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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.


TRIM52 mediates NS2A degradation of NS2A.(a) Co-transfection of 293T cells with a fixed amount of NS2A-expressing plasmid and with increasing amounts of TRIM52-encoding plasmid, empty vector was used to equalize the doses of DNA plasmid. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (b) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132 or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (c) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of wild type or RING domain lacking TRIM52-encoding plasmid or with empty vector. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated by Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (d) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132, NH4Cl (20 mM) or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel).
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f4: TRIM52 mediates NS2A degradation of NS2A.(a) Co-transfection of 293T cells with a fixed amount of NS2A-expressing plasmid and with increasing amounts of TRIM52-encoding plasmid, empty vector was used to equalize the doses of DNA plasmid. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (b) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132 or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (c) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of wild type or RING domain lacking TRIM52-encoding plasmid or with empty vector. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated by Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (d) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132, NH4Cl (20 mM) or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel).

Mentions: TRIM52 interacted with JEV NS2A, and an in vivo self-ubiquitylation assay indicated that TRIM52 demonstrated E3 ligase activity. We thus investigated whether TRIM52 could influence the stability of NS2A protein. For this purpose, 293T cells were co-transfected with a fixed amount of Flag-tagged NS2A-expressing plasmid (50 ng) and with increasing amounts of HA-tagged TRIM52-encoding plasmids (500, 1000, and 2000 ng), equalizing the DNA doses with empty vector. The cells were harvested at 30 hpt, and the expression of NS2A was analyzed by Western blot. Figure 4a shows that an increasing dose of TRIM52 reduced the NS2A protein level. We subsequently, evaluated whether TRIM52 mediated NS2A protein degradation via the proteasome-dependent pathway. We transfected 293T cells with a fixed amount of NS2A-expressing plasmids (50 ng) plus TRIM52-expressing plasmid (2000 ng) or with empty vector control. The proteasome inhibitor MG132 (10 μM) or dimethyl sulphoxide (DMSO) was added at 24 hpt, and the cells were collected after 10 h. Figure 4b shows that NS2A protein levels significantly decreased in the presence of TRIM52 without MG132 treatment (Fig. 4b, right side, black bars). By contrast, NS2A protein levels were rescued by MG132 treatment.


TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A
TRIM52 mediates NS2A degradation of NS2A.(a) Co-transfection of 293T cells with a fixed amount of NS2A-expressing plasmid and with increasing amounts of TRIM52-encoding plasmid, empty vector was used to equalize the doses of DNA plasmid. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (b) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132 or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (c) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of wild type or RING domain lacking TRIM52-encoding plasmid or with empty vector. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated by Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (d) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132, NH4Cl (20 mM) or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel).
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f4: TRIM52 mediates NS2A degradation of NS2A.(a) Co-transfection of 293T cells with a fixed amount of NS2A-expressing plasmid and with increasing amounts of TRIM52-encoding plasmid, empty vector was used to equalize the doses of DNA plasmid. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (b) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132 or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (c) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of wild type or RING domain lacking TRIM52-encoding plasmid or with empty vector. The cells were collected at 30 hpt, and the protein levels of TRIM52, and NS2A were evaluated by Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel). (d) The 293T cells were co-transfected with 50 ng of NS2A-expressing plasmid plus 2000 ng of TRIM52-encoding plasmid or with empty vector. MG132, NH4Cl (20 mM) or DMSO was added at 24 hpt, and the cells were collected after 10 h of treatment. The protein levels of TRIM52, and NS2A were evaluated through Western blot analysis. GAPDH was used as normalizer, and relative quantification of detected signal was analyzed using Image J software (right panel).
Mentions: TRIM52 interacted with JEV NS2A, and an in vivo self-ubiquitylation assay indicated that TRIM52 demonstrated E3 ligase activity. We thus investigated whether TRIM52 could influence the stability of NS2A protein. For this purpose, 293T cells were co-transfected with a fixed amount of Flag-tagged NS2A-expressing plasmid (50 ng) and with increasing amounts of HA-tagged TRIM52-encoding plasmids (500, 1000, and 2000 ng), equalizing the DNA doses with empty vector. The cells were harvested at 30 hpt, and the expression of NS2A was analyzed by Western blot. Figure 4a shows that an increasing dose of TRIM52 reduced the NS2A protein level. We subsequently, evaluated whether TRIM52 mediated NS2A protein degradation via the proteasome-dependent pathway. We transfected 293T cells with a fixed amount of NS2A-expressing plasmids (50 ng) plus TRIM52-expressing plasmid (2000 ng) or with empty vector control. The proteasome inhibitor MG132 (10 μM) or dimethyl sulphoxide (DMSO) was added at 24 hpt, and the cells were collected after 10 h. Figure 4b shows that NS2A protein levels significantly decreased in the presence of TRIM52 without MG132 treatment (Fig. 4b, right side, black bars). By contrast, NS2A protein levels were rescued by MG132 treatment.

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.