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SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.

Su CI, Tseng CH, Yu CY, Lai MM - J. Virol. (2016)

Bottom Line: By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation.SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling.Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

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Related in: MedlinePlus

DENV NS5 protein is a target for SUMO modification. (A) HEK293T cells were cotransfected with Ubc9 and each HA-tagged DENV viral protein, with or without EGFP-tagged SUMO1, for 48 h. The cell lysates were subjected to immunoprecipitation (IP) by anti-HA agarose and WB analysis with anti-HA antibody. (B) The in vitro SUMOylation assay was reconstituted with recombinant SUMO E1 (Aos1/Uba2), SUMO E2 (Ubc9), His-tagged SUMO1, and immunopurified NS5 in the presence (+) or absence (−) of ATP as described in Materials and Methods. The reaction mixture was analyzed by WB with anti-NS5 antibody. (C) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 or left untransfected, as indicated, for 48 h. Transfectants were collected and subjected to analysis by IP-WB with the indicated antibodies. (D) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 together with SENP1 or SENP2 for 48 h as indicated. Cell lysates were harvested for IP-WB analysis with anti-HA antibody. Arrow, SUMOylated NS5 protein. (E) N18 cells were infected with DENV (MOI of 10) for 6 h and then transfected with EGFP-tagged SUMO1 or HA-tagged GFP. After 18 h of transfection, cells were lysed and analyzed by IP-WB with anti-GFP and anti-NS5 antibodies. (F) HEK293T cells were cotransfected with NS5 and Ubc9 plus Flag-tagged SUMO1 or SUMO2 for 48 h. Transfectants were harvested and subjected to analysis by IP-WB with the indicated antibodies. IB, immunoblot.
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Figure 2: DENV NS5 protein is a target for SUMO modification. (A) HEK293T cells were cotransfected with Ubc9 and each HA-tagged DENV viral protein, with or without EGFP-tagged SUMO1, for 48 h. The cell lysates were subjected to immunoprecipitation (IP) by anti-HA agarose and WB analysis with anti-HA antibody. (B) The in vitro SUMOylation assay was reconstituted with recombinant SUMO E1 (Aos1/Uba2), SUMO E2 (Ubc9), His-tagged SUMO1, and immunopurified NS5 in the presence (+) or absence (−) of ATP as described in Materials and Methods. The reaction mixture was analyzed by WB with anti-NS5 antibody. (C) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 or left untransfected, as indicated, for 48 h. Transfectants were collected and subjected to analysis by IP-WB with the indicated antibodies. (D) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 together with SENP1 or SENP2 for 48 h as indicated. Cell lysates were harvested for IP-WB analysis with anti-HA antibody. Arrow, SUMOylated NS5 protein. (E) N18 cells were infected with DENV (MOI of 10) for 6 h and then transfected with EGFP-tagged SUMO1 or HA-tagged GFP. After 18 h of transfection, cells were lysed and analyzed by IP-WB with anti-GFP and anti-NS5 antibodies. (F) HEK293T cells were cotransfected with NS5 and Ubc9 plus Flag-tagged SUMO1 or SUMO2 for 48 h. Transfectants were harvested and subjected to analysis by IP-WB with the indicated antibodies. IB, immunoblot.

Mentions: Since Ubc9 previously has been shown to interact with several DENV proteins (32, 34, 38), we asked whether any viral protein could be modified by SUMO. To this end, all 10 of the DENV proteins each were cloned with an HA tag and used in an in vivo SUMOylation assay to screen the potential SUMOylated DENV proteins. By cotransfection with EGFP-tagged SUMO1, each HA-tagged viral protein was immunoprecipitated by anti-HA agarose and separated by SDS-PAGE to reveal any additional protein bands showing a higher molecular mass than those expected of each viral protein. Among all of the viral proteins we examined, a higher-molecular-mass band was found only with NS5 (Fig. 2A), which suggested that DENV NS5 can be SUMOylated. E and NS4A also showed several higher-molecular-mass bands, both in the absence and in the presence of SUMO. The nature of these proteins is not known. For the current study, we focused on NS5.


SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication.

