Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry.
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.
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
TO
Vega-Almeida
Mariana
M
Salas-Benito
Mónica Ascensión
MA
De Nova-Ocampo
Rosa María
RM
Del Angel
Juan Santiago
JS
Salas-Benito
D000330:Aedes / Q000166:cytology / Q000502:physiology / Q000821:virology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003715:Dengue / Q000821:virology*; D003716:Dengue Virus / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000502:physiology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D050883:HSC70 Heat-Shock Proteins / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000502:physiology; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins / Q000502:physiology; D053585:Virus Attachment; D053586:Virus Internalization
D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins