Temporal regulation of the Mre11-Rad50-Nbs1 complex during adenovirus infection

Abstract

Adenovirus infection induces a cellular DNA damage response that can inhibit viral DNA replication and ligate viral genomes into concatemers. It is not clear if the input virus is sufficient to trigger this response or if viral DNA replication is required. Adenovirus has evolved two mechanisms that target the Mre11-Rad50-Nbs1 (MRN) complex to inhibit the DNA damage response. These include E4-ORF3-dependent relocalization of MRN proteins and E4-ORF6/E1B-55K-dependent degradation of MRN components. The literature suggests that degradation of the MRN complex due to E4-ORF6/E1B-55K does not occur until after viral DNA replication has begun. We show that, by the time viral DNA accumulates, the MRN complex is inactivated by either of the E4-induced mechanisms and that, with E4-ORF6/E1B-55K, this inactivation is due to MRN degradation. Our data are consistent with the conclusion that input viral DNA is sufficient to induce the DNA damage response. Further, we demonstrate that when the DNA damage response is active in E4 mutant virus infections, the covalently attached terminal protein is not cleaved from viral DNAs, and the viral origins of replication are not detectably degraded at a time corresponding to the onset of viral replication. The sequences of concatemeric junctions of viral DNAs were determined, which supports the conclusion that nonhomologous end joining mediates viral DNA ligation. Large deletions were found at these junctions, demonstrating nucleolytic procession of the viral DNA; however, the lack of terminal protein cleavage and terminus degradation at earlier times shows that viral genome deletion and concatenation are late effects.

FIG. 1.

MRN degradation does not occur until late during Ad infection. Cell lysates from mock-infected A549 cells and mutant inORF3-infected (E4-ORF3 mutant) A549 cells were harvested at 6, 8, 10, 12, 14, and 18 hpi. (A) MRN degradation was visualized by immunoblot analysis of each component of the MRN complex, along with γ-tubulin as a loading control. (B) Each band was quantified and normalized to γ-tubulin. The values for mock-infected samples are set at 1. Relative levels of Mre11, Rad50, and Nbs1 in infected cell lysates are shown at the indicated time points. The results represent the average of three independent experiments.

FIG. 2.

The onset of viral DNA replication occurs at 10 to 12 hpi with viruses that inactivate MRN. Cells were infected with dl309 (phenotypically wild-type Ad5), dl355 (E4-ORF6 mutant), inORF3, and dl355/inORF3 (E4-ORF6/E4-ORF3 mutant). High-molecular-weight DNA was prepared from infected cell lysates at 4, 6, 8, 10, 12, 15, 18, and 24 hpi. Levels of DNA in each sample were quantified by slot blot analysis and plotted on the graph. Equivalent levels of viral DNA (vDNA) were observed with the 4 and 6 hpi samples compared to the level of the 8 hpi sample. The results represent the average of three independent experiments.

FIG. 3.

The MRN complex is inactivated by 10 hpi in wild-type and single-mutant infections. A549 cells were mock infected (A, E) or infected with dl309 (B, F); dl355, inORF3, dl1520 (E1B-55K mutant), and dl355/inORF3 (C, G); or dl1520/dl341 (E1B-55K/E4-ORF3 mutant) for 10 h. The cells were then fixed (A to D) or treated with 1 Gy of ionizing radiation and fixed 1 h postirradiation (E to H). The cells were stained with antibodies against DBP and pATM-S1981 in Alexa Fluor 350 (blue) or FITC (green), respectively. (A to C, E, and F) Representative immunofluorescence images. (D, H) The number of pATM foci were counted in DBP-expressing cells for 50 cells infected with each virus in triplicate, and the average number was plotted in the graphs. The results represent the cumulative value of pATM foci observed: i.e., 0 represents the number of cells with 0 pATM foci, 1 represents the number of cells with 0 and 1 pATM foci, 2 represents the number of cells with 0, 1 and 2 pATM foci, etc.

FIG. 4.

Immunofluorescence shows a disappearance of Mre11 signal by 8 hpi that is due to degradation by E4-ORF6 and E1B-55K. A549 cells were infected with inORF3 (A to C), dl355/inORF3 (D to F), or dl1520/dl341 (G to I) or cotransfected with CUL5-NTD and DsRed2-Mito expression plasmids overnight and infected with inORF3 (J to M) for 8 h. The cells were then stained with antibodies for Mre11 (green, FITC) and DBP (blue, Alexa Fluor 350). (Bottom) Cells were scored positive or negative for Mre11 for staining, and the percentages of 50 cells in triplicate were plotted on the graph.

FIG. 5.

TP cleavage is not the basis for MRN inhibition of Ad replication. A549 cells were infected with dl309 (lanes 3 and 4), dl355 (lanes 5 and 6), inORF3 (lanes 7 and 8), dl1520 (lanes 9 and 10), dl355/inORF3 (lanes 11 and 12), and dl1520/dl341 (lanes 13 and 14) and harvested at 10 hpi to prepare nuclear viral DNA (vDNA). Part of the samples were treated with proteinase K (prot. K; odd lanes, +), while the others were left untreated (even lanes, −). The samples were digested with BglII, run on a 1% agarose gel, and subjected to Southern blot analysis with a ³²P-labeled whole-genome probe. Standards were run alongside the samples (lanes 1 and 2).