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. 2008 Dec;82(24):12543-54.
doi: 10.1128/JVI.01215-08. Epub 2008 Oct 15.

Human cytomegalovirus protein pp71 displaces the chromatin-associated factor ATRX from nuclear domain 10 at early stages of infection

Vera Lukashchuk  1 , Steven McFarlane, Roger D Everett, Chris M Preston
Affiliations

Human cytomegalovirus protein pp71 displaces the chromatin-associated factor ATRX from nuclear domain 10 at early stages of infection

Vera Lukashchuk et al. J Virol. 2008 Dec.

Abstract

The human cytomegalovirus (HCMV) tegument protein pp71, encoded by gene UL82, stimulates viral immediate-early (IE) transcription. pp71 interacts with the cellular protein hDaxx at nuclear domain 10 (ND10) sites, resulting in the reversal of hDaxx-mediated repression of viral transcription. We demonstrate that pp71 displaces an hDaxx-binding protein, ATRX, from ND10 prior to any detectable effects on hDaxx itself and that this event contributes to the role of pp71 in alleviating repression. Introduction of pp71 into cells by transfection, infection with a pp71-expressing herpes simplex virus type 1 vector, or by generation of transformed cell lines promoted the rapid relocation of ATRX from ND10 to the nucleoplasm without alteration of hDaxx levels or localization. A pp71 mutant protein unable to interact with hDaxx did not affect the intranuclear distribution of ATRX. Infection with HCMV at a high multiplicity of infection resulted in rapid displacement of ATRX from ND10, the effect being observed maximally by 2 h after adsorption, whereas infection with the UL82-null HCMV mutant ADsubUL82 did not affect ATRX localization even at 7 h postinfection. Cell lines depleted of ATRX by transduction with shRNA-expressing lentiviruses supported increased IE gene expression and virus replication after infection with ADsubUL82, demonstrating that ATRX has a role in repressing IE transcription. The results show that ATRX, in addition to hDaxx, is a component of cellular intrinsic defenses that limit HCMV IE transcription and that displacement of ATRX from ND10 by pp71 is important for the efficient initiation of viral gene expression.

