Serotype-specific reorganization of the Mre11 complex by adenoviral E4orf3 proteins

Abstract

The early transcriptional region 4 (E4) of adenovirus type 5 (Ad5) encodes gene products that modulate splicing, apoptosis, transcription, DNA replication, and repair pathways. Viruses lacking both E4orf3 and E4orf6 have a severe replication defect, partially characterized by the formation of genome concatemers. Concatemer formation is dependent upon the cellular Mre11 complex and is prevented by both the E4orf3 and E4orf6 proteins. The Mre11/Rad50/Nbs1 proteins are targeted for proteasome-mediated degradation by the Ad5 viral E1b55K/E4orf6 complex. The expression of Ad5 E4orf3 causes a redistribution of Mre11 complex members and results in their exclusion from viral replication centers. For this study, we further analyzed the interactions of E4 proteins from different adenovirus serotypes with the Mre11 complex. Analyses of infections with serotypes Ad4 and Ad12 demonstrated that the degradation of Mre11/Rad50/Nbs1 proteins is a conserved feature of the E1b55K/E4orf6 complex. Surprisingly, Nbs1 and Rad50 were localized to the replication centers of both Ad4 and Ad12 viruses prior to Mre11 complex degradation. The transfection of expression vectors for the E4orf3 proteins of Ad4 and Ad12 did not alter the localization of Mre11 complex members. The E4orf3 proteins of Ad4 and Ad12 also failed to complement defects in both concatemer formation and late protein production of a virus with a deletion of E4. These results reveal surprising differences among the highly conserved E4orf3 proteins from different serotypes in the ability to disrupt the Mre11 complex.

FIG. 1.

Effect of E4orf3 on localization of components of PODs/ND10. HeLa cells were transfected with an expression vector for Ad5 E4orf3, and the localization of proteins was assessed by immunofluorescence. E4orf3 induced the reorganization of SUMO1 (A) and DAXX (B) into nuclear track-like structures that partially colocalized with Rad50. (C) Neither BLM nor TopBP1 was recruited to the structures induced for Rad50 and PML by E4orf3 expression.

FIG. 2.

Localization of Mre11 complex during infection with different Ad serotypes. (A) In uninfected HeLa cells, the Nbs1 and RPA32 proteins were localized diffusely throughout the cellular nucleoplasm. In Ad-infected cells, the RPA32 protein was found to be relocalized in a pattern that overlapped with that of viral replication centers detected by staining with an antibody to the viral DBP. This shows that RPA32 relocalization can be used as a surrogate marker for viral replication centers. Localization of the Mre11 complex was determined by staining for Nbs1 during infections of HeLa cells with Ad5 (B), Ad4 (C), and Ad12 (D). Cells infected with Ad5 (MOI of 25), Ad 4 (MOI of 100), and Ad12 (MOI of 25) were fixed and analyzed by immunofluorescence at 12 and 24 h postinfection. Staining with RPA32 was used to localize the replication centers because the Ad5 DBP monoclonal antibody does not cross-react with the Ad4 DBP and Ad12 DBP proteins. (B) Nbs1 was excluded from Ad5 replication centers after 12 h and 24 h of infection. Nbs1 localized to sites of viral replication during Ad4 (C) and Ad12 (D) infections.

FIG. 3.

Degradation of the Mre11 complex is conserved among Ad serotypes. (A) Transfection of HeLa cells that stably express the Ad5 E1b55K protein (6) with plasmids encoding the E4orf6 proteins of Ad5, Ad4, and Ad12. All E4or6 proteins resulted in the nuclear accumulation of Ad5 E1b55K and the degradation of Nbs1. (B) Immunoblotting of 293 cells (expressing Ad5 E1b55K) transfected with cDNA expression vectors (4 μg) for E4orf6 proteins of Ad5, Ad4, and Ad12. Negative controls included untransfected cells (mock) and cells transfected with the empty vector construct. The expression of all three E4orf6 proteins led to the down-regulation of Mre11, Rad50, and Nbs1. The p53 protein was also degraded with each E4orf6 protein, and γ-tubulin served as a loading control.

FIG. 4.

