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. 2006 Oct;80(20):10218-28.
doi: 10.1128/JVI.00375-06.

Functional interaction between human herpesvirus 6 immediate-early 2 protein and ubiquitin-conjugating enzyme 9 in the absence of sumoylation

Andru Tomoiu  1 , Annie Gravel, Robert M Tanguay, Louis Flamand
Affiliations

Functional interaction between human herpesvirus 6 immediate-early 2 protein and ubiquitin-conjugating enzyme 9 in the absence of sumoylation

Andru Tomoiu et al. J Virol. 2006 Oct.

Abstract

The immediate-early 2 (IE2) protein of human herpesvirus 6 is a potent transactivator of cellular and viral promoters. To better understand the biology of IE2, we generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for proteins that could interact with IE2. The most frequently isolated IE2-interacting protein was the human ubiquitin-conjugating enzyme 9 (Ubc9), a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using deletion mutants of IE2, we mapped the IE2-Ubc9-interacting region to residues 989 to 1037 of IE2. The interaction was found to be of functional significance to IE2, as Ubc9 overexpression significantly repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited a similar effect on IE2, indicating that the E2 SUMO-conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus sumoylation sites or evidence of IE2 conjugation to SUMO could be demonstrated under in vivo or in vitro conditions. Moreover, expression levels and nuclear localization of IE2 were not altered by Ubc9 overexpression, suggesting that Ubc9's repressive function likely occurs at the transcriptional complex level. Overall, our results indicate that Ubc9 influences IE2's function and provide new information on the complex interactions that occur between herpesviruses and the sumoylation pathway.

