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. 2014 Nov;88(22):13469-81.
doi: 10.1128/JVI.02538-14. Epub 2014 Sep 10.

Adenovirus E1A Targets the DREF Nuclear Factor to Regulate Virus Gene Expression, DNA Replication, and Growth

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Adenovirus E1A Targets the DREF Nuclear Factor to Regulate Virus Gene Expression, DNA Replication, and Growth

Sandi Radko et al. J Virol. .
Free PMC article

Abstract

The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication.

Importance: This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to viral promoters during infection together with E1A. These results suggest that DREF contributes to activation of viral gene expression. However, like several other PML-associated proteins, DREF also appears to function as a growth restriction factor for adenovirus infection.

Figures

FIG 1
FIG 1
E1A and DREF schematic diagrams. (A) Schematic diagram of HAdV5 E1A showing the two major splice variants and conserved regions. The location of E1A exon 2 is indicated, with the residues used in MS analysis shown. (B) Schematic diagram of DREF showing key features.
FIG 2
FIG 2
E1A interacts with DREF. (A) Subconfluent human HT1080 fibrosarcoma cells were infected with HAdV5 dl309, pm975, or dl520 expressing genomic E1A, E1A289R, or E1A243R, respectively, or Ad-LacZ with the E1 region deleted, for 24 h. Cells were subsequently harvested, lysed, and subjected to IP using M73 monoclonal anti-E1A antibody. IPs were resolved on 4 to 12% gradient Novex Bolt gels, and DREF was detected using rabbit polyclonal anti-DREF antibody. Part of the cell lysate used in the IPs (1.5%) was used as the input control. (B) Schematic representation of E1A289R and fragments fused to GFP. (C) Human HA-tagged DREF was cotransfected together with plasmids expressing the indicated GFP-E1A fragment fusions or GFP alone as a control into HT1080 human fibrosarcoma cells. Twenty-four hours after transfections cells were harvested and immunoprecipitated for GFP. IPs were resolved on 4 to 12% gradient Novex Bolt gels, and DREF was detected with anti-HA (3F10) monoclonal antibody. The input control was 1.5% of the cell lysate used in the IPs. (D) Human HA-tagged DREF was cotransfected together with plasmids expressing the indicated E1A243R single-amino-acid-substitution mutants into HT1080 human fibrosarcoma cells. An empty plasmid (pcDNA3) was used in the “no E1A” control transfection. Twenty-four hours after transfections cells were harvested and immunoprecipitated for E1A (M73). IPs were resolved on 4 to 12% gradient Novex Bolt gels, and DREF was detected with anti-HA (3F10) monoclonal antibody. The input control was 1.5% of the cell lysate used in the IPs. (E) Human HA-tagged DREF was cotransfected together with plasmids expressing indicated GFP-E1A fusions from various HAdV species into HT1080 human fibrosarcoma cells. GFP plasmid was used as a control. Twenty-four hours after transfections cells were harvested and immunoprecipitated for GFP. IPs were resolved on 4 to 12% gradient Novex Bolt gels, and DREF was detected with anti-HA (3F10) monoclonal antibody. The input control was 1.5% of the cell lysate used in the IPs.
FIG 3
FIG 3
E1A directly interacts with DREF. Bacterially purified and GST-fused fragments of DREF were mixed with bacterially expressed and purified His-tagged E1A289R and collected using Ni-nitrilotriacetic acid resin. Pulldowns were resolved on 4 to 12% gradient Novex Bolt gels. DREF was detected using a polyclonal anti-DREF antibody, GST was detected using a polyclonal anti-GST antibody, and input E1A was detected using the M73 antibody.
FIG 4
FIG 4
Identification of a new MoRF in the E1A C terminus that contributes to enhanced DREF SUMOylation. (A) Multiple-sequence alignment fragments of CR4 from HAdV E1As from different serotypes together with cellular protein USPL1 highlighting amino acid conservation. Black shading indicates conservation across all species and proteins, whereas gray shading indicates only partial conservation. (B) pCAN-Myc-DREF expressing myc-tagged DREF was cotransfected into HT1080 cells together with plasmids expressing the indicated HA-tagged SUMO and genomic E1A or a control plasmid expressing no E1A. Total DREF was immunoprecipitated using the anti-myc (9E10) monoclonal antibody. IPs were resolved on a 4 to 12% gradient Novex Bolt gel, and SUMOylated isoforms of DREF were detected using anti-HA (3F10) antibody. The input control was 1.5% of the cell lysate used in the IPs. (C) Same as panel B except that the indicated isoforms of HAdV5 E1As were used in addition to two mutants: E1A289R T123H, which does not bind UBC9, and E1A243R L272/273A, which does not bind to DREF. The input control was 1.5% of the cell lysate used in the IPs. (D) Densitometric quantification of results presented in panel C normalized to the negative control (no E1A; n = 3; error bars represent standard deviations of results for the biological replicates). (E) Subconfluent HT1080 cells were infected with either Ad-LacZ with the E1 region deleted or HAdV5 dl309 as indicated. Twenty-four hours after infection endogenous DREF was immunoprecipitated using polyclonal anti-DREF antibody and resolved on a 4 to 12% gradient Novex Bolt gel. SUMOylated DREF was subsequently detected using rabbit monoclonal anti-SUMO-1 antibody. DREF was detected using polyclonal anti-DREF antibody, and E1A was detected using monoclonal M73 anti-E1A antibody. The input control was 1.5% of the cell lysate used in the IPs.
