Limits...
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.

Show MeSH

Related in: MedlinePlus

Putative SUMO-interacting motif (SIM) located in the N-terminal domain of NS5 is crucial for NS5 SUMOylation. (A) Schematic diagram of NS5 and its truncation derivatives. The predicted sizes and the ability of NS5 derivatives to undergo SUMOylation are summarized on the right. NLS, nuclear localization signal. (B) HEK293T cells were cotransfected with Ubc9 and each NS5 construct with (+) or without (−) EGFP-tagged SUMO1 for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (C) Amino acid sequence of the N-terminal 71 to 300 residues in DENV2 NS5. The lysine (K) residues are shown in boldface, whereas the conserved K residues among four serotypes of DENV are underlined. (D) In HEK293T cells, plasmid expressing NS5-WT or the indicated mutants containing single, double, or triple K-R substitution(s) were cotransfected with or without EGFP-SUMO1 and Ubc9. The whole-cell extracts then were collected and subjected to IP-WB analysis by anti-HA antibody. (E) HEK293T cells were cotransfected with Ubc9, EGFP-SUMO1, and each NS5 construct containing multiple K-R substitutions as indicated for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (F) Protein alignment of the N-terminal sequences from four different serotypes of DENV NS5. The putative SIM of DENV NS5 is shown in the gray square. (G) HEK293T cells were cotransfected with Ubc9 and/or EGFP-SUMO1 plus NS5-WT or its mutant containing 12 K-R substitutions [termed 12(K-R)] or VVDL-to-AAAA substitution at the putative SIM motif (SIMmut). Cells were harvested at 48 h posttransfection for IP-WB analysis using anti-HA antibody. Arrow, SUMOylated NS5 proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836324&req=5

Figure 3: Putative SUMO-interacting motif (SIM) located in the N-terminal domain of NS5 is crucial for NS5 SUMOylation. (A) Schematic diagram of NS5 and its truncation derivatives. The predicted sizes and the ability of NS5 derivatives to undergo SUMOylation are summarized on the right. NLS, nuclear localization signal. (B) HEK293T cells were cotransfected with Ubc9 and each NS5 construct with (+) or without (−) EGFP-tagged SUMO1 for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (C) Amino acid sequence of the N-terminal 71 to 300 residues in DENV2 NS5. The lysine (K) residues are shown in boldface, whereas the conserved K residues among four serotypes of DENV are underlined. (D) In HEK293T cells, plasmid expressing NS5-WT or the indicated mutants containing single, double, or triple K-R substitution(s) were cotransfected with or without EGFP-SUMO1 and Ubc9. The whole-cell extracts then were collected and subjected to IP-WB analysis by anti-HA antibody. (E) HEK293T cells were cotransfected with Ubc9, EGFP-SUMO1, and each NS5 construct containing multiple K-R substitutions as indicated for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (F) Protein alignment of the N-terminal sequences from four different serotypes of DENV NS5. The putative SIM of DENV NS5 is shown in the gray square. (G) HEK293T cells were cotransfected with Ubc9 and/or EGFP-SUMO1 plus NS5-WT or its mutant containing 12 K-R substitutions [termed 12(K-R)] or VVDL-to-AAAA substitution at the putative SIM motif (SIMmut). Cells were harvested at 48 h posttransfection for IP-WB analysis using anti-HA antibody. Arrow, SUMOylated NS5 proteins.

Mentions: SUMOpylation occurs on lysine residues of the targeted substrates. There are 64 lysines in DENV NS5 protein that might serve as the SUMO acceptors. To delineate the SUMOylated residues in NS5, various HA-tagged NS5 truncation mutants were generated (Fig. 3A) and subjected to in vivo SUMOylation assay. Given that the SUMO modification machinery is predominantly nuclear and that NLS are important for the SUMOylation of nuclear proteins (19, 41), we preserved the NLS in all of these deletion mutants. By in vivo SUMOylation assay, we found that EGFP-tagged SUMO1 was only detected on the NS5 fragments containing the 71 to 300 residues at the N terminus (Fig. 3B, lanes 1 to 8), whereas the C-terminal half of NS5 seemed not to be critical for SUMOylation (Fig. 3B, lanes 9 to 14). These results suggested that two acceptor lysine sites for SUMOylation are located in the N-terminal 71 to 300 residues of NS5.


