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Human Parechovirus 1 Infection Occurs via αVβ1 Integrin - PubMed

  • ️Fri Jan 01 2016

Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

Pirjo Merilahti et al. PLoS One. 2016.

Abstract

Human parechovirus 1 (HPeV-1) (family Picornaviridae) is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to αVβ1, αVβ3 and αVβ6 integrin receptor(s), the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of αVβ1, αVβ3, αVβ6 and α5β1 in susceptible cell lines (A549, HeLa and SW480) to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express αVβ1 integrin but not αVβ3 or αVβ6 integrins. To further study the role of β1 integrin, we used a mouse cell line, GE11-KO, which is deficient in β1 expression, and its derivate GE11-β1 in which human integrin β1 subunit is overexpressed. HPeV-1 (Harris strain) and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-β1 supported the internalization process. An integrin β1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with β1 integrin on the cell surface, and HPeV-1 and β1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of αVβ1 integrin without visible receptor clustering.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. HPeV-1 infects the cell lines that do not express αVβ3 and αVβ6 integrins.

(A) A549, HeLa, and SW480 cell lines were inoculated with HPeV-1 at MOI of 10, unbound viruses were removed followed by incubation at 37°C for 6 h, and processed for immunofluorescence staining and imaging (with a 20×objective). HPeV-1 particles and nuclei are shown in green and blue, respectively. (B) The receptor profile of A549, HeLa and SW480 cells was analyzed with flow cytometry. Cells were fixed, and cell surface integrins (αV, β1, αVβ3, αVβ6 and α5β1) were stained using specific antibodies, and 20 000 cells/antibody was measured by flow cytometry.

Fig 2
Fig 2. HPeV-1 infection is inhibited by integrin function-blocking antibodies.

(A) Immunofluorescence microscopy of antibody-treated SW480 cells infected with HPeV-1. SW480 cells were treated with 15 μg/ml of anti-integrin antibodies for 1 h and subsequently infected with HPeV-1 at a MOI of 10. The cells were incubated with HPeV-1 for 1 h on ice followed by wash and incubation for 6 h at 37°C. The cells were fixed and stained with nuclear stain Hoechst (blue) and HPeV-1 specific antiserum (green). (B) Relative infection efficiency of HPeV-1 in integrin-treated cells. SW480 cells were processed as in Fig 2A. The infection efficiency of HPeV-1 in total of 10 000 cells was counted from microscopic images, and p values were calculated. Percentage of HPeV-1 infection in mock-treated (control) cells was set as 100%. Standard deviations are shown, and ** indicates p<0.001. The cells were imaged with a 10×objective.

Fig 3
Fig 3. HPeV-1 internalization is inhibited in mouse cells lacking β1 integrin expression.

(A) HPeV-1 internalizes into GE11-β1 but not GE11-KO cells. GE11-KO and -β1 cells were inoculated with HPeV-1 at a MOI of 5 on ice for 1 h followed by incubation for 6 h at 37°C. The cells were fixed, permeabilized, and stained with appropriate stains or antibodies as described in Materials and Methods. Actin filaments are shown in red, HPeV-1 in green and β1 integrin in blue. The β1 integrin expression on the cell surface was also analyzed by flow cytometry (right panel). (B) Clinical HPeV-1 isolates with low passage numbers (152231, 152478 and 452252) act similarly to the prototype. The cells were treated as in (A). (C) CV-A9 infects both GE11-KO and GE11-β1 cells. The CV-A9 infection was performed similarly to the HPeV-1 assay, followed by staining with anti-CV-A9 antibody (green) and DAPI (blue). Microscopic imaging was performed with Zeiss LSM780 confocal microscopy using a Plan-Apochromat objectives (63× / 1.2 oil/water [panels A and B] or with 40×/ 1.2. oil/water [panel C]). Bar 10 μm.

Fig 4
Fig 4. Activation of β1 integrins enhances HPeV-1 infectivity without visible receptor clustering.

(A and B) SW480 cells were treated with β1 integrin activating antibody (TS2/16) at 37°C for 1 h after which the cells were inoculated on ice with HPeV-1 at a MOI of 10 followed by incubation for 6 h at 37°C. The cells were fixed, permeabilized and stained. The relative infection efficiency of HPeV-1 was calculated from nine parallel images (totally 20 000 to 40 000 cells per each antibody concentration) obtained with Zeiss Axiovert 200M (10×objective). The percentage of infection of HPeV-1 in mock-treated (control) cells was set as 100%. The error bars indicate standard deviation, * indicates p<0.01 and ** indicates p<0.001. (C) Receptor clustering is not detected during HPeV-1 infection in SW480 cell line. The cells were incubated with activating anti-β1 antibody for 15 min after which a secondary antibody was added, and incubation was continued for another 15 minutes before fixing of the cells (positive control, left panel). HPeV-1 was allowed to bind to SW480 cells for 15 minutes before fixing and staining (middle panel). SW480 cells were incubated with β1 primary antibody for 15 minutes prior to fixation of the cells. Integrin β1 is shown in red, HPeV-1 in green and nuclei in blue. Microscopic imaging was performed using Zeiss LSM780 confocal microscopy using a Plan-Apochromat objective (63× / 1.2 oil/water). Bar 10 μm.

Fig 5
Fig 5. HPeV-1 co-localizes with β1 integrin during the early stages of virus entry.

GE11-β1 cells were inoculated on ice for 1 h with HPeV-1 at a MOI of 5 and incubated for 0 (non-permeabilized), 5 and 30 minutes (permeabilized) at 37°C before fixing and staining. HPeV-1 is shown in green and β1 integrin in red. The right panel shows the co-localization analysis performed by BioImageXD software. The software calculates co-localizing voxels between two channels (HPeV-1 and β1 integrin), and these (co-localizing voxels) are shown in white. Microscopic imaging was performed with Zeiss LSM780 confocal microscopy using a Plan-Apochromat objective (63× / 1.2 oil/water). Bar 10 μm.

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This work was supported by Turku Doctoral Programme of Molecular Medicine, The Finnish Cultural Foundation, European Union (AIROPico, FP7-PEOPLE-2013-IAPP Grant no. 612308)(http://airopico.eu), Academy of Finland (grant no. 128539 to P.S.)(www.aka.fi/en-GB/A/) and Jane and Aatos Erkko Foundation (to S.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.