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. 2014 Nov;88(22):13221-30.
doi: 10.1128/JVI.02078-14. Epub 2014 Sep 3.

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells

Aleksandra Milewska  1 , Miroslaw Zarebski  2 , Paulina Nowak  1 , Karol Stozek  1 , Jan Potempa  3 , Krzysztof Pyrc  4
Affiliations

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells

Aleksandra Milewska et al. J Virol. 2014 Nov.

Abstract

Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63.

Importance: ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.

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Figures

FIG 1
FIG 1
Human cell lines overexpressing ACE2 protein. (A) Lysates of A549_ACE2+ (A549 +) and A549_WT (A549 −) cells and of 293T_ACE2+ (293T +) and 293T_WT (293T −) cells were tested for the presence of the ACE2 protein with Western blotting using antibodies specific to the ectodomain of the human ACE2 protein. Concomitantly, β-actin protein levels were assessed in each sample. Numbers on the left side represent molecular mass (kDa) assessed with a size marker. The results shown are representative of at least three independent experiments. (B) A549_ACE2+, A549_WT, 293T_ACE2+, and 293T_WT cells were tested for the surface expression of the ACE2 protein with flow cytometry using antibodies specific to the ectodomain of the human ACE2 protein. The results shown are representative of at least three independent experiments.
FIG 2
FIG 2
Cytopathic effect on A549_ACE2+ cells during HCoV-NL63 infection. ACE2-overexpressing (ACE2+) and wild-type (WT) A549 and 293T cells were infected with HCoV-NL63 or mock inoculated and cultured for 6 days. Cytopathic effect was observed only on HCoV-NL63-infected A549_ACE2+ cells. Magnification, ×200. The results shown are representative of at least three independent experiments.
FIG 3
FIG 3
HCoV-NL63 nucleocapsid protein expression in ACE2+ cells. ACE2+ and WT 293T and A549 cells were infected with HCoV-NL63 (+) or mock infected (−). HCoV-NL63 nucleocapsid protein was detected at 6 days p.i. in A549_ACE2+ and 293T_ACE2+ cell lysates, suggesting viral replication. No signal from the NL63-N protein was observed in mock-infected cells. A sample containing lysate of LLC-Mk2 cells infected with HCoV-NL63 was used as a positive control (PC). The position of the 55-kDa molecular mass marker is shown on the left side. The results shown are representative of at least three independent experiments.
FIG 4
FIG 4
HCoV-NL63 replication in ACE2-overexpressing cells. ACE2-overexpressing and wild-type cells were infected with HCoV-NL63 (+) or mock infected (−) and cultured for 6 days. (A) Genomic RNA (1a) and a set of HCoV-NL63 sgmRNAs, including spike (S), ORF3, envelope (E), membrane (M), and nucleocapsid (N), were detected in A549_ACE2+ and 293T_ACE2+ cells. No HCoV-NL63 sgmRNAs were detected in WT cells. LLC-Mk2 cells infected with HCoV-NL63 (+) or mock infected (−) were used as controls. Positions of nucleotide size markers are shown on the left side of each panel. The results shown are representative of at least three independent experiments. (B) HCoV-NL63 replication on A549 and 293T cells was evaluated with real-time PCR. A marked increase in virus yield was observed on A549_ACE2+ cells and, to a much lesser extent, on 293T_ACE2+ cells. No increase in virus yield was observed on HCoV-NL63-infected A549 and 293T WT cells. Data on virus replication are presented as the number of HCoV-NL63 RNA copies/ml. All assays were performed in triplicate, and average values with standard errors (error bars) are presented.
FIG 5
FIG 5
Directed expression of the ACE2 protein on A549 cells does not alter HCoV-NL63 adhesion. Analysis of HCoV-NL63 adherence to ACE2-overexpressing (ACE2+) or wild-type (WT) A549 cells was conducted with flow cytometry. The results shown are representative of at least three independent experiments.
FIG 6
FIG 6
Adherence of HCoV-NL63 to LLC-Mk2 cells depleted of the ACE2 protein. LLC-Mk2 cells were depleted of surface ACE2 protein by incubation with 1 μM PMA and subsequently incubated with purified HCoV-NL63 or mock incubated. DMSO-treated cells were used as a control. (A) HCoV-NL63 replication on LLC-Mk2 ACE2+ and ACE2− cells was evaluated with real-time PCR. A significant decrease in viral replication was observed on LLC-Mk2 cells pretreated with PMA compared to control cells. Data on virus replication are presented as the number of HCoV-NL63 RNA copies/ml. (B) Analysis of HCoV-NL63 adherence to ACE2+ (+ DMSO) and ACE2− (+ PMA) LLC-Mk2 cells. HCoV-NL63 was labeled with specific antibodies, and virus adhesion was analyzed by flow cytometry. (C) A decrease in surface expression of the ACE2 protein on LLC-Mk2 cells after PMA treatment was confirmed using flow cytometry. (D) HCoV-NL63 adhesion to ACE2+ (DMSO-treated) and ACE2− (PMA-treated) LLC-Mk2 cells was confirmed by confocal microscopy. LLC-Mk2 cells were pretreated with PMA or DMSO and incubated with purified HCoV-NL63. HCoV-NL63 virions are presented in green, while the blue denotes DNA. Each image is a single confocal plane (xy) with two orthogonal views (xz and yz) created by maximum projection of axial planes (thickness, 0.7 μm). Scale bar, 10 μm. The results shown are representative of at least three independent experiments.
FIG 7
FIG 7
HCoV-NL63 adhesion to neuraminidase-treated cells and in the presence of sugar moieties. LLC-Mk2 cells were treated with DMSO (A), 1 μM PMA (B), or 1 μM PMA and 200 mU/ml type V neuraminidase (Neu) (C) and further incubated with purified HCoV-NL63 or mock incubated. Virus adhesion was assessed also in the presence of 50 mM sugar monomers: galactose (D), mannose (E), N-acetylglucosamine (F), fucose (G), or glucose (H), as a negative control. Virus adhesion was analyzed by flow cytometry. The results shown are representative of at least three independent experiments.
FIG 8
FIG 8
HCoV-NL63 adhesion to ACE2+/ACE2− cells in the presence of heparan sulfate. (A) Flow cytometry analysis of HCoV-NL63 adhesion. The ACE2 protein was removed from the surface of LLC-Mk2 cells by incubation with 1 μM PMA (ACE2−), while control cells were treated with DMSO (ACE2+). Adhesion of HCoV-NL63 was assessed on ACE2+ and ACE2− cells in the presence of 300 μg/ml HS or control PBS. (B) Confocal microscopy analysis of HCoV-NL63 adhesion. LLC-Mk2 cells were stimulated with 1 μM PMA or DMSO and incubated with purified HCoV-NL63 (NL63) in the presence or absence of heparan sulfate (HS). NC, cells incubated with the mock sample. HCoV-NL63 virions are presented in green, while the blue denotes DNA. Each image is a single confocal plane (xy) with two orthogonal views (xz and yz) created by maximum projection of axial planes (thickness, 4.8 μm). Scale bar, 5 μm. Bars in the graph represent the mean numbers of virions from 10 cells per sample ± standard errors.
FIG 9
FIG 9
HCoV-NL63 replication in the presence of heparan sulfate. LLC-Mk2 cells were infected with HCoV-NL63 in the presence of increasing concentrations of HS or PBS. Virus replication in cell culture supernatants was evaluated using real-time PCR on day 4 p.i. Data on virus replication are presented as the number of HCoV-NL63 RNA copies/ml. All assays were performed in triplicate, and average values with standard errors (error bars) are presented. For all concentrations, the decrease in virus yield is statistically significant (Student's t test; P < 0.05).
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References

