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. 2014:2014:976015.
doi: 10.1155/2014/976015. Epub 2014 Jul 10.

Voltage dependent anion channel is redistributed during Japanese encephalitis virus infection of insect cells

Chanida Fongsaran  1 , Narumon Phaonakrop  2 , Sittiruk Roytrakul  2 , Chutima Thepparit  1 , Atichat Kuadkitkan  1 , Duncan R Smith  3
Affiliations

Voltage dependent anion channel is redistributed during Japanese encephalitis virus infection of insect cells

Chanida Fongsaran et al. ScientificWorldJournal. 2014.

Abstract

Despite the availability of an effective vaccine, Japanese encephalitis remains a significant cause of morbidity and mortality in many parts of Asia. Japanese encephalitis is caused by the Japanese encephalitis virus (JEV), a mosquito transmitted flavivirus. Many of the details of the virus replication cycle in mosquito cells remain unknown. This study sought to determine whether GRP78, a well-characterized flavivirus E protein interacting protein, interacted with JEV E protein in insect cells, and whether this interaction was mediated at the cell surface. GRP78 was shown to interact with JEV E protein by coimmunoprecipitation, and was additionally shown to interact with voltage dependent anion protein (VDAC) through the same methodology. Antibody inhibition experiments showed that neither GRP78 nor VDAC played a role in JEV internalization to insect cells. Interestingly, VDAC was shown to be significantly relocalized in response to JEV infection, and significant levels of colocalization between VDAC and GRP78 and VDAC and ribosomal L28 protein were seen in JEV infected but not uninfected cells. This is the first report of relocalization of VDAC in response to JEV infection and suggests that this may be a part of the JEV replication strategy in insect cells.

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Figures

Figure 1
Figure 1
Coimmunoprecipitation analysis of GRP78 and JEV E protein and GRP78 and VDAC. C6/36 cells were mock and or infected with JEV for V days, after which cells were lysed and either E protein (a) or VDAC (b) immunoprecipitated with an appropriate antibody. Coimmunoprecipitation of GRP78 was determined by electrophoresis of the precipitated proteins and transfered to solid matrix support after which membrane was probed with an antibody against GRP78.
Figure 2
Figure 2
Antibody inhibition analysis of JEV entry to insect cells. C6/36 cells were pretreated with antibodies against GRP78, VDAC, VDAC, and GRP78 and integrin αV/β5 or no antibody before infection with JEV at MOI of 10. After 8 hours the percent infection was determined by flow cytometry. Representative raw data profiles (a) and tabular analysis (b) from three independent experiments in duplicate are shown.
Figure 3
Figure 3
Location of JEV E protein, VDAC, and GRP78 in JEV infected C6/36 cells. C6/36 cells grown on glass cover slips were mock infected or infected with JEV and examined for location of JEV E protein (green), VDAC (red), and GRP78 (blue) using an Olympus FluoView 1000 confocal microscope. Representative individual and merged images are shown, and one panel has bright field added.
Figure 4
Figure 4
Location of VDAC and GRP78 in JEV infected C6/36 cells. Signal of VDAC and GRP78 extracted from Figure 3 for improved visualization purposes with enlargement.
Figure 5
Figure 5
Location of JEV E protein, VDAC, and ribosomal L28 protein in JEV infected C6/36 cells. C6/36 cells grown on glass cover slips were mock infected or infected with JEV and examined for location of JEV E protein (blue), ribosomal L28 protein (red), and VDAC (green) using an Olympus FluoView 1000 confocal microscope. Representative individual and merged images are shown, and one panel has bright field added.
Figure 6
Figure 6
Location of JEV E protein, VDAC, and ribosomal L28 protein in JEV infected C6/36 cells. Signal of VDAC and ribosomal L28 protein extracted from Figure 5 for improved visualization purposes with enlargement.
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