvate (5 mM) were added to the ND96, and pH was adjusted to 7.5 by addition of NaOH. The control superfusate (ND96) contained 96 mM NaCl, 2 mM KCl,

1.8 mM CaCl2,1 mM MgCl2 and 5 mM HEPES, pH was adjusted to 7.4 by addition of NaOH [47]. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [48,49]. 2.4. Statistical analysis Data are provided as means ± SEM, n represents the number of oocytes investigated. All experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using analysis of variance (ANOVA) or t-test, as appropriate. Results with p < 0.05 were considered statistically significant. 3. Results Dual electrode voltage clamp experiments in Xenopus oocytes were performed in order to explore whether the parvovirus B19 capsid protein VP1 is capable to alter the activity of the epithelial Naþ channel ENaC. Prior to measurements, ENaC was expressed in Xenopus oocytes with or without additional expression of VP1 and amiloride sensitive current taken as a measure of in ENaC activity. As illustrated in Fig. 1, amiloride sensitive current in ENaC expressing Xenopus oocytes was markedly increased by coexpression of wild type VP1 but not by coexpression of the H153AVP1 mutant lacking PLA2 activity. Accordingly, in ENaC expressing Xenopus oocytes the amiloride sensitive current was significantly higher following coexpression of wild type VP1 than following coexpression of H153AVP1 (Fig. 1). As phospholipase A2 of VP1 generates lysophosphatidylcholine, additional experiments were performed to explore whether lysophosphatidylcholine influences amiloride sensitive current in ENaC expressing Xenopus oocytes. As illustrated in Fig. 2, treatment of ENaC expressing Xenopus oocytes with lysophosphatidylcholine (1 mg/ml) for 5 min was indeed followed by a marked increase of amiloride sensitive current. Additional experiments tested whether the effects of VP1 coexpression and lysophosphatidylcholine treatment were additive. As illustrated in Fig. 3, the coexpression of VP1 again significantly increased the amiloride sensitive current. The treatment of ENaC and VP1 expressing Xenopus oocytes with lysophosphatidylcholine (1 mg/ml) for 5 min was without additional effect on the amiloride sensitive current. A powerful negative regulator of ENaC is the ubiquitin ligase Nedd4-2. Thus, VP1 could have been effective by reversing the effect of Nedd4-2 on ENaC. In order to test this possibility, Nedd4-2 was coexpressed in ENaC or VP1 and ENaC expressing Xenopus oocytes. As illustrated in Fig. 4, coexpression of Nedd4-2 downregulated the amiloride sensitive current to similarly low levels in Xenopus oocytes expressing ENaC alone and in Xenopus oocytes expressing ENaC together with VP1. 4. Discussion The present study discloses a novel action of the B19V capsid protein VP1, i.e. the up-regulation of the epithelial Naþ channel ENaC. The effect of VP1 is disrupted by replacement of the histidine by alanine in the putative catalytic site of the PLA2 motif (H153AVP1) [26,28]. The same mutation has previously been shown to abrogate the effect of VP1 on Ca2þ entry [29], Naþ/Kþ ATPase activity [30], and Kþ channels [31,32]. Moreover, the effect of VP1 expression was mimicked by the phospholipase A2 product lysophosphatidylcholine. The present observation may shed new light on the negative effect of B19V on endothelial function. The virus is known to enter

M. Ahmed et al. / Biochemical and Biophysical Research Communications 468 (2015) 179e184

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ENaC Fig. 1. Up-regulation of amiloride sensitive current in ENaC expressing Xenopus oocytes by coexpression of VP1 but not of H153AVP1. A. Original tracings recorded in Xenopus oocytes injected with cRNA encoding ENaC alone (a), cRNAs encoding both ENaC and wild type VP1 (b) and cRNAs encoding both ENaC and PLA2-negative H153AVP1 mutant (c). The currents were recorded at a holding potential of 80 mV. B. Arithmetic means ± SEM (n ¼ 13e14) of the amiloride sensitive current in Xenopus oocytes injected with cRNA encoding ENaC alone (white bar), cRNAs encoding both ENaC and wild type VP1 (black bar) and cRNAs encoding both ENaC and PLA2-negative H153AVP1 mutant (grey bar). *** (p < 0.001) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding ENaC alone, ### (p < 0.001) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding ENaC and wild type VP1 (ANOVA-one way).

