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. 2010 Jul;84(14):7185-94.
doi: 10.1128/JVI.02231-09. Epub 2010 May 5.

Human cytomegalovirus IE72 protein interacts with the transcriptional repressor hDaxx to regulate LUNA gene expression during lytic infection

Matthew Reeves  1 , David Woodhall, Teresa Compton, John Sinclair
Affiliations

Human cytomegalovirus IE72 protein interacts with the transcriptional repressor hDaxx to regulate LUNA gene expression during lytic infection

Matthew Reeves et al. J Virol. 2010 Jul.

Abstract

A putative latency-associated transcript (LUNA) complementary to the human cytomegalovirus (HCMV) UL81-82 region previously identified in seropositive donors' monocytes is also expressed during lytic infection. Thus, the LUNA promoter is active during both lytic and latent infection. Consequently, the mechanisms regulating this promoter may provide further insight into factors that determine whether the outcome of HCMV infection is latent or lytic. By transfection, the LUNA promoter exhibited low but reproducible activity. Substantial activation by virus infection suggested that a viral factor was important for LUNA expression during lytic infection. IE72, a known transactivator of viral promoters, activated the LUNA promoter in cotransfection assays. Furthermore, coinfection with wild-type HCMV but not an IE72 deletion virus (CR208) also activated the LUNA promoter. Finally, diminished LUNA gene expression in CR208 virus-infected cells supported a role for IE72 in LUNA gene expression. The initial regulation of herpesvirus immediate-early gene expression is associated with proteins found at cellular nuclear domain 10 (ND10) bodies, such as PML, hDaxx, and ATRX. hDaxx transfection repressed LUNA promoter activity. Furthermore, we observed binding of hDaxx to the LUNA promoter, which was abrogated by IE72 gene expression via direct interaction. Finally, we show that small interfering RNA (siRNA) knockdown of the hDaxx interaction partner ATRX rescued LUNA gene expression in CR208-infected cells. Overall, these data show that hDaxx/ATRX-mediated repression of LUNA during lytic infection absolutely requires IE72 gene expression. It also suggests that the targeting of cellular factors by IE72 is important throughout the different phases of HCMV gene expression during productive infection.

