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. 2009 Jun;83(11):5408-18.
doi: 10.1128/JVI.02188-08. Epub 2009 Mar 11.

NS5 of dengue virus mediates STAT2 binding and degradation

Joseph Ashour  1 , Maudry Laurent-Rolle, Pei-Yong Shi, Adolfo García-Sastre
Affiliations

NS5 of dengue virus mediates STAT2 binding and degradation

Joseph Ashour et al. J Virol. 2009 Jun.

Abstract

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response. As such, viral pathogens have devised multiple mechanisms to antagonize this pathway and thus facilitate infection. Dengue virus (DENV) encodes several proteins (NS2a, NS4a, and NS4b) that have been shown individually to inhibit the IFN response. In addition, DENV infection results in reduced levels of expression of STAT2, which is required for IFN signaling (M. Jones, A. Davidson, L. Hibbert, P. Gruenwald, J. Schlaak, S. Ball, G. R. Foster, and M. Jacobs, J. Virol. 79:5414-5420, 2005). Translation of the DENV genome results in a single polypeptide, which is processed by viral and host proteases into at least 10 separate proteins. To date, no single DENV protein has been implicated in the targeting of STAT2 for decreased levels of expression. We demonstrate here that the polymerase of the virus, NS5, binds to STAT2 and is necessary and sufficient for its reduced level of expression. The decrease in protein level observed requires ubiquitination and proteasome activity, strongly suggesting an active degradation process. Furthermore, we show that the degradation of but not binding to STAT2 is dependent on the expression of the polymerase in the context of a polyprotein that undergoes proteolytic processing for NS5 maturation. Thus, the mature form of NS5, when not expressed as a precursor, was able to bind to STAT2 but was unable to target it for degradation, establishing a unique role for viral polyprotein processing in providing an additional function to a viral polypeptide. Therefore, we have identified both a novel mechanism by which DENV evades the innate immune response and a potential target for antiviral therapeutics.