Redistribution of the Mre11 complex is a specific activity of E4orf3 from serotype Ad5. HeLa cells were transfected with plasmids carrying FLAG-tagged cDNAs encoding the E4orf3 proteins of Ad5, Ad4, and Ad12. After 24 h, the localization of E4orf3 and cellular proteins was analyzed by immunofluorescence. (A) E4orf3 proteins from all three serotypes disrupted PML bodies and generated track-like structures. The expression of FLAG-tagged E4orf3 from Ad5 (top), Ad4 (middle), and Ad12 (bottom) caused a dispersion of the usually punctate PML staining. (B) Expression of FLAG-tagged Ad5 E4orf3 leads to disruption of the Mre11 complex, and E4orf3 colocalizes with Rad50 and Nbs1. (C) Nbs1 localization was unaffected by the expression of FLAG-tagged E4orf3 proteins from Ad4 and Ad12.

FIG. 5.

E1b55K is recruited into nuclear PML track-like structures by E4orf3 proteins. HeLa cells expressing the E1b55K viral protein were transfected with E4orf3 expression vectors. (A) In mock-transfected cells, E1b55K was located predominantly in cytoplasmic speckles and PML was localized to PODs/ND10. The expression of E4orf3 from Ad4, Ad5, and Ad12 resulted in the import of E1b55K into the nucleus, where it partially colocalized with PML in track-like structures. (B) Nuclear tracks of E1b55K colocalized with Nbs1 when induced by Ad5 E4orf3 but not when induced by the Ad4 E4orf3 and Ad12 E4orf3 proteins.

FIG. 6.

Expression of Ad5 E4orf3 prevents accumulation of the Mre11 complex at virus replication centers during infection with a virus with an E4 deletion. HeLa cells were transfected with expression vectors for E4orf3 proteins or with an empty vector (0.8 μg). The left column shows images of transfected cells stained for PML localization after 24 h, demonstrating track-like structures in cells expressing all of the E4orf3 proteins. The images on the right are from transfected cells that were also infected with the virus dl1004, which has an E4 deletion, for a further 24 h and then stained for Nbs1 and RPA32 (marks viral replication centers). The Nbs1 protein was excluded from virus centers by Ad5 E4orf3 but not by the E4orf3 proteins of Ad4 and Ad12.

FIG. 7.

Ability of E4orf3 proteins to complement a virus with an E4 deletion. (A) HeLa cells were transfected with expression vectors for the E4orf3 proteins of Ad5, Ad4, and Ad12. After 24 h, the cells were infected with the virus dl1004, containing an E4 deletion, and DNAs were harvested for PFGE analysis after a further 48 h. Concatemerization was abrogated only by wild-type Ad5 E4orf3. An empty vector and the N82A mutant of Ad5 E4orf3 served as negative controls. The production of late proteins was assessed by immunoblotting with an antifiber antibody. Infection with Ad5 served as a positive control for this experiment. (B) Transcription of the different E4orf3 constructs was confirmed by RT-PCR with lysates from transfected cells, using serotype-specific primer sets.

FIG. 8.

The E4orf3 protein of Ad5 exhibits divergence from the E4orf3 proteins of other adenovirus species. (A) The relationships among E4orf3 proteins of the indicated human and monkey adenoviruses were calculated by application of the PHYLIP suite of software as described in Materials and Methods. The uprooted tree obtained by this method was presented with the Ad12 subgroup A protein as the outgroup. The length of each branch corresponds to the expected number of amino substitutions, with the scale bar corresponding to a rate of change of 0.25 substitutions per residue. Simian viruses are identified as “sAd,” and human viruses are identified as “Ad.” The vertical bar to the right of the virus names identifies viruses of the indicated subgroup, as suggested by the analysis of Kovacs and associates (21). (B) The divergence of E4orf3, E4orf6, E4orf2, and E1b55K proteins from representative human adenoviruses is presented as branching graphs in which the length of each branch corresponds to the expected number of amino substitutions at each position. All graphs are presented on the same scale. The specific viruses represented in this analysis are indicated, as are their subgroup designations (A to F). Bar, rate of change of 0.25 substitutions per residue.