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Figures

FIG. 1.
FIG. 1.
Mapping of the IE2-Ubc9 interaction domain. Deletion mutants of IE2 were generated by restriction digest or mutagenesis of wild-type IE2. R1 is the SSRA/SSRD amino acid repeat region of IE2.
FIG. 2.
FIG. 2.
(A) HHV-6A IE2 protein relative localization with Ubc9 in infected cells. HSB-2 cells were infected with HHV-6A and processed for immunofluorescence as described in Materials and Methods. Left panels represent cells reacted with anti-IE2 antibody (IE2 in green), and middle panels show the same fields colored with anti-Ubc9 antibody (Ubc9 in red). Colocalizing proteins are represented in the merged panels by the yellow color. (B) HHV-6A IE2 protein relative localization with SUMO-1 in infected cells. Left panels represent cells reacted with anti-IE2 antibody (IE2 in green), and middle panels show the same fields colored with anti-SUMO-1 antibody (SUMO-1 in red). (C) HHV-6A IE2 protein relative localization with Ubc9 in transfected cells. HEK293T cells were transfected with the pcDNA4-IE2 construct and processed for immunofluorescence as described in Materials and Methods. Left panels represent cells reacted with anti-IE2 antibody (IE2 in green), and middle panels show the same fields colored with anti-Ubc9 antibody (Ubc9 in red). (D) HHV-6A IE2 protein relative localization with SUMO-1 in transfected cells. Left panels represent cells reacted with anti-IE2 antibody (IE2 in green), and middle panels show the same fields colored with anti-SUMO-1 antibody (SUMO-1 in red).
FIG. 3.
FIG. 3.
HHV-6 IE1 but not IE2 is sumoylated in vivo. HHV-6A-infected HSB-2 cells and HHV-6B-infected Molt-3 cells were lysed as described in Materials and Methods. After immunoprecipitation (IP) of IE1B or IE2A, samples were analyzed by electrophoresis and Western blotted (WB) with anti-IE1, anti-IE2, or anti-SUMO-1 antibodies. (A) Immunoprecipitated samples were analyzed by SDS-PAGE and probed for IE1B or IE2A using appropriate antibodies. MI, mock-infected cells. (B) The same samples were probed using an anti-SUMO-1 antibody.
FIG. 4.
FIG. 4.
Inhibitory effect of Ubc9 on HIV-1 LTR promoter transactivation by HHV-6A IE2. Molt-3 cells were transfected as described in Materials and Methods with 2 μg pLTR-Luc reporter construct, 4 μg pcDNA4-IE2, and 0, 4, 8, or 12 μg of either pcDNA3.1-Ubc9 (A) or pcDNA3.1-Ubc9 C93S (B). Cells were lysed 48 h after electroporation. Transactivation of the pLTR-Luc reporter was quantified by measurement of luciferase activity, expressed as transactivation (n-fold) relative to control (pcDNA-transfected cells). Results are means ± standard deviations (SD) from four distinct experiments and were normalized for protein content in each sample. *, P < 0.05 compared with IE2-transfected cells.
FIG.5.
FIG.5.
(A to F) Inhibitory effect of Ubc9 on promoter transactivation by HHV-6A IE2. HEK293T cells were transfected as described in Materials and Methods with 1 μg reporter construct (pLTR-Luc encoding the HIV-1 LTR promoter or p2-1900 encoding the human COX-2 promoter), 0.1 μg transactivator (pcDNA4-IE2 or control pcDNA4-Tat), and 0, 0.12, 0.25, 0.5, or 1 μg of either pcDNA3.1-Ubc9 or pcDNA3.1-Ubc9 C93S. Cells were lysed 48 h after transfection. Transactivation of the reporters was quantified by measurement of luciferase activity expressed as transactivation (n-fold) relative to control (pcDNA-transfected cells). Results are means ± SD from three distinct experiments of duplicate transfections and were normalized for protein content in each sample. *, P < 0.05 compared with IE2- or Tat-transfected cells. (G) Effect of the deletion of the Ubc9-interacting domain of HHV-6A IE2 on HIV-1 LTR promoter transactivation. HEK293T cells were transfected with 1 μg pLTR-Luc reporter construct, 0.1 μg pcDNA4-IE2 or pcDNA4-IE2Δ993-1037, and 1 μg pcDNA3.1-Ubc9 or pcDNA3.1-Ubc9 C93S. Luciferase activity measurement is described in Materials and Methods. *, P < 0.05 compared with IE2-transfected cells.
FIG.5.
FIG.5.
(A to F) Inhibitory effect of Ubc9 on promoter transactivation by HHV-6A IE2. HEK293T cells were transfected as described in Materials and Methods with 1 μg reporter construct (pLTR-Luc encoding the HIV-1 LTR promoter or p2-1900 encoding the human COX-2 promoter), 0.1 μg transactivator (pcDNA4-IE2 or control pcDNA4-Tat), and 0, 0.12, 0.25, 0.5, or 1 μg of either pcDNA3.1-Ubc9 or pcDNA3.1-Ubc9 C93S. Cells were lysed 48 h after transfection. Transactivation of the reporters was quantified by measurement of luciferase activity expressed as transactivation (n-fold) relative to control (pcDNA-transfected cells). Results are means ± SD from three distinct experiments of duplicate transfections and were normalized for protein content in each sample. *, P < 0.05 compared with IE2- or Tat-transfected cells. (G) Effect of the deletion of the Ubc9-interacting domain of HHV-6A IE2 on HIV-1 LTR promoter transactivation. HEK293T cells were transfected with 1 μg pLTR-Luc reporter construct, 0.1 μg pcDNA4-IE2 or pcDNA4-IE2Δ993-1037, and 1 μg pcDNA3.1-Ubc9 or pcDNA3.1-Ubc9 C93S. Luciferase activity measurement is described in Materials and Methods. *, P < 0.05 compared with IE2-transfected cells.
FIG. 6.
FIG. 6.
Nuclear localization of IE2 in transfected cells is not altered by overexpression of Ubc9. HEK293T cells were transfected as described in Materials and Methods with 1 μg pLTR-Luc reporter construct, 2 μg pcDNA4-IE2 and 0, 1, 2, or 3 μg pcDNA3.1-Ubc9. Cells were harvested 48 h after transfection and split for the following assays. (A) Transactivation of the pLTR-Luc reporter was quantified by measurement of luciferase activity, expressed as transactivation (n-fold) relative to control (pcDNA-transfected cells). Results (±SD) represent one typical experiment out of three and were normalized for protein content in each sample. (B) The HEK293T-transfected cells were also processed for cellular fractionation into cytoplasmic (C) and nuclear (N) protein fractions as described in Materials and Methods. Samples were analyzed by SDS-PAGE and probed using anti-IE2 or anti-Ubc9 antibodies.
FIG. 7.
FIG. 7.
(A) Overexpression of HHV-6A IE2 does not alter Ubc9 expression or the sumoylation pattern of total cellular proteins. HEK293T cells were transfected with 2, 4, or 6 μg pcDNA4-IE2 and 2 μg HA-tagged construct pCMV-SUMO-1. Cells were harvested 48 h after transfection. Total lysates were analyzed by SDS-PAGE and probed for Ubc9, IE2, HA-tagged sumoylated proteins (designated by a bracket), and actin. (B) Overexpression of HHV-6A IE2 does not alter the sumoylation pattern of RanGAP1. HEK293T cells were transfected with 2 or 4 μg pcDNA4-IE2, 2 μg pCMV-SUMO-1, and 2 μg pcDNA3-Myc-RanGAP1. Total lysates were analyzed by SDS-PAGE and probed for RanGAP1 using an anti-Myc antibody. Arrows indicate (from top to bottom) HA-SUMO-1-modified Myc-RanGAP1, endogenous SUMO-1-modified Myc-RanGAP1, and unconjugated Myc-RanGAP1. HA-tagged SUMO-1 is 26 kDa, while endogenous SUMO-1 is 18 kDa.
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