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TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A

View Article: PubMed Central - PubMed

ABSTRACT

The members of tripartite-motif containing (TRIM) protein participate in various cellular processes and play an important role in host antiviral function. TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity, or indirectly by promoting host innate immunity. This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus (JEV) infection. Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293T cells. In addition, JEV nonstructural protein 2A (NS2A) is a protein that interacts with TRIM52. Their interaction degraded NS2A in a proteasome-dependent manner via the E3 ligase activity of TRIM52. Thus, TRIM52 is a novel antiviral TRIM protein, and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2A.

No MeSH data available.


Interaction of TRIM52 with JEV NS2A protein.(a) 293T cells in six-well plates were co-transfected with expression plasmids encoding HA-TRIM52 plus the indicated plasmids expressing Flag-tagged JEV viral proteins. The cells were harvested at 36 hpt, and the cell lysates were prepared for Western blot analysis using the indicated antibodies. Immunoprecipitation (IP) was performed using anti-Flag affinity gel. WCL, whole cell lysates. (b) Co-localization of TRIM52 with NS2A. 293T cells were co-transfected with plasmids expressing HA-TRIM52 plus NS2A-Flag. The cells were fixed at 24 hpt and were subjected to immunofluorescence to detect HA-TRIM52 (green) and NS2A-Flag (red). The nuclei were stained with DAPI (blue). Images were obtained using confocal microscope (Carl Zeiss MicroImaging, Inc.). (c) Purified proteins of GST and GST-TRIM52 were analyzed by SDS-PAGE. (d) Western blot analysis of the anti-Flag affinity NS2A from 293T-NS2A cell lysates. (e) GST pull-down and Western blot analysis of the interaction between TRIM52 and NS2A. (f) IP using anti-Flag affinity gel and Western blot analysis for the interaction of NS2A with TRIM52 and RING domain deleted TRIM52 in 293T cells co-transfected with NS2A-Flag or with empty vector plus HA-TRIM52 and HA-TRIM52dR.
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f2: Interaction of TRIM52 with JEV NS2A protein.(a) 293T cells in six-well plates were co-transfected with expression plasmids encoding HA-TRIM52 plus the indicated plasmids expressing Flag-tagged JEV viral proteins. The cells were harvested at 36 hpt, and the cell lysates were prepared for Western blot analysis using the indicated antibodies. Immunoprecipitation (IP) was performed using anti-Flag affinity gel. WCL, whole cell lysates. (b) Co-localization of TRIM52 with NS2A. 293T cells were co-transfected with plasmids expressing HA-TRIM52 plus NS2A-Flag. The cells were fixed at 24 hpt and were subjected to immunofluorescence to detect HA-TRIM52 (green) and NS2A-Flag (red). The nuclei were stained with DAPI (blue). Images were obtained using confocal microscope (Carl Zeiss MicroImaging, Inc.). (c) Purified proteins of GST and GST-TRIM52 were analyzed by SDS-PAGE. (d) Western blot analysis of the anti-Flag affinity NS2A from 293T-NS2A cell lysates. (e) GST pull-down and Western blot analysis of the interaction between TRIM52 and NS2A. (f) IP using anti-Flag affinity gel and Western blot analysis for the interaction of NS2A with TRIM52 and RING domain deleted TRIM52 in 293T cells co-transfected with NS2A-Flag or with empty vector plus HA-TRIM52 and HA-TRIM52dR.

Mentions: Studies have reported that JEV infection blocks interferon (IFN) induced Janus kinase (JAK) signal transducer and activation of transcription (STAT) signaling pathway to evade the antiviral actions of IFN2021. We thus speculated that TRIM52 interacts with certain JEV viral proteins, restricting JEV replication. Co-immunoprecipitation (IP)-Western blot analysis was used to detect the interaction between TRIM52 and JEV viral proteins. The plasmids expressing individual Flag-tagged JEV proteins were co-transfected with HA-tagged TRIM52. The results shown in Fig. 2a indicates that NS2Ainteracts with TRIM52. Furthermore, confocal microscopy results indicated that TRIM52 co-localized with NS2A in the cytosol (Fig. 2b).


TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A
Interaction of TRIM52 with JEV NS2A protein.(a) 293T cells in six-well plates were co-transfected with expression plasmids encoding HA-TRIM52 plus the indicated plasmids expressing Flag-tagged JEV viral proteins. The cells were harvested at 36 hpt, and the cell lysates were prepared for Western blot analysis using the indicated antibodies. Immunoprecipitation (IP) was performed using anti-Flag affinity gel. WCL, whole cell lysates. (b) Co-localization of TRIM52 with NS2A. 293T cells were co-transfected with plasmids expressing HA-TRIM52 plus NS2A-Flag. The cells were fixed at 24 hpt and were subjected to immunofluorescence to detect HA-TRIM52 (green) and NS2A-Flag (red). The nuclei were stained with DAPI (blue). Images were obtained using confocal microscope (Carl Zeiss MicroImaging, Inc.). (c) Purified proteins of GST and GST-TRIM52 were analyzed by SDS-PAGE. (d) Western blot analysis of the anti-Flag affinity NS2A from 293T-NS2A cell lysates. (e) GST pull-down and Western blot analysis of the interaction between TRIM52 and NS2A. (f) IP using anti-Flag affinity gel and Western blot analysis for the interaction of NS2A with TRIM52 and RING domain deleted TRIM52 in 293T cells co-transfected with NS2A-Flag or with empty vector plus HA-TRIM52 and HA-TRIM52dR.
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f2: Interaction of TRIM52 with JEV NS2A protein.(a) 293T cells in six-well plates were co-transfected with expression plasmids encoding HA-TRIM52 plus the indicated plasmids expressing Flag-tagged JEV viral proteins. The cells were harvested at 36 hpt, and the cell lysates were prepared for Western blot analysis using the indicated antibodies. Immunoprecipitation (IP) was performed using anti-Flag affinity gel. WCL, whole cell lysates. (b) Co-localization of TRIM52 with NS2A. 293T cells were co-transfected with plasmids expressing HA-TRIM52 plus NS2A-Flag. The cells were fixed at 24 hpt and were subjected to immunofluorescence to detect HA-TRIM52 (green) and NS2A-Flag (red). The nuclei were stained with DAPI (blue). Images were obtained using confocal microscope (Carl Zeiss MicroImaging, Inc.). (c) Purified proteins of GST and GST-TRIM52 were analyzed by SDS-PAGE. (d) Western blot analysis of the anti-Flag affinity NS2A from 293T-NS2A cell lysates. (e) GST pull-down and Western blot analysis of the interaction between TRIM52 and NS2A. (f) IP using anti-Flag affinity gel and Western blot analysis for the interaction of NS2A with TRIM52 and RING domain deleted TRIM52 in 293T cells co-transfected with NS2A-Flag or with empty vector plus HA-TRIM52 and HA-TRIM52dR.
Mentions: Studies have reported that JEV infection blocks interferon (IFN) induced Janus kinase (JAK) signal transducer and activation of transcription (STAT) signaling pathway to evade the antiviral actions of IFN2021. We thus speculated that TRIM52 interacts with certain JEV viral proteins, restricting JEV replication. Co-immunoprecipitation (IP)-Western blot analysis was used to detect the interaction between TRIM52 and JEV viral proteins. The plasmids expressing individual Flag-tagged JEV proteins were co-transfected with HA-tagged TRIM52. The results shown in Fig. 2a indicates that NS2Ainteracts with TRIM52. Furthermore, confocal microscopy results indicated that TRIM52 co-localized with NS2A in the cytosol (Fig. 2b).

View Article: PubMed Central - PubMed

ABSTRACT

The members of tripartite-motif containing (TRIM) protein participate in various cellular processes and play an important role in host antiviral function. TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity, or indirectly by promoting host innate immunity. This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus (JEV) infection. Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293T cells. In addition, JEV nonstructural protein 2A (NS2A) is a protein that interacts with TRIM52. Their interaction degraded NS2A in a proteasome-dependent manner via the E3 ligase activity of TRIM52. Thus, TRIM52 is a novel antiviral TRIM protein, and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2A.

No MeSH data available.