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ENaC Fig. 2. Up-regulation of amiloride sensitive current in ENaC expressing Xenopus oocytes by treatment with lysophosphatidylcholine. A. Original tracings recorded in Xenopus oocytes injected with cRNA encoding ENaC in the absence (a) or presence (b) of lysophosphatidylcholine (1 mg/ml). The currents were recorded at a holding potential of 80 mV. B. Arithmetic means ± SEM (n ¼ 13e14) of the amiloride sensitive current in Xenopus oocytes injected with cRNA encoding ENaC without (white bar) or with (black bar) subsequent lysophosphatidylcholine treatment. *** (p < 0.001) indicates statistically significant difference from untreated Xenopus oocytes (ANOVA-one way).

myocardial endothelial cells [22,23] and trigger a clinical condition similar to myocardial infarction [22,23]. Stimulation of ENaC could lead to cell swelling and endothelial stiffnes [34,35] which impairs NO release and thus endothelial derived vascular relaxation

[34,35]. Excessive ENaC activity is known to trigger stiff endothelial cell syndrome [35] and could thus compromise myocardial perfusion following B19V infection. The effect of VP1 on ENaC is expected to be compounded by inhibition of Naþ/Kþ-ATPase [30], which

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ENaC Fig. 3. Lysophosphatidylcholine insensitivity of amiloride sensitive current in ENaC and VP1 expressing Xenopus oocytes. A. Original tracings recorded in Xenopus oocytes injected with cRNA encoding ENaC alone (a), and cRNAs encoding both ENaC and wild type VP1 in the absence (b) or presence (c) of lysophosphatidylcholine (1 mg/ml). The currents were recorded at a holding potential of 80 mV. B. Arithmetic means ± SEM (n ¼ 13e14) of the amiloride sensitive current in Xenopus oocytes injected with cRNA encoding ENaC alone (white bar), and cRNAs encoding both ENaC and wild type VP1 in the absence (black bar) or presence (grey bar) of lysophosphatidylcholine (1 mg/ml). *** (p < 0.001) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding ENaC alone (ANOVA-one way).

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ENaC Fig. 4. Down-regulation of amiloride sensitive current by Nedd4-2 in Xenopus oocytes expressing ENaC with or without VP1. A. Original tracings recorded in Xenopus oocytes injected with cRNA encoding ENaC alone (a), cRNAs encoding both ENaC and Nedd4-2 (b) and cRNAs encoding both ENaC and VP1without (c) and with (d) additional coexpression of Nedd4-2. The currents were recorded at a holding potential of 80 mV. B. Arithmetic means ± SEM (n ¼ 9e11) of the amiloride sensitive current in Xenopus oocytes injected with cRNA encoding ENaC alone (white bar), cRNAs encoding both ENaC and Nedd4-2 (light grey bar) and cRNAs encoding both ENaC and VP1without (black bar) and with (dark grey bar) additional coexpression of Nedd4-2. The currents were recorded at a holding potential of 80 mV *(p < 0.05), ** (p < 0.01) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding ENaC alone, ### (p < 0.001) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding ENaC and wild type VP1 (ANOVA-one way).

would dissipate the ion gradients across the cell membrane thus further compromising the ability of the cell to maintain cell volume constancy [30]. Moreover, endothelial cell swelling may be fostered by VP1-dependent down-regulation of Kþ channels [31,32], which would curtail Kþ exit through Kþ channels thus leading to depolarization and thus decrease of the electrical driving force for Cl

exit [50]. In view of the present data it is tempting to speculate that patients suffering from cardiac ischemia due to parvovirus B19 infection may benefit from treatment with aldosterone receptor antagonist spironolactone, as ENaC is upregulated by aldosterone [34,35] which thus sets the stage for lysophosphatidylcholine