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Figures

FIG. 1.
FIG. 1.
Reduced levels of STAT2 in cells expressing the nonstructural region of the DENV polyprotein. (A) Vero cells were infected for 24 h with DENV at an MOI of 10 and subsequently lysed and examined by Western blotting. (B) U6A cells stably expressing STAT2-GFP were infected with DEN2 at an MOI of 40 for 24 h prior to fixation. Cells were then probed with antibody against NS5 and stained for DNA (DAPI [4′,6′-diamidino-2-phenylindole]). (C) U6A-STAT2-GFP cells were infected with DEN2 at an MOI of 40 and measured for a loss of GFP by live microscopy at the given time points. White arrows indicate cells in the infected samples at 9 h p.i. that have lost the GFP signal. (D) Vero cells were infected with DENV at an MOI of 10, subsequently lysed at the given time points, and examined by Western blotting. (E) Vero cells stably expressing a DEN1 replicon (NS1-5) were lysed and examined by Western blotting. (F) wtVero cells or Vero cells stably expressing a DEN1 replicon were treated with the indicated amounts of type I IFN (IFN-α/β) for 24 h. Cells were subsequently challenged with NDV-GFP and assayed via fluorescence microscopy for GFP expression at 14 h p.i. (G) Vero cells or Vero cells stably expressing a DEN1 replicon were treated with 1,000 units of type I (IFN-α/β) or type II (IFN-γ) IFN for 24 h. Cells were subsequently challenged with NDV-GFP and assayed via fluorescence microscopy for GFP expression at 14 h p.i. (H) 293T cells were cotransfected with plasmids expressing NS1-5-HA and GFP or NS1-4b-HA and GFP. Twenty-four hours posttransfection, cells were sorted by FACS for GFP-positive cells and subsequently lysed and examined via Western blotting. Densitometry analysis of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with the value of 1 in the case of NS5 levels being indicative of no detection (background levels).
FIG. 2.
FIG. 2.
DENV NS5 interacts with STAT2. (A) 293T cells were cotransfected with plasmids expressing FLAG-tagged STAT1 (STAT1-FLAG) and STAT2 (STAT2-FLAG) and empty plasmid (empty) or plasmid expressing HA-tagged DENV NS5 (NS5-HA), DENV core (CORE-HA), or NiV-V (HA-NiV-V) proteins. Lysates were then immunoprecipitated with anti-HA antibody (IP HA), and Western blotting was performed using anti-HA and anti-FLAG antibodies. Asterisks mark the heavy and light chains from the HA antibody. (B) 293T cells were cotransfected with plasmids expressing NS5-HA and either STAT1-FLAG or STAT2-FLAG. Lysates were then immunoprecipitated with anti-FLAG antibody (IP FLAG), and Western blotting was performed using anti-HA and anti-FLAG antibodies. TCE, total cell extracts were subjected to Western blotting using anti-HA, anti-FLAG, and anti-GAPDH antibodies.
FIG. 3.
FIG. 3.
Expression of a precursor form of DENV NS5 cleaved by the DENV protease results in reduced STAT2 levels. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. Schematics of the transfected constructs are shown at the bottom. ORFs that contain an “sp” at the N terminus have a signal peptide which directs the entire polyprotein to the surface of the ER for translation. Black arrows and red arrows indicate cleavage sites for cellular and the DEN2 viral proteases, respectively. The DEN2 active protease is highlighted in red. Densitometry analysis of the levels of STAT2 and NS5 are included on the far right, and levels are calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A). Mutation of the DENV protease recognition site at the N terminus of NS5 is indicated by the blue dashes. The DENV protease labeled in blue indicates a serine-to-alanine mutation within the catalytic site of the DENV protease.
FIG. 4.
FIG. 4.
Cleaved NS5 is sufficient for reduced STAT2 levels and does not require an N-terminal glycine. (A) 293T cells were transfected with the indicated constructs. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, subsequently lysed, subjected to 4-to-20% SDS-polyacrylamide gel electrophoresis, and examined via Western blotting using GFP-, HA-, STAT1-, STAT2-, and GAPDH-specific antibodies. The TEV protease and its cleavage site are indicated by the green text and arrow, respectively. Cleavage sequences targeted by endogenous deubiquitinases are noted by a yellow arrow. Densitometry analyses of the levels of STAT2 and NS5 are included at the bottom, and levels were calculated relative to the levels in lane 1, with a value of 1 in the case of NS5 levels being indicative of no detection (background levels). (B) Same as above (A) except that lysates were run on a 7.5% SDS-polyacrylamide gel and subsequently analyzed by Western blotting using GFP-, STAT2-, and tubulin-specific antibodies.
FIG. 5.
FIG. 5.
Inhibitors of the ubiquitin-proteasome pathway prevent STAT2 degradation by DENV NS5. (A) wtVero or Vero cells stably expressing the DEN1 replicon were treated with the indicated amounts of MG132. Sixteen hours posttreatment, cells were lysed and examined for ubiquitin, STAT2, STAT1, NS5, and GAPDH levels via Western blotting. (B) STAT2-deficient U6A cells were transfected with HA-ubiquitin, STAT2-FLAG, STAT1-GFP, NS2b-3, and either E-clvNS5-HA or an empty vector plasmid. Cells were then treated with lactacystin for 8 h and subsequently lysed and examined by Western blotting using ubiquitin, STAT2, STAT1, HA, and tubulin antibodies. (C) 293T cells were cotransfected with NS2b-3-HA and the plasmids indicated at the top. Ten hours posttransfection, cells were treated with the indicated amounts of lactacystin. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined by Western blotting using ubiquitin-, GFP-, HA-, and GAPDH-specific antibodies. (D) STAT2-deficient U6A cells were transfected with 0.1 or 1 μg of FLAG-STAT2-FLAG, 0.1 μg NS2b-3, and either 1 μg E-clvNS5-HA or an empty vector plasmid. Cells were then lysed and examined by Western blotting using FLAG antibody and long-term film exposure to detect any additional low-intensity bands. The arrow indicates the expected size of FLAG-STAT2-FLAG. The asterisk marks a nonspecific band running at the same mobility of FLAG-STAT2-FLAG. (E) 293T cells were cotransfected with NS2b-3 and the plasmids indicated at the top. Twenty-four hours posttransfection, cells were sorted for GFP-positive cells by FACS, lysed, and examined via Western blotting using STAT2-, GFP-, HA-, FLAG-, and GAPDH-specific antibodies.
FIG. 6.
FIG. 6.
Truncations in the DENV NS5 protein affect its ability to associate with STAT2, decrease STAT2 levels, and inhibit IFN signaling. (A) 293T cells were transfected with the indicated constructs. Numbering refers to the glycine start position within the NS5 protein. All numbered forms of NS5 are expressed within the context of the E-Ub-NS5-GFP construct (i.e., 10-900, NS5 residues 10 to 900 fused to the C terminus of the E-Ub cassette). Twenty-four hours posttransfection, cells were sorted for GFP-positive cells using FACS, lysed, and examined by Western blotting using STAT2, GFP, and tubulin antibodies. (B) 293T cells were cotransfected with the indicated plasmids (the numbering system is identical to that described in A), STAT1-FLAG, and STAT2-FLAG. In order to detect STAT2 binding by NS5, STAT2-FLAG plasmid was transfected in excess with respect to E-Ub-NS5-GFP plasmids, resulting in a not-detectable degradation of STAT2-FLAG. Lysates were immunoprecipitated with a polyclonal GFP antibody (pGFP), and Western blots were performed using FLAG, GFP, and GAPDH antibodies. TCE, total cell extracts. (C) 293T cells were transfected with the ISRE-54-CAT reporter, a constitutively expressing firefly luciferase plasmid, and the indicated HA-tagged viral protein. Twenty-four hours posttransfection, cells were treated with 1,000 U/ml of type I IFN. Twenty-four hours posttreatment, cells were lysed and measured for CAT and luciferase activity. Data are represented with the standard deviations from three independent experiments. Samples showing P values of less than 0.05 compared with the empty control sample are indicated.
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