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Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry

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Abstract

Dengue virus (DENV) is the causative agent of the most important mosquito-borne viral disease, which is endemic to over 100 countries in tropical and subtropical areas of the world. It is transmitted to humans by Aedes mosquitoes. The first step in the viral infection of host cells is virion attachment to the plasma membrane, which is mediated by specific surface molecules. There are several molecules that participate in DENV infection of mosquitoes, but only a few have been identified. In this work, we co-purified 4 proteins from C6/36 cells using a recombinant DENV 4 E protein and identified them as 70 kDa Heat Shock and 70 kDa Heat Shock cognate proteins (HSP70/HSc70), Binding immunoglobulin protein (BiP), Thioredoxin/protein disulphide isomerase (PDI), and 44 kDa Endoplasmic reticulum resident protein (ERp44) via matrix-assisted laser desorption/ionisation time of flight (Maldi-ToF) analysis. Using immunofluorescence and flow cytometry assays, we observed re-localisation of HSP70/HSc70 and, to a lesser extent, BiP to the plasma membrane under stress conditions, such as during DENV infection. By performing binding and infection assays independently, we found that all 4 proteins participate in both processes, but to differing extents: HSP70/HSc70 is the most critical component, while ERp44 is less important. Viral infection was not inhibited when the cells were incubated with antibodies against all of the surface proteins after virus binding, which suggests that DENV entry to C6/36 cells is mediated by these proteins at the same step and not sequentially.

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Acknowledgments

We thank Matilde García Espitia for technical assistance, Victor Hugo Rosales G. for flow cytometry assistance, Ana Laura Luna Torres for statistical analysis, Mary Ann Gawinowicz for Maldi-ToF analysis, and Juan Ernesto Ludert for his critical comments on the manuscript.

This project was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT-60883) and the Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional (SIP-20070712 and 20120932).

Tania Vega has scholarships from the Programa Institucional de Formación de Investigadores (PIFI-IPN), the Secretaría de Investigación y Posgrado (IPN) and the Instituto de Ciencia y Tecnología (DF).

Dr. Juan Salas and Dr. Mónica De Nova have fellowships from the Comisión de Operación y Fomento a las Actividades Académicas (COFAA) and the Estímulo al Desempeño de los Investigadores (EDI).

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  1. Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Guillermo Massieu Helguera 239, Fraccionamiento La Escalera-Ticomán, Mexico, DF, CP 07320, Mexico

    Tania Olivia Vega-Almeida, Mariana Salas-Benito, Mónica Ascensión De Nova-Ocampo & Juan Santiago Salas-Benito

  2. Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional. Av. Instituto Politécnico Nacional 2508 San Pedro Zacatenco, Mexico, DF, CP 07360, Mexico

    Rosa María del Angel

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  1. Tania Olivia Vega-Almeida
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Correspondence to Juan Santiago Salas-Benito.

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705_2012_1596_MOESM1_ESM.ppt

Supplemental material 1. Viral binding determined in immunofluorescence assay. C6/36 cells were incubated at 4 °C with specific antibodies against heat shock, PDI, and ERp44 proteins, with an unrelated antibody (IgG), or without antibodies (2° Ab and C6-DENV4). Then, DENV 4 was added, followed by incubation at 4 °C and detection with an anti-DENV complex antibody in non-permeabilised cells. C6/36 cells without virus were included as a control (2° Ab). The nuclei were stained with DAPI, and the cells were visualised using an immunofluorescence microscope.

705_2012_1596_MOESM2_ESM.ppt

Supplemental material 2. Viral infection as determined by immunofluorescence assay. A, C6/36 cells were incubated at 4 °C with specific antibodies (indicated in each photograph), with an unrelated antibody (IgG), or without antibodies (2° Ab and C6-DENV4). Then, DENV 4 was added and detected 24 h post-infection with an anti-DENV-complex antibody. Uninfected cells were included as a control (2° Ab). The nuclei were stained with DAPI, and the cells were visualised with an immunofluorescence microscope

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Vega-Almeida, T.O., Salas-Benito, M., De Nova-Ocampo, M.A. et al. Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry. Arch Virol 158, 1189–1207 (2013). https://doi.org/10.1007/s00705-012-1596-0

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