Su CI, Tseng CH, Yu CY, Lai MM - J. Virol. (2016)

DENV NS5 protein is a target for SUMO modification. (A) HEK293T cells were cotransfected with Ubc9 and each HA-tagged DENV viral protein, with or without EGFP-tagged SUMO1, for 48 h. The cell lysates were subjected to immunoprecipitation (IP) by anti-HA agarose and WB analysis with anti-HA antibody. (B) The in vitro SUMOylation assay was reconstituted with recombinant SUMO E1 (Aos1/Uba2), SUMO E2 (Ubc9), His-tagged SUMO1, and immunopurified NS5 in the presence (+) or absence (−) of ATP as described in Materials and Methods. The reaction mixture was analyzed by WB with anti-NS5 antibody. (C) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 or left untransfected, as indicated, for 48 h. Transfectants were collected and subjected to analysis by IP-WB with the indicated antibodies. (D) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 together with SENP1 or SENP2 for 48 h as indicated. Cell lysates were harvested for IP-WB analysis with anti-HA antibody. Arrow, SUMOylated NS5 protein. (E) N18 cells were infected with DENV (MOI of 10) for 6 h and then transfected with EGFP-tagged SUMO1 or HA-tagged GFP. After 18 h of transfection, cells were lysed and analyzed by IP-WB with anti-GFP and anti-NS5 antibodies. (F) HEK293T cells were cotransfected with NS5 and Ubc9 plus Flag-tagged SUMO1 or SUMO2 for 48 h. Transfectants were harvested and subjected to analysis by IP-WB with the indicated antibodies. IB, immunoblot.
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Figure 2: DENV NS5 protein is a target for SUMO modification. (A) HEK293T cells were cotransfected with Ubc9 and each HA-tagged DENV viral protein, with or without EGFP-tagged SUMO1, for 48 h. The cell lysates were subjected to immunoprecipitation (IP) by anti-HA agarose and WB analysis with anti-HA antibody. (B) The in vitro SUMOylation assay was reconstituted with recombinant SUMO E1 (Aos1/Uba2), SUMO E2 (Ubc9), His-tagged SUMO1, and immunopurified NS5 in the presence (+) or absence (−) of ATP as described in Materials and Methods. The reaction mixture was analyzed by WB with anti-NS5 antibody. (C) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 or left untransfected, as indicated, for 48 h. Transfectants were collected and subjected to analysis by IP-WB with the indicated antibodies. (D) HEK293T cells were cotransfected with NS5, SUMO1, and Ubc9 together with SENP1 or SENP2 for 48 h as indicated. Cell lysates were harvested for IP-WB analysis with anti-HA antibody. Arrow, SUMOylated NS5 protein. (E) N18 cells were infected with DENV (MOI of 10) for 6 h and then transfected with EGFP-tagged SUMO1 or HA-tagged GFP. After 18 h of transfection, cells were lysed and analyzed by IP-WB with anti-GFP and anti-NS5 antibodies. (F) HEK293T cells were cotransfected with NS5 and Ubc9 plus Flag-tagged SUMO1 or SUMO2 for 48 h. Transfectants were harvested and subjected to analysis by IP-WB with the indicated antibodies. IB, immunoblot.
Mentions: Since Ubc9 previously has been shown to interact with several DENV proteins (32, 34, 38), we asked whether any viral protein could be modified by SUMO. To this end, all 10 of the DENV proteins each were cloned with an HA tag and used in an in vivo SUMOylation assay to screen the potential SUMOylated DENV proteins. By cotransfection with EGFP-tagged SUMO1, each HA-tagged viral protein was immunoprecipitated by anti-HA agarose and separated by SDS-PAGE to reveal any additional protein bands showing a higher molecular mass than those expected of each viral protein. Among all of the viral proteins we examined, a higher-molecular-mass band was found only with NS5 (Fig. 2A), which suggested that DENV NS5 can be SUMOylated. E and NS4A also showed several higher-molecular-mass bands, both in the absence and in the presence of SUMO. The nature of these proteins is not known. For the current study, we focused on NS5.

Bottom Line: By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation.SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling.Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Show MeSH
Related in: MedlinePlus