FIG 5
FIG 5
E1A alters the subcellular distribution of DREF. (A) HT1080 cells were transfected with a plasmid expressing HA-tagged DREF alone or together with pcDNA3.1-E1A expressing genomic HAdV5 E1A. Cells were fixed 24 h after transfection and stained for DREF using anti-HA antibody (3F10) and for E1A using anti-E1A (M73) antibody. DAPI was used as a nuclear counterstain. (B) Same as in panel A except DREF was costained with anti-PML antibody in the presence or absence of wild-type E1A243R or the indicated point mutants. (C) Quantification of the phenotype observed in panel B. Seven random nuclei from HT1080 cells cotransfected with plasmids expressing DREF and the indicated E1A243R substitution point mutants were scored for the total number of PML bodies and the number of PML bodies with overlapping DREF staining. Data are represented as percentages of PML bodies with overlapping DREF staining; error bars represent the standard deviations between results for the nuclei.
FIG 6
FIG 6
DREF restricts HAdV growth and colocalizes with viral replication centers. (A) DREF was knocked down in HT1080 cells using DREF-specific siRNA (siDREF); nonspecific siRNA was used as a negative control (siControl). Twenty-four hours after siRNA transfection, cells were infected with HAdV5 (dl309) at an MOI of 1, and viral titers were determined on 293 cells at the indicated time points (n = 4; error bars represent standard deviations of results for the biological replicates). (B) HT1080 cells were transfected with a plasmid expressing HA-tagged DREF, infected with HAdV5 (dl309) 24 h later, and imaged a further 24 h later. DREF was visualized using anti-HA (3F10) antibody, and 72k DBP was detected using monoclonal anti-72k DBP antibody. PML was visualized using polyclonal anti-PML antibody. The top row represents cells treated only with control siRNA and stained for 72k DBP and DREF; the middle and bottom rows represents cells that were stained for 72k DBP and PML and were treated with either DREF siRNA or control siRNA.
FIG 7
FIG 7
DREF coregulates viral gene expression and suppresses viral genome replication. (A) HT1080 cells were treated with siRNA for DREF or a negative-control siRNA. Twenty-four hours after siRNA transfection cells were infected with HAdV5 (dl309) at an MOI of 10, and total RNA was extracted 20 and 40 h after infection using TRIzol reagent. mRNA levels were quantified using real-time quantitative PCR and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The levels were plotted as fold changes versus the level for control siRNA-treated cells, set to 1 (n = 7; error bars represent standard deviations of the results for biological replicates). (B) Representative Western blots of E1A, 72k DBP, and viral structural proteins at 20 and 40 h after HAdV5 infection with dl309 at an MOI of 10 in DREF knockdown and control knockdown cells. E1A was detected using M73 anti-E1A monoclonal antibody, 72k DBP was detected using anti-DBP monoclonal antibody, and viral structural proteins were detected using polyclonal anti-adenovirus 5 antibody. Twenty micrograms of total cell lysate was loaded per lane, and samples were resolved on a 4 to 12% gradient Novex Bolt gel. (C) HT1080 cells were treated with siRNA for DREF or a negative-control siRNA. Twenty-four hours after siRNA transfection cells were infected with HAdV5 (dl309) at an MOI of 10, and low-molecular-weight DNA was extracted at the indicated time points after infection as described in Materials and Methods. Viral genomes were quantified using qPCR with primers recognizing the E1B region, with the pXC1 plasmid used to generate the standard curve. The data are plotted as genome copies per cell, and error bars represent standard deviations of results for biological replicates (n = 4). (D) Same cells as in panel C prior to harvest for viral DNA content analysis imaged at 40 h after HAdV5 (dl309) infection showing cellular morphology. Magnification, ×100.
FIG 8
FIG 8
DREF is recruited to viral promoters. HT1080 cells were infected with HAdV5 (dl309) at an MOI of 10, and ChIPs were carried out 16 h after viral infection. E1A was immunoprecipitated using a cocktail of anti-E1A antibodies (M73 and M58), and DREF was immunoprecipitated using anti-DREF polyclonal antibody. 12CA5 anti-HA monoclonal antibody was used as a negative IgG control. Data are plotted as % of input sample (n = 4; error bars represent standard deviations of results for biological replicates).

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