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

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

Putative SUMO-interacting motif (SIM) located in the N-terminal domain of NS5 is crucial for NS5 SUMOylation. (A) Schematic diagram of NS5 and its truncation derivatives. The predicted sizes and the ability of NS5 derivatives to undergo SUMOylation are summarized on the right. NLS, nuclear localization signal. (B) HEK293T cells were cotransfected with Ubc9 and each NS5 construct with (+) or without (−) EGFP-tagged SUMO1 for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (C) Amino acid sequence of the N-terminal 71 to 300 residues in DENV2 NS5. The lysine (K) residues are shown in boldface, whereas the conserved K residues among four serotypes of DENV are underlined. (D) In HEK293T cells, plasmid expressing NS5-WT or the indicated mutants containing single, double, or triple K-R substitution(s) were cotransfected with or without EGFP-SUMO1 and Ubc9. The whole-cell extracts then were collected and subjected to IP-WB analysis by anti-HA antibody. (E) HEK293T cells were cotransfected with Ubc9, EGFP-SUMO1, and each NS5 construct containing multiple K-R substitutions as indicated for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (F) Protein alignment of the N-terminal sequences from four different serotypes of DENV NS5. The putative SIM of DENV NS5 is shown in the gray square. (G) HEK293T cells were cotransfected with Ubc9 and/or EGFP-SUMO1 plus NS5-WT or its mutant containing 12 K-R substitutions [termed 12(K-R)] or VVDL-to-AAAA substitution at the putative SIM motif (SIMmut). Cells were harvested at 48 h posttransfection for IP-WB analysis using anti-HA antibody. Arrow, SUMOylated NS5 proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836324&req=5

Figure 3: Putative SUMO-interacting motif (SIM) located in the N-terminal domain of NS5 is crucial for NS5 SUMOylation. (A) Schematic diagram of NS5 and its truncation derivatives. The predicted sizes and the ability of NS5 derivatives to undergo SUMOylation are summarized on the right. NLS, nuclear localization signal. (B) HEK293T cells were cotransfected with Ubc9 and each NS5 construct with (+) or without (−) EGFP-tagged SUMO1 for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (C) Amino acid sequence of the N-terminal 71 to 300 residues in DENV2 NS5. The lysine (K) residues are shown in boldface, whereas the conserved K residues among four serotypes of DENV are underlined. (D) In HEK293T cells, plasmid expressing NS5-WT or the indicated mutants containing single, double, or triple K-R substitution(s) were cotransfected with or without EGFP-SUMO1 and Ubc9. The whole-cell extracts then were collected and subjected to IP-WB analysis by anti-HA antibody. (E) HEK293T cells were cotransfected with Ubc9, EGFP-SUMO1, and each NS5 construct containing multiple K-R substitutions as indicated for 48 h. Transfectants were harvested and analyzed by IP-WB using anti-HA antibody. (F) Protein alignment of the N-terminal sequences from four different serotypes of DENV NS5. The putative SIM of DENV NS5 is shown in the gray square. (G) HEK293T cells were cotransfected with Ubc9 and/or EGFP-SUMO1 plus NS5-WT or its mutant containing 12 K-R substitutions [termed 12(K-R)] or VVDL-to-AAAA substitution at the putative SIM motif (SIMmut). Cells were harvested at 48 h posttransfection for IP-WB analysis using anti-HA antibody. Arrow, SUMOylated NS5 proteins.
Mentions: SUMOpylation occurs on lysine residues of the targeted substrates. There are 64 lysines in DENV NS5 protein that might serve as the SUMO acceptors. To delineate the SUMOylated residues in NS5, various HA-tagged NS5 truncation mutants were generated (Fig. 3A) and subjected to in vivo SUMOylation assay. Given that the SUMO modification machinery is predominantly nuclear and that NLS are important for the SUMOylation of nuclear proteins (19, 41), we preserved the NLS in all of these deletion mutants. By in vivo SUMOylation assay, we found that EGFP-tagged SUMO1 was only detected on the NS5 fragments containing the 71 to 300 residues at the N terminus (Fig. 3B, lanes 1 to 8), whereas the C-terminal half of NS5 seemed not to be critical for SUMOylation (Fig. 3B, lanes 9 to 14). These results suggested that two acceptor lysine sites for SUMOylation are located in the N-terminal 71 to 300 residues of 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