Volume 20, Issue 5, Pages 631-641 (November 2016) ICAM-5/Telencephalin Is a Functional Entry Receptor for Enterovirus D68  Wei Wei, Haoran Guo, Junliang Chang, Yingzi Yu, Guanchen Liu, Nannan Zhang, Stephen H. Willard, Shu Zheng, Xiao-Fang Yu  Cell Host & Microbe  Volume 20, Issue 5, Pages 631-641 (November 2016) DOI: 10.1016/j.chom.2016.09.013 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Enterovirus D68 Binds Specifically to ICAM-5 (A) Induction of CPEs by EV-D68 in various cell lines. +, more than 50% cell death in virus-infected cultures; −, no observed cell death in virus-infected cultures compared with uninfected cultures. (B) Detection of endogenous ICAM-1 and ICAM-5 mRNA in HEK293T cells and RD cells. Levels of ICAM-1 and ICAM-5 mRNA in HEK293T and RD cells were assessed by qRT-PCR using ICAM-1- or ICAM-5-specific primers. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. (C) The immunoglobulin domains are depicted as half circles, and extra- and intra-cellular orientations are indicated by NH2 and COOH on the protein chains, respectively. Red lines indicate the regions of ICAM-1 or ICAM-5 fragments used in this study. N-glycosylation sites on domain 1 (D1) of each ICAM molecule were labeled by green markers. The regions targeted by the shRNA or siRNA are labeled by blue lines. (D) Amino acid sequence alignment of D1 of ICAM-1 and ICAM-5. Amino acids in ICAM-1 that are required for rhinovirus binding are boxed. (E) Schematic of the virion capture assay for the detection of interaction between EV-D68 and ICAM-5. (F) EV-D68 viruses were mixed with 0.1, 1, or 5 μg ICAM-5-Fc or 5 μg Ig-Fc soluble protein at 4°C for 1 hr. Subsequently, 50 μL protein G agarose (Roche) was mixed with each sample for 2 hr. Samples were then washed twice with PBS, and RNA was extracted for qRT-PCR to detect captured EV-D68 RNA. Here and in all other qRT-PCR assays, error bars denote SEM; ANOVA test, n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 ICAM-5 Is Essential for EV-D68 Infection (A) Stable silencing of ICAM-5 expression. HEK293T cells were transduced with retroviral vectors expressing a control shRNA or an ICAM-5-specific shRNA. Levels of ICAM-5 mRNA in the transduced HEK293T cells were assessed by qRT-PCR using ICAM-5-specific primers. GAPDH mRNA was used as an internal control. The relative level of ICAM-5 mRNA in the control HEK293T cells was set to 100%. (B) Silencing of ICAM-5 expression attenuates EV-D68 replication. HEK293T cells expressing a control shRNA or an ICAM-5-specific shRNA were infected with EV-D68 at a multiplicity of infection (MOI) of 0.5. Cells were harvested 0, 6, 12, and 24 hr post-infection, and RNA samples were analyzed by qRT-PCR to detect EV-D68 RNA. The non-specific signal for EV-D68 RNA at 0 hr post-infection was set to 1. (C) Silencing of ICAM-5 expression inhibits EV-D68-induced CPEs. Transduced HEK293T cells were infected with equal amounts of EV-D68. 72 hr post-infection, cells were imaged via light microscopy. (D–F) Soluble ICAM-5 inhibits EV-D68 virus infection in permissive cells. EV-D68 viruses were incubated with control-Fc or ICAM5-Fc soluble proteins for 2 hr before infection. Soluble ICAM-5 inhibits EV-D68 viral RNA (vRNA) accumulation (D), viral titers (E), and EV-D68-induced CPEs (F) in HEK293T cells. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 EV-D68 Replicates Efficiently in Vero Cells Expressing ICAM-5 (A) Generation of Vero cells expressing ICAM-5. Vero cells were transduced with a control CDH or an ICAM-5 expression vector using lentiviral transduction systems. Levels of ICAM-5 mRNA (left) were assessed by qRT-PCR using ICAM-5-specific primers. GAPDH mRNA was used as an internal control. The level of ICAM-5 mRNA in control Vero-CDH cells was set to 1. Immunoblot analysis indicates the expression of ICAM-5 proteins (right) in stably transduced Vero-ICAM-5 but not control Vero-CDH cells. (B) Cell surface expression of ICAM-5 in transduced Vero-ICAM-5 cells. Vero-ICAM-5 and control Vero-CDH cells were separately incubated with 1 μg/ml anti-ICAM-5 goat antibody (R&D) for 2 hr and then incubated for 1 hr with Alexa Fluor 488-conjugated donkey anti-goat IgG secondary antibody (Thermo Fisher Scientific). Cells were washed three times and then analyzed by flow cytometry. (C) Vero-ICAM-5 and Vero-CDH cells were infected with EV-D68 at an MOI of 0.5. Cells were harvested 6 and 12 hr post-infection. RNA samples were analyzed by qRT-PCR to detected EV-D68 RNA. GAPDH mRNA was used as a control. The signal for the EV-D68 RNA in Vero-CDH cells 6 hr post-infection was set to 1. (D) Relative binding of EV-D68 to Vero-ICAM5 cells. Vero-ICAM5 and Vero-CDH cells were incubated with diluted EV-D68 at 4°C for 2 hr. EV-D68 vRNA of bound viruses was measured by using quantitative real-time RT-PCR. The experiments were done in triplicate. (E) Detection of EV-D68-induced CPE. Vero-ICAM-5 or control Vero-CDH cells were infected with equal amounts of EV-D68, and the CPE was imaged via light microscopy. (F) Determination of infectious viral titers in the culture supernatants of EV-D68-infected Vero-ICAM-5 (purple columns) or Vero-CDH (green columns) cells. (G) Soluble ICAM-5 inhibits EV-D68 virus infection. Vero-ICAM-5 cells were treated with different amounts of ICAM-5-Fc, ICAM-1-Fc, or Ig-Fc and subsequently infected with equal amounts of EV-D68. Cell-associated viral RNA was detected by qRT-PCR 6 hr after infection. The levels of the housekeeping gene GAPDH were used as a control. The level of EV-D68 RNA in the infected Vero-ICAM-5 cells in the presence of 10 μg/mL Ig-Fc control was set to 100%. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 EV-D68 Infection of Primary Cells Is Inhibited by Soluble ICAM-5 Proteins (A) The soluble ICAM-5 fragment inhibits EV-D68 virus infection in primary human bronchial epithelial cells. EV-D68 viruses were incubated with control-Fc or ICAM5-Fc soluble protein for 2 hr before infection. EV-D68 RNA from infected cell samples was determined by qRT-PCR. The levels of the housekeeping gene GAPDH were used as an internal control. The level of EV-D68 RNA in the infected cells treated with Ig-Fc control was set to 100%. Non-specific adsorption of EV-D68-associated RNA with non-permissive Vero cells was used as a negative control. (B) The soluble ICAM-5 fragment inhibits EV-D68 virus infection in primary rat neurons. (C) Schematic of the newborn mouse infection assay for the detection of EV-D68 replication in brain tissue. (D) Soluble ICAM-5 inhibits EV-D68 virus infection in vivo. EV-D68 viruses were incubated with control-Fc or ICAM5-Fc soluble protein for 2 hr before cerebral injection into newborn mice. EV-D68 RNA from the infected tissue was collected at various time points as indicated and determined by qRT-PCR. The level of the mouse housekeeping gene GAPDH was used as an internal control. The level of EV-D68 RNA in the infected tissue treated with the Ig-Fc control was set to 100%. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Cell Surface Sialic Acid Has a Crucial Role in EV-D68 Infection (A–C) HEK293T cells or A172 cells treated with NA or control buffer were infected with EV-D68 viruses. Virus titers (A) and EV-D68-induced CPE in NA-treated HEK293T cells (B) or A172 cells (C) were determined 48 hr post-infection. (D–F) NA-treated Vero-ICAM5 and Vero-CDH cells were challenged by EV-D68 viruses. Virus titers (D) and EV-D68-induced CPE in NA-treated Vero-CDH cells (E) or Vero-ICAM5 cells (F) were determined at 48 hr post-infection. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Asn54-Linked Glycan Is Critical for ICAM-5-Mediated EV-D68 Infection (A) Vero cells stably expressing ICAM-5 wild-type (WT), N54A, or N74A were infected with EV-D68 viruses. Viral titers were measured 48 hr post-infection. (B) Relative binding of EV-D68 to Vero-ICAM5 cells. Vero-ICAM5 WT, Vero-ICAM5 N54A, or Vero-ICAM5 N74A cells were incubated with diluted EV-D68 at 4°C for 2 hr. EV-D68 RNA of bound viruses was measured by real-time qRT-PCR. The experiments were done in triplicate (top). Also shown are immunoblotting results of ICAM5 WT, N54A, and N74A (bottom). (C) Detection of EV-D68-induced CPE. Vero-ICAM-5 WT or mutants were infected with equal amounts of EV-D68, and CPE was imaged via light microscopy. (D) Subcellular location of ICAM-5 WT and N54A protein. Vero-ICAM5 WT cells and Vero-ICAM5 N54A cells were fixed, permeabilized, and stained with a mouse anti-ICAM5 antibody followed by a secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody. The nuclei were stained with DAPI. The cells were examined by deconvolution microscopy. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 ICAM-5 Is a Functional Receptor for Circulating EV-D68 Strains (A) Effect of neuraminidase treatment on EV-D68 infection. RD cells treated with NA or control buffer were infected with the EV-D68 viruses Fermon, MO, or KY. EV-D68-induced CPE was determined at 48 hr post-infection. (B) Effect of neuraminidase treatment on EV-D68 infection. RD cells treated with NA or control buffer were infected with the EV-D68 viruses Fermon, MO, or KY. The levels of EV-D68 RNA in the infected cells were assessed by qRT-PCR 3 hr after infection. The level of GAPDH mRNA was used as an internal control. The level of EV-D68 RNA in virus-infected control RD cells was set to 100%. (C) Induction of CPE by circulating MO or KY strains of EV-D68 in Vero-ICAM-5 cells. (D and E) ICAM-5 expression enhances EV-D68 MO or KY infection. Vero cells expressing ICAM-5 or ICAM-1 or CDH control cells were challenged with EV-D68 MO or KY separately. The levels of EV-D68 RNA in the infected cells were assessed by qRT-PCR 6 hr after infection with MO (D) or KY (E). The level of GAPDH mRNA was used as an internal control. The background level of EV-D68 RNA in virus-infected Vero-CDH cells was set to 1. (F and G) Inhibition of EV-D68 replication by soluble ICAM-5-Fc. EV-D68 viruses were incubated with ICAM-5-Fc, ICAM-1-Fc, or Ig-Fc control protein for 1 hr before viral infection. Cell-associated EV-D68 RNA was determined at various time points after infection. The level of GAPDH mRNA was used as an internal control. The background level of EV-D68 RNA in Vero-ICAM-5 cells treated with Ig-Fc at 0 hr post-infection was set to 1. Cell Host & Microbe 2016 20, 631-641DOI: (10.1016/j.chom.2016.09.013) Copyright © 2016 Elsevier Inc. Terms and Conditions