Adenovirus core protein VII protects the viral genome from a DNA damage response at early times after infection

Abstract

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity.

Fig. 1.

Mutually exclusive binding interactions of core protein VII and pATM to Ad genomes. A549 cells were infected for 10 h with dl355/inORF3 mutant virus, fixed, and immunostained for pATM (FITC) (A) and protein VII (TRITC) (B). A merge of panels A and B is shown in panel C. dl355/inORF3 virus-infected cells were also stained for pATM (FITC) (D) and γH2AX (TRITC) (E). A merge of panels D and E is shown in panel F.

Fig. 2.

pATM foci form on the Ad genome. A549 cells were infected for 10 h with dl355/inORF3 mutant virus containing genome incorporation of EdU and fixed, and a Click-iT reaction was performed to label EdU-incorporated viral genomes (A, D). Subsequently, the cells were immunostained for pATM (TRITC) (B). A merge of panels A and B is shown in panel C. dl355/inORF3 virus-infected cells were also immunostained for protein VII (TRITC) (E). A merge of panels D and E is shown in panel F.

Fig. 3.

An inverse correlation is observed between a decrease in protein VII foci and an increase in pATM foci. (A) A549 cells were mock infected or infected with dl355/inORF3 virus over a time course of 2 to 14 h. Cells were fixed and immunostained for protein VII and pATM. The number of foci for each protein was counted in the same 50 cells for each experiment in triplicate, and the average numbers are plotted. (B) The same data from panel A are presented as a stacked bar graph, with the total number of foci detected per time point plotted in comparison to protein VII and pATM foci.

Fig. 4.

Inhibition of transcription during Ad infection inhibits the formation of pATM foci. A549 cells were infected with dl355/inORF3 or dl312 virus for 10 h. Cells infected with dl355/inORF3 virus were left untreated, or AraC or α-amanitin was added to the medium immediately after infection. Cells were fixed and immunostained for protein VII and pATM. The number of foci for each protein was counted in 50 cells each in triplicate experiments, and the average numbers are plotted. The error bars represent 95% confidence intervals, and the values for cells treated with α-amanitin or infected with dl312 virus are statistically significant (P < 0.001).

Fig. 5.

Expression of E1A and Mre11. (A) Whole-cell extracts were prepared from uninfected A549 cells (lane 1) or A549 cells infected with wild-type Ad5 in the absence (lane 2) or presence (lane 3) of α-amanitin in the culture medium. Equal amounts of protein extracts were separated by SDS-PAGE, and Western blot analyses were performed using an antibody that recognizes E1A (arrow). The band labeled with an asterisk represents a cross-reactive cellular protein that serves as a loading control. (B) Whole-cell extracts were prepared from uninfected HeLa (lane 1) and A549 (lane 2) cells and human diploid fibroblasts (HDF) (lane 3) and ATLD1 cells (lane 4) as well as from ATLD1 cells infected with adenoviruses that express Mre11-WT (lane 5) or Mre11-3 (lane 6). Equal amounts of protein extracts were separated by SDS-PAGE, and Western blot analyses were performed using an antibody that recognizes Mre11.

Fig. 6.

Mre11 nuclease activity is not required to inhibit virus replication during a DNA damage response. ATLD1 cells were mock infected (A) or infected with recombinant adenoviruses that express Mre11-WT (B) or Mre11-3 (C). At 48 hpi, cells were fixed and immunostained for Mre11. ATLD1 cells were infected with recombinant adenoviruses that express Mre11-WT (D to F) or Mre11-3 (G to I). At 48 hpi, cells were reinfected with dl355/inORF3 virus containing genome incorporation of EdU. (D, G) Cells were fixed at 6 hpi with dl355/inORF3 virus, and a Click-iT reaction was performed to label EdU-incorporated viral genomes. (E, H) Subsequently, the cells were immunostained for Mre11. A merge of panels D and E and panels G and H are shown in panels F and I, respectively. ATLD1 cells were infected with recombinant adenoviruses that express Mre11-WT (J to M) or Mre11-3 (N to Q). At 48 hpi, cells were reinfected with dl355/inORF3 virus. Cells were fixed at 48 hpi with dl355/inORF3 virus and immunostained for Mre11 (J, L, N, and P) and Ad DBP (K, M, O, and Q).

Fig. 7.

Competition model for Ad replication. The viral genome enters the nucleus bound to core protein VII, which masks the viral genomic termini from recognition by MRN. When early gene expression takes place, core protein VII is released from the genome, exposing the genomic termini. If E1B and E4 proteins inhibit MRN activity, then the replication preinitiation complex can form and replication ensues. If a DNA damage response takes place, then MRN and associated DNA repair components bind to the Ad genomic termini and physically mask the origins of DNA replication. Pol, polymerase; NF-I, nuclear factor I.