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Virus-Cell Interactions

Simian Virus 5 V Protein Acts as an Adaptor, Linking DDB1 to STAT2, To Facilitate the Ubiquitination of STAT1

B. Precious, K. Childs, V. Fitzpatrick-Swallow, S. Goodbourn, R. E. Randall
B. Precious
1School of Biology, University of St. Andrews, Fife KY16 9TS, United Kingdom
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K. Childs
2Department of Basic Medical Sciences, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom
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V. Fitzpatrick-Swallow
2Department of Basic Medical Sciences, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom
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S. Goodbourn
2Department of Basic Medical Sciences, St. George's Hospital Medical School, University of London, London SW17 0RE, United Kingdom
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R. E. Randall
1School of Biology, University of St. Andrews, Fife KY16 9TS, United Kingdom
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  • For correspondence: rer@st-and.ac.uk
DOI: 10.1128/JVI.79.21.13434-13441.2005
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  • FIG. 1.
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    FIG. 1.

    SV5 V binds directly to DDB1, STAT2 but not STAT1. (a). Saccharomyces cerevisiae CG1945 was transformed with a plasmid directing the expression of a GAL4 DNA binding domain fusion with SV5 V (pGBKT7.SV5 V) or the parental nonfusion vector pGBKT7, and a plasmid directing the expression of either a GAL4 activation domain fusion with DDB1 (pHON3.DDB1), STAT1 (pHON3.STAT1), or STAT2 (pGADT7.STAT2), or the parental vector pHON3 or pGADT7. The double transformants were selected and replated sequentially on SD without leucine and tryptophan or leucine, tryptophan, and histidine as described in the Materials and Methods. All combinations of plasmids should confer growth on medium lacking leucine and tryptophan (SD-LT), whereas only combinations of proteins that can interact confer growth on medium lacking leucine, tryptophan, and histidine and containing 2 mM 3-aminotriazole (SD-LTH). (b). S. cerevisiae PJ69-4α was transformed with a plasmid directing the expression of a GAL4 DNA binding domain fusion with DDB1 (pHON1.DDB1) or STAT1 (pHON1.STAT1) or the parental nonfusion vector pHON1, and a plasmid directing the expression of either a GAL4 activation domain fusion with STAT2 (pGADT7.STAT2) or DDB1 (pHON3.DDB1) or the parental vectors pHON3 or pGADT7. The double transformants were selected and replated sequentially on SD-LT and SD-LTH. All combinations of plasmids should confer growth on medium lacking leucine and tryptophan (SD-LT), whereas only combinations of proteins that can interact confer growth on medium lacking leucine, tryptophan adenine and histidine and containing 2 mM 3-aminotriazole (SD-LTH). (c). We incubated 500 μl of soluble cell extracts of Sf9 cells that had, or had not, been infected with recombinant baculoviruses which express DDB1, STAT2 or STAT1 with 10 μl of glutathione beads saturated with either GST-SV5 V (lanes 2 to 5) or GST (lanes 7 to 10). Bound proteins were visualized by Coomassie blue staining of the proteins following their separation through a 4 to 12% SDS-PAGE gel. Lanes 1 and 6 are controls of GST-SV5 V and GST, respectively incubated with buffer only (the bands seen below GST-SV5 V have been identified by Western blotting as truncations of GST-SV5 V[data not shown]) Lanes 11 to 14 represent 5 μl of the soluble cell extracts (i.e., 100 times this amount was mixed with the GST beads). Asterisks on lanes 11 to 14 indicate the positions of DDB1 STAT2 and STAT1, respectively, in the Sf9 cell extracts.

  • FIG. 2.
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    FIG. 2.

    Assembly of DDB/SV5 V/STAT2/STAT1 complexes. Protein G beads were saturated with anti-STAT2 antibody and 40-μl aliquots of the resulting suspension of beads (50% vol/vol) were incubated with 500 μl of soluble Sf9 cell extract infected with a recombinant baculovirus that expressed STAT2 (Fig. 1, lane 12). These STAT2-saturated beads were then incubated with various mixtures of soluble extracts of; purified bacterially expressed SV5 V, soluble extracts of SF9 cells infected with recombinant baculoviruses that express either DDB1 or STAT1 (Fig. 1, lanes 11 and 13, respectively), or preformed and purified DDB1/SV5 V complexes. Samples equivalent to 5 μl of the original bead suspension were subjected to 4 to 12% SDS-PAGE; the SV5 V protein was detected by Western blot analysis, and other bound proteins were detected by Coomassie blue staining of the gel (note that the V protein was detected by Western blot analysis because it comigrated with antibody light chain on SDS-PAGE). The + symbol above each lane indicates which proteins or cell extracts were incubated with the immobilized anti-STAT2-saturated beads (Note that all comparable lanes had exactly the same amount of expressed protein cell extracts in the same volume added to the beads, the volume being corrected, where appropriate, with uninfected Sf9 cell extract.)