    1. Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Rancaniello VR, Roizman B. (ed) 2013. Fields virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
    1. Peiris JS, Yuen KY, Osterhaus AD, Stohr K. 2003. The severe acute respiratory syndrome. N. Engl. J. Med. 349:2431–2441. 10.1056/NEJMra032498. - DOI - PubMed
    1. Stadler K, Masignani V, Eickmann M, Becker S, Abrignani S, Klenk HD, Rappuoli R. 2003. SARS—beginning to understand a new virus. Nat. Rev. Microbiol. 1:209–218. 10.1038/nrmicro775. - DOI - PMC - PubMed
    1. de Groot RJ, Baker SC, Baric RS, Brown CS, Drosten C, Enjuanes L, Fouchier RA, Galiano M, Gorbalenya AE, Memish ZA, Perlman S, Poon LL, Snijder EJ, Stephens GM, Woo PC, Zaki AM, Zambon M, Ziebuhr J. 2013. Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J. Virol. 87:7790–7792. 10.1128/JVI.01244-13. - DOI - PMC - PubMed
    1. Fouchier RA, Hartwig NG, Bestebroer TM, Niemeyer B, de Jong JC, Simon JH, Osterhaus AD. 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. U. S. A. 101:6212–6216. 10.1073/pnas.0400762101. - DOI - PMC - PubMed

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