The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5

Abstract

The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.

FIG. 1.

Panels a and b, respectively; 2fTGH, 2f/PIV2-V, and 2f/SV5-V cells were (+) or were not (-) infected with 5 PFU of SV5/cell (a) or hPIV2/cell (b) as indicated. At 16 h p.i. the cells were harvested, and the presence of STAT1 or STAT2 was detected by immunoblot analysis. (c) 293 cells were transiently transfected with combinations of plasmids expressing V, myc-STAT1, STAT2, and DDB1 (as shown), with a total of 8 μg of plasmid per transfection, 2 μg of each plasmid together with the appropriate amount of carrier plasmid (pEFplink2). At 48 h posttransfection the cells were harvested, and the presence of myc-STAT1 was detected by immunoblot analysis.

FIG. 2.

(a) DDB1 is coimmunoprecipitated with the V protein of the W3 and CPI+ strains of SV5 but not the CPI− strain. Flasks (75 cm²) of 2fTGH, 2f/SV5-V(CPI+/m), 2fSV5-V(CPI-/m), and 2f/SV5-V(W3) cells were metabolically labeled with [³⁵S]methionine for 5 h, harvested, and immunoprecipitated with anti-myc (lanes 1 to 3) or anti-Pk (lanes 4 to 6) antibodies. (b) DDB1 is coimmunoprecipitated with CPI+ V but not CPI− V in 293 cells transiently transfected with plasmids expressing DDB1 together with CPI+ or CPI− myc-tagged V (2 μg of each plasmid per 25-cm² flask). Cells were labeled with [³⁵S]methionine for 4 h at 48 h posttransfection, harvested, and immunoprecipitated with anti-myc antibody. The position of V is indicated with an asterisk. (c) The anti-C-terminus antibody (V mAb 11) immunoprecipitates the V protein from 2f/SV5-V(CPI−) cells but not from 2f/SV5(W3) or 2f/SV5(CPI+) cells. Cells were labeled with [³⁵S]methionine for 4 h, harvested, and immunoprecipitated with V mAb 11 antibody. (d) Immunoblot assays of total cell lysates of the cells reveals that the anti-Pk MAb interacts with the V proteins of W3 and CPI+ but not CPI− while the anti-C-term antibody (V mAb 11) interacts with the V proteins of W3, CPI+, and CPI−. (e) The anti-C terminal (V mAb 11) antibody immunoprecipitates V proteins of W3, CPI+, and CPI− from infected cell lysates. 2fTGH cells were mock infected or were infected with SV5 strains W3, CPI+, or CPI− at a multiplicity of infection of 1, and at 16 h postinfection they were metabolically labeled for 4 h with [³⁵S]methionine and immunoprecipitated with anti-C-terminus (V mAb 11) or anti-Pk MAbs as indicated.

FIG. 3.

(A) Examples of the ability of altered SV5 V proteins to block IFN signaling are shown. HeLa cells were transfected with plasmids as indicated, a luciferase reporter plasmid, and a β-galactosidase expression vector. Cells were or were not treated with 1,000 U of IFN-á for 4 h; luciferase levels in extracts were normalized to β-galactosidase levels, and the relative expression values were plotted; a value of 1.0 was assigned to the basal expression level of the mock-treated vector-only sample. Data shown represent the average from at least three independent transfection experiments. (B) Summary of the phenotypes of SV5 V or altered forms of SV5 V. The protein domains present in each form of SV5 V are shown on the left. Interaction with DDB1 was determined using the yeast two-hybrid assay; a strongly positive interaction (+++) was scored as the ability of transformants to give colonies in excess of 2 mm after 5 days' growth at 30°C on SDM lacking histidine plus 5 mM 3-aminotriazole and was comparable to that seen for the interaction between the SV40 T antigen and p53 (plasmids pVA3 and pTD1; Clontech). Colonies of 1 to 2 mm after 5 days are indicated by ++. No interaction (−) was indicated by colonies of less than 0.2 mm and was equivalent to the growth rate of the untransformed yeast strain or yeast transformed with control plasmid pGBT9 or pGBT9.SV5-V or doubly transformed with pGBT9.SV5-V and pHON3 (data not shown). Inhibition of IFN signaling was determined using the approach exemplified in panel A. Intact SV5 V and a truncation expressing only amino acids 20 to 222 limit IFN signaling to twofold or less (and are scored as +) in comparison to the 30- to 40-fold induction seen with vector alone. All other forms of SV5 V examined failed to inhibit IFN signaling and are therefore scored as −.

FIG. 4.

HeLa and HeLa/SV5-V cells were or were not treated with siRNA or control RNA twice at 24-h intervals. At 100 h posttreatment, IFN was or was not added to the culture medium, and 20 h later the cells were metabolically labeled with [³⁵S]methionine for 5 h in the presence or absence of IFN as appropriate. Cells were then harvested, and the levels of DDB1 estimated by coimmunoprecipitation with V (a) (note that DDB1 was detected in HeLa/SV5-V cells treated with control RNA but not when they were treated with siRNAs and that DDB1 was not immunoprecipitated from HeLa cells as they do not express V) and immunoblot analysis (b). Levels of STAT1 in the cell samples were also examined by immunoprecipitation (c) and immunoblot analysis (d). Note the increase in levels of STAT1 in HeLa/SV5-V cells treated with siRNAs, especially following incubation with IFN (lanes 3 and 4), but not in cells treated with control RNA (lanes 5 and 6).

FIG. 5.

STAT1 is degraded in cells from a group E patient with xeroderma pigmentosum. GM02415 cells were infected with SV5 (strain W3) at a multiplicity of infection of 5. At the time of infection, the cells were (+) or were not (−) treated with IFN throughout the course of infection. At 24 h p.i. the cells were harvested, and the presence of STAT1 was detected by immunoblot analysis.