  • FIG. 3.
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    FIG. 3.

    Interaction of SV5 V with STAT2 is stabilized by the presence of both DDB1 and STAT1. Complexes of SV5 V and STAT2 were assembled on protein G beads as described for Fig. 2. These complexes were subsequently incubated for 180 min with 500 μl of soluble antigen extracts of Sf9 cells that had or had not been infected with recombinant baculoviruses that expressed DDB1 or STAT1 (as shown in Fig. 1, lanes 11, 13, and 14). After incubation the beads were centrifuged and supernatant fluids were collected. Samples from washed beads and the supernatants were analyzed by SDS-PAGE followed by Coomassie blue staining of the gel (a) or by Western blotting with anti-Pk SV5 V antibody (b). The presence of SV5 V in the supernatant fluids was also detected by Western blot analysis (c). It is important to note that lane 1 represents the composition of the STAT2/SV5 V beads prior to incubation with the indicated cell extracts. Also note all comparable lanes had exactly the same amount of expressed protein cell extracts in the same volume added to the beads, the volume being corrected, where appropriate, with uninfected Sf9 cell extract.

  • FIG. 4.
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    FIG. 4.

    SV5 V, DDB1, STAT2 and STAT1 complexes recruit Cul4a from 293T cell extracts. We incubated 40 μl of DDB1/SV5 V/STAT2/STAT1 complexes captured on protein G beads (as described and shown in Fig. 2, lane 6) with 500 μl of either buffer (lanes 1) or soluble antigen extracts of 293T cells (lanes 2) as described in Materials and Methods. The proteins captured on the beads were separated by electrophoresis through a 4 to 12% PAGE and either visualized by staining the gel with Coomassie blue (panel a) or subjected to Western blot analysis using a pool of antibodies to STAT1 and DDB1 (panel b) or an anti-Cul4 antibody (panel c). Note that the Coomassie blue-stained band marked * in panel A was analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF-mass spectrometry and found to contain DDB1 and bovine immunoglobulin G (heavy and light chains, which presumably was captured from the 293T cell extract).

  • FIG. 5.
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    FIG. 5.

    SV5 V, DDB1, STAT2, STAT1, Cul4a containing complexes can induce ubiquitination after of STAT1. Complexes containing various combinations of DDB1, SV5 V, STAT2, STAT1 and proteins captured from 293T cells (including Cul4a) were assembled on beads as previously described; 5 μl of washed beads were then used for ubiquitination assays in the presence and absence of E1 and UBCH5a. The presence of polyubiquitinated STAT1 in the samples was detected by Western blot analysis using anti-STAT1 antibody followed by horseradish peroxidase-labeled protein A. Labeled proteins were then visualized on x-ray film following exposure to ECL reagents. (Note that lanes 2 and 6 and lanes 1 and 5 are the same complexes as shown in Fig. 4, lanes 1 and 2, respectively.)

  • FIG. 6.
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    FIG. 6.

    Model for the role of SV5 V in the ubiquitination of STAT1. (a). In uninfected cells DDB1 and Cul4a complexes do not associate with STAT1 and STAT2, which can associate together in the absence of IFN stimulated phosphorylation. (b). After infection with SV5, the V protein acts as a linker bringing DDB1/Cul4a complexes into a close and stable association with STAT1/STAT2 complexes. (c). An E3 ligase complex is formed by the recruitment of other cellular proteins (including E1 and UBCH5a) and STAT1 becomes polyubiquitinated. (d). STAT1 is degraded by the proteasome. The DDB1/SV5 V/STAT2 complex is destabilized. While SV5 V remains bound to DDB1/Cul4a complex, STAT2 either captures STAT1 to form a new degradation complex or dissociates from the complex (perhaps through competition with STAT2/STAT1 complexes) and can thus bind any free STAT1 before the resulting STAT1/STAT2 complex is recaptured by the DDB1/SV5 V-containing E3 ligase. Note that the model does not attempt to predict the stoichiometry of the components of the complex, only their relative positions.

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Simian Virus 5 V Protein Acts as an Adaptor, Linking DDB1 to STAT2, To Facilitate the Ubiquitination of STAT1
B. Precious, K. Childs, V. Fitzpatrick-Swallow, S. Goodbourn, R. E. Randall
Journal of Virology Oct 2005, 79 (21) 13434-13441; DOI: 10.1128/JVI.79.21.13434-13441.2005

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Simian Virus 5 V Protein Acts as an Adaptor, Linking DDB1 to STAT2, To Facilitate the Ubiquitination of STAT1
B. Precious, K. Childs, V. Fitzpatrick-Swallow, S. Goodbourn, R. E. Randall
Journal of Virology Oct 2005, 79 (21) 13434-13441; DOI: 10.1128/JVI.79.21.13434-13441.2005
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KEYWORDS

DNA-binding proteins
parainfluenza virus 5
Trans-Activators
viral structural proteins

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