M. Ahmed et al. / Biochemical and Biophysical Research Communications 468 (2015) 179e184

induced ENaC activation and subsequent presumed endothelial stiffening. In conclusion, parvovirus B19 capsid protein VP1 up-regulates the epithelial Naþ channel ENaC, an effect lacking following disruption of the PLA2 motif and mimicked by the PLA2 product lysophosphatidylcholine. As B19 enters endothelial cells, the virus could up-regulate endothelial ENaC activity leading to endothelial swelling and stiffening. 5. Disclosure Statement The funder played no role neither in study design, collection, analysis and interpretation of data, nor in the writing of the report and the decision to submit the article for publication. Acknowledgments The authors acknowledge the technical assistance of E. Faber and the meticulous preparation of the manuscript by Tanja Loch. This study was supported by the Deutsche Forschungsgemeinschaft, SFB-Transregio 19 (project TP B5). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.10.137. References [1] E.D. Heegaard, K.E. Brown, Human parvovirus B19, Clin. Microbiol. Rev. 15 (2002) 485e505. [2] K.E. Brown, N.S. Young, J.M. Liu, Molecular, cellular and clinical aspects of parvovirus B19 infection, Crit. Rev. Oncol. Hematol. 16 (1994) 1e31. [3] W.C. Koch, Fifth (human parvovirus) and sixth (herpesvirus 6) diseases, Curr. Opin. Infect. Dis. 14 (2001) 343e356. [4] H.W. Lehmann, A. Knoll, R.M. Kuster, S. Modrow, Frequent infection with a viral pathogen, parvovirus B19, in rheumatic diseases of childhood, Arthr. Rheum. 48 (2003) 1631e1638. [5] M.J. Anderson, S.E. Jones, S.P. Fisher-Hoch, E. Lewis, S.M. Hall, C.L. Bartlett, B.J. Cohen, P.P. Mortimer, M.S. Pereira, Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet 1 (1983) 1378. [6] N.S. Young, K.E. Brown, Parvovirus B19, N. Engl. J. Med. 350 (2004) 586e597. [7] D. Dingli, D.H. Pfizenmaier, E. Arromdee, P. Wennberg, P.C. Spittell, A. ChangMiller, B.L. Clarke, Severe digital arterial occlusive disease and acute parvovirus B19 infection, Lancet 356 (2000) 312e314. [8] S. Trapani, M. Ermini, F. Falcini, Human parvovirus B19 infection: its relationship with systemic lupus erythematosus, Semin. Arthr. Rheum. 28 (1999) 319e325. [9] Y.V. Karetnyi, P.R. Beck, R.S. Markin, A.N. Langnas, S.J. Naides, Human parvovirus B19 infection in acute fulminant liver failure, Arch. Virol. 144 (1999) 1713e1724. [10] E.M. Sokal, M. Melchior, C. Cornu, A.T. Vandenbroucke, J.P. Buts, B.J. Cohen, G. Burtonboy, Acute parvovirus B19 infection associated with fulminant hepatitis of favourable prognosis in young children, Lancet 352 (1998) 1739e1741. [11] L.C. Corman, D.J. Dolson, Polyarteritis nodosa and parvovirus B19 infection, Lancet 339 (1992) 491. [12] T.H. Finkel, T.J. Torok, P.J. Ferguson, E.L. Durigon, S.R. Zaki, D.Y. Leung, R.J. Harbeck, E.W. Gelfand, F.T. Saulsbury, J.R. Hollister, Chronic parvovirus B19 infection and systemic necrotising vasculitis: opportunistic infection or aetiological agent? Lancet 343 (1994) 1255e1258. [13] K.E. Brown, J.R. Hibbs, G. Gallinella, S.M. Anderson, E.D. Lehman, P. McCarthy, N.S. Young, Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen), N. Engl. J. Med. 330 (1994) 1192e1196. [14] U. Kuhl, M. Pauschinger, T. Bock, K. Klingel, C.P. Schwimmbeck, B. Seeberg, L. Krautwurm, W. Poller, H.P. Schultheiss, R. Kandolf, Parvovirus B19 infection mimicking acute myocardial infarction, Circulation 108 (2003) 945e950. [15] J. Crane, Parvovirus B19 infection in pregnancy, J. Obstet. Gynaecol. Can. 24 (2002) 727e743. [16] C.E. Oyer, E.H. Ongcapin, J. Ni, N.E. Bowles, J.A. Towbin, Fatal intrauterine adenoviral endomyocarditis with aortic and pulmonary valve stenosis: diagnosis by polymerase chain reaction, Hum. Pathol. 31 (2000) 1433e1435. [17] A. Telerman, M. Tuynder, T. Dupressoir, B. Robaye, F. Sigaux, E. Shaulian, M. Oren, J. Rommelaere, R. Amson, A model for tumor suppression using H-1 parvovirus, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 8702e8706. [18] K.E. Brown, S.M. Anderson, N.S. Young, Erythrocyte P antigen: cellular

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