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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 induces ubiquitination of NS2A.(a) NS2A-Flag (2000 ng) was co-expressed with HA-Ubi (1000 ng) and with increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were harvested at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-HA, anti-V5 and anti-GAPDH antibodies. (b) Co-transfection of NS2A-Flag (2000 ng) and increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were collected at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-V5 and anti-K48-ubiquitin antibodies.
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f5: TRIM52 induces ubiquitination of NS2A.(a) NS2A-Flag (2000 ng) was co-expressed with HA-Ubi (1000 ng) and with increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were harvested at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-HA, anti-V5 and anti-GAPDH antibodies. (b) Co-transfection of NS2A-Flag (2000 ng) and increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were collected at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-V5 and anti-K48-ubiquitin antibodies.

Mentions: We evaluated whether TRIM52 can mediate ubiquitination of NS2A protein. First, 293T cells were co-transfected with plasmid encoding NS2A-Flag plus V5-TRIM52 or with empty vector control in the presence or absence of HA-ubi. After 30 hpt, the cells were harvested and an IP was performed using anti-Flag affinity gel to analyze the ubiquitination of NS2A protein. Figure 5a shows that when NS2A-Flag was co-expressed with V5-TRIM52 in the presence of HA-ubi, smear signals were significantly increased in a TRIM52 dose-dependent manner. In addition, we determined the effect of TRIM52 on the K48-linked ubiquitination of NS2A. We co-transfected 293T cells with NS2A-Flag plus V5-TRIM52 or with vector control. The cells were collected at 30 hpt, and NS2A from cell lysates was precipitated using anti-Flag affinity gel. Western blot assay was subsequently performed using the indicated antibodies. Figure 5c shows that overexpression of TRIM52 significantly increased the K48-linked ubiquitination of NS2A. These results demonstrate that TRIM52 expression promotes NS2A ubiquitination.


TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A
TRIM52 induces ubiquitination of NS2A.(a) NS2A-Flag (2000 ng) was co-expressed with HA-Ubi (1000 ng) and with increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were harvested at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-HA, anti-V5 and anti-GAPDH antibodies. (b) Co-transfection of NS2A-Flag (2000 ng) and increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were collected at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-V5 and anti-K48-ubiquitin antibodies.
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f5: TRIM52 induces ubiquitination of NS2A.(a) NS2A-Flag (2000 ng) was co-expressed with HA-Ubi (1000 ng) and with increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were harvested at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-HA, anti-V5 and anti-GAPDH antibodies. (b) Co-transfection of NS2A-Flag (2000 ng) and increasing amounts of TRIM52-V5 (500 ng, 2000 ng) in 293T cells. The cells were collected at 30 hpt, and IP was performed using anti-Flag affinity gel. The precipitates were analyzed through Western blot analysis using anti-Flag, anti-V5 and anti-K48-ubiquitin antibodies.
Mentions: We evaluated whether TRIM52 can mediate ubiquitination of NS2A protein. First, 293T cells were co-transfected with plasmid encoding NS2A-Flag plus V5-TRIM52 or with empty vector control in the presence or absence of HA-ubi. After 30 hpt, the cells were harvested and an IP was performed using anti-Flag affinity gel to analyze the ubiquitination of NS2A protein. Figure 5a shows that when NS2A-Flag was co-expressed with V5-TRIM52 in the presence of HA-ubi, smear signals were significantly increased in a TRIM52 dose-dependent manner. In addition, we determined the effect of TRIM52 on the K48-linked ubiquitination of NS2A. We co-transfected 293T cells with NS2A-Flag plus V5-TRIM52 or with vector control. The cells were collected at 30 hpt, and NS2A from cell lysates was precipitated using anti-Flag affinity gel. Western blot assay was subsequently performed using the indicated antibodies. Figure 5c shows that overexpression of TRIM52 significantly increased the K48-linked ubiquitination of NS2A. These results demonstrate that TRIM52 expression promotes NS2A ubiquitination.

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.