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Figures

FIG. 1.
FIG. 1.
HCMV infection activates the LUNA promoter in permissive cells. (A) The putative LUNA promoter was cloned into pGL3-Basic using the primer sequences underlined. The TATA box is highlighted in bold. (B) Primary fibroblasts transfected with pLUNA (LUNAprom), pUL112/113, or pMIEP were analyzed for luciferase gene expression. Results are from triplicate samples from 3 independent experiments. (C) Primary fibroblasts transfected for 24 h with pLUNA or pMIEP were either mock infected (−) or infected with Towne at an MOI of 5 (+) and analyzed for luciferase gene expression 24 h postinfection. Results are from triplicate samples from 3 independent experiments (results for the LUNA promoter, ×103; results for the MIEP, ×106). Error bars indicate standard deviations. (D) RT-PCR analysis was performed for LUNA, IE gene, and GAPDH RNA expression from HCMV-infected fibroblasts at 0 to 8 hpi.
FIG. 2.
FIG. 2.
IE72 activates the LUNA promoter but does not bind it directly. (A) Primary fibroblasts transfected with pUL112/113 or pLUNA were mock transfected (M) or cotransfected with pON2203 (pIE72) or pME18S (con [control]) and analyzed for luciferase gene expression 48 h posttransfection. (B) Primary fibroblasts transfected for 24 h with pLUNA or pMIEP were subsequently mock infected or infected with Towne, CR208, or CR208Q virus at an MOI of 5. Luciferase gene expression was quantified 24 h postinfection and is expressed as the fold increase over the level for mock-infected cells. (C and D) RT-PCR for LUNA and IE gene expression in mock-, Towne (MOI of 5)-, or CR208 (MOI of 5)-infected cells was performed 8 h after infection of fibroblasts. (E) Concomitant with the RT-PCR analysis, DNA isolated from mock-, Towne-, or CR208-infected fibroblasts was harvested 4 h postinfection and analyzed using IE gene (“viral”)- and GAPDH (“cell”)-specific PCRs. (F and G) ChIP assays were performed with Towne-infected fibroblasts at 12 h postinfection using an anti-IE protein antibody (ab) or a mouse IgG control. DNA purified before (input [Inp]) and after IP was amplified in LUNA promoter- and MIEP-specific PCRs. Error bars indicate standard deviations.
FIG. 3.
FIG. 3.
TSA does not rescue LUNA gene expression in CR208-infected cells. (A) Primary fibroblasts incubated with TSA (330 nM) or DMSO 24 h posttransfection were transfected with pMIEP or pLUNA. Luciferase activity was measured 24 h posttransfection, and the fold change in luciferase expression over the level observed with the DMSO control was calculated in the presence (+) of TSA. (B) Fibroblasts were preincubated overnight with 330 nM TSA (+) or DMSO (−) and then infected with Towne or CR208. RT-PCR analysis for IE gene, LUNA, UL44, and GAPDH expression was then performed. Error bars indicate standard deviations.
FIG. 4.
FIG. 4.
IE72 physically interacts with hDaxx in vitro and in vivo. (A) The LexA/B42AD system was used to screen for interactions between IE72 and hDaxx. Induction of β-galactosidase activity was determined by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining. The known interaction between the SV40 T antigen (LexATag) and p53 (B42ADp53) was used as a positive control. (B) Nuclear extracts from uninfected (−CMV) and infected (+CMV) U373 cells were harvested 18 hpi, immunoprecipitated with an anti-IE protein antibody (IE72) or an isotype-matched control antibody (IgG), and analyzed by Western blotting for IP of hDaxx. (C) Nuclear extracts from uninfected cells or cells infected with either the K450R virus (K450Rmut) or its revertant (K450Rrev) were harvested 18 hpi, immunoprecipitated with an anti-IE protein antibody or an isotype-matched control antibody, and analyzed by Western blotting for IP of hDaxx. (D) RT-PCR for LUNA, IE gene, and GAPDH expression was performed with K450Rmut (Mut)- and K450Rrev (Rev)-infected fibroblasts at 8 hpi.
FIG. 5.
FIG. 5.
hDaxx binds and represses the LUNA promoter in transfection and infection assays. (A) Fibroblasts transfected with pLUNA or pUL112/113 were cotransfected with either pcDNA3 (−) or pcDNA-hDaxx (+) and were assayed 48 h later for luciferase gene expression. (B) Fibroblasts transfected with LUNA promoter for 24 h were mock infected (HFF) or infected with Towne or CR208 and then harvested for ChIP assays 24 h postinfection. Alternatively, an HFF line constitutively expressing IE72 was transfected with pLUNA in parallel (ihfie1.3). Following lysis, immunoprecipitation was performed with either anti-Daxx or a rabbit IgG serum control overnight, and then the purified DNA was amplified with LUNA promoter-specific primers. Band densitometry was performed on PCR products, and the corrected hDaxx samples were expressed as percentages of the input (Inp) controls. Error bars indicate standard deviations.
FIG. 6.
FIG. 6.
ATRX represses the LUNA promoter during infection by binding to the LUNA promoter. (A) Fibroblasts were transfected with a siRNA-ATRX cocktail (ATRX) or a mismatch control (Scr) and analyzed 24 h posttransfection by Western blotting for ATRX and GAPDH expression. (B and C) Fibroblasts were transfected with a siRNA-ATRX cocktail or a mismatch control; 24 h posttransfection, cells were infected (MOI of 5) with Towne or CR208, and RNA (B) and DNA (C) were harvested 12 h postinfection. RT-PCR was performed on the RNA using LUNA-, actin-, UL44-, and ATRX-specific primers. Alternatively, RNA was amplified in the LUNA-specific PCR without prior RT. An IE gene-specific PCR was used to amplify viral DNA to confirm equivalent numbers of genomes in the infected cells. (D) Fibroblasts infected with CR208 or Towne at an MOI of 3 were harvested at 3 and 6 h postinfection and analyzed by ChIP for ATRX binding. Sonicated cell lysates were immunoprecipitated with an anti-ATRX antibody or a goat IgG control serum, and the DNA was amplified in a LUNA promoter-specific PCR. The IgG control was used to correct the ATRX samples, which were then expressed as percentages of the input. Results from 2 ChIP experiments are shown. Error bars indicate standard deviations.
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