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Figures

FIG. 1.
FIG. 1.
ATRX depletion in fibroblasts. The site within the hATRX gene from which the shA90 sequence was derived (A) and the level of depletion determined in a Western blot assay (B) are shown. Anti-ATRX antibodies H300 and 39F, specific for the C terminus or N terminus, respectively, were used. USP7 acted as a loading control.
FIG. 2.
FIG. 2.
Effects of depleting ATRX or hDaxx on the localization of ND10 proteins. Control (ShLuci), ATRX-depleted (ShA90), or hDaxx-depleted (ShDaxx) cell cultures were analyzed by IF, using antibodies specific for PML, ATRX, or hDaxx.
FIG. 3.
FIG. 3.
Dissociation of ATRX from ND10 by pp71. HFFF2 monolayers were infected or transfected and analyzed by IF. (A and B) Dispersal and partial dispersal, respectively, of ATRX in cells at 3 h postinfection at 38.5°C with in1316 (MOI, 0.1). (C) Colocalization of YFPpp71 with hDaxx at 3 h after infection at 38.5°C with in1316. (D) Dispersal of both YFPpp71 and hDaxx in an in1316-infected cell expressing greater amounts of YFPpp71 at 7 h postinfection. (E) Simultaneous detection of YFPpp71, ATRX, and hDaxx at 3 h postinfection at 38.5°C with in1316, demonstrating dispersal of ATRX but punctate hDaxx. (F and G) Distributions of ATRX and Daxx, respectively, in cells expressing untagged pp71 3 h postinfection at 38.5°C with in1360 (MOI of 0.1). (H) Dispersal of ATRX but punctate hDaxx 4 h posttransfection of pYFPpp71 into HFFF2 cells. (I) ATRX remains in punctate foci at 3 h postinfection at 38.5°C with in0125. (J) A mixture of C4N and CV-1(F) cultures. In cells with low-level expression of YFPpp71, the protein colocalized with Daxx foci but ATRX was dispersed.
FIG. 4.
FIG. 4.
Dissociation of ATRX from ND10 in HCMV-infected cells. (A to D) HFFF2 cells at 1 h pa (A and B) and at 2 h pa (C and D) after infection with HCMV at an MOI of 2. (E) Cells at 1 h pa after infection with HCMV, at an MOI of 2, detecting pp71, ATRX, and hDaxx, demonstrating dispersed ATRX but punctate hDaxx in a cell containing pp71. (F and G) Cells infected with UV-irradiated HCMV at 1 h pa. (H) Cells infected with HCMV, at an MOI of 0.25, at 3 h pa. (I) HFFF2 cells infected with ADsubUL82 at 7 h postinfection, demonstrating punctate ATRX and hDaxx in an IE protein-expressing cell.
FIG. 5.
FIG. 5.
Multiplicity dependence of ATRX dispersal. Cells were infected with HCMV and stained for ATRX and hDaxx. Fluorescence patterns were scored as dispersed if one or no discrete foci were detectable. Open squares, ATRX at 1 h pa; filled squares, hDaxx at 1 h pa; open circles, ATRX at 2 h pa; filled circles, hDaxx at 2 h pa. The data were taken from three independent experiments, one of which used different primary antibodies.
FIG. 6.
FIG. 6.
ATRX and hDaxx levels in HCMV-infected cells. HFFF2 monolayers were mock infected (lane 5) or infected with HCMV at an MOI of 2 (lanes 1 to 4) or 0.5 (lanes 6 to 9). Extracts were prepared at 1, 2, 3, or 4 h pa and protein contents analyzed.
FIG. 7.
FIG. 7.
IE production after infection of ATRX-depleted cultures with ADsubUL82. Control (ShLuci) and ATRX-depleted (ShA90) cells were mock infected or infected with ADsubUL82 (0.2 infectious units per cell), AD169 (MOI of 0.3), AD169UV (MOI of 3 based on the original titer) alone, or coinfected with ADsubUL82 and AD169UV, and harvested for Western blot analysis of ATRX, HCMV IE proteins, hDaxx, and actin at 18 h postinfection or pp65 and actin at 2 h postinfection.
FIG. 8.
FIG. 8.
Increased replication of ADsubUL82 in ATRX-depleted cells. HF-shLuci or HF-shA90 cells were infected with ADsubUL82 (0.2 infectious units per cell) and incubated at 37°C for 11 days. Replicating virus was detected by fluorescence due to the green fluorescent protein (GFP) sequences inserted into the UL82 coding region, and cultures were also stained with propidium iodide.
FIG. 9.
FIG. 9.
HCMV IE gene expression and ATRX localization. (A and B) HFFF2 cells infected with ADsubUL82 at 17 h postinfection, giving examples of low-level (A) and high-level (B) IE expression. (C and D) HF-shLuci and HF-shA90 cultures 12 h after infection with ADsubUL82 (0.2 infectious units per cell); the images are representative of those used to generate the data in Table 1. (E to H) HFFF2 cells infected with wild-type HCMV, at an MOI of 0.5, analyzed at 2.5 h pa. Images were selected to include cells with both low-level and undetectable IE protein content. (E and F) IE production in cells with dispersed or punctate ATRX, respectively; (G and H) IE production in cells with dispersed or punctate hDaxx, respectively.
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References

    1. Ahn, J. H., and G. S. Hayward. 2000. Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection. Virology 27439-55. - PubMed
    1. Ahn, J.-H., and G. S. Hayward. 1997. The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells. J. Virol. 714599-4613. - PMC - PubMed
    1. Argentaro, A., J.-C. Yang, L. Chapman, M. S. Kowalczyk, R. J. Gibbons, D. R. HIggs, D. Neuhaus, and D. Rhodes. 2007. Structural consequences of disease-causing mutations in the ATRX-DNMT3L (ADD) domain of the chromatin-associated protein ATRX. Proc. Natl. Acad. Sci. USA 10411939-11944. - PMC - PubMed
    1. Baldick, C. J., A. Marchini, C. E. Patterson, and T. Shenk. 1997. Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J. Virol. 714400-4408. - PMC - PubMed
    1. Baumann, C., A. Schmidtmann, K. Muegge, and R. De La Fuente. 2008. Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia. BMC Mol. Biol. 929. - PMC - PubMed

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