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 doublestrand 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.Adenovirus (Ad) has a linear, double-stranded DNA genome with inverted terminal repeats at each end that contain the origins of viral DNA replication. The cellular Mre11-Rad50-Nbs1 (MRN) complex can recognize the termini of the linear Ad genome as double-strand breaks (DSBs) and elicit a DNA damage response (reviewed in reference 42). The repair program leads to activation of a checkpoint signaling cascade and the ligation of the Ad genomes into concatemers (6,7,18,33,41). There are several reasons this response may inhibit viral DNA replication. First, Ad genome concatenation would bury the viral origins of DNA replication within the multimer, inhibiting efficient replication (11). Second, deletion of viral DNA sequences at the concatemeric junctions resulted in the loss of functional origins of replication (18). Finally, multimeric Ad genomes would be too large to package into the viral capsid (27).There are three Ad proteins that function to inactivate the MRN complex and thereby inhibit the DNA damage response. The E4-ORF3 protein induces the rearrangement of promyelocytic leukemia (PML) nuclear bodies into track structures in the nucleus and sequesters the MRN complex in these tracks by 6 h postinfection (hpi) (12, 33), a time prior to the onset of viral DNA replication. The viral proteins E1B-55K and E4-ORF6 work in concert to recruit a CUL5-containing E3 ubiquitin ligase c...

Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

“…8). During the early phase of infection, the pVII(Δ24) protein is tethered to the HAdV-5 DNA to protect the viral genome from the cellular DNA damage response (18,39). As infection proceeds, the pVII(Δ24) protein is gradually removed from the viral DNA to allow proper HAdV-5 DNA replication and gene expression (20,40).…”

“…Notably, MLTU encodes an abundant histone-like protein, pVII, which is proteolytically processed into the mature protein VII during the final steps of virion maturation (13). The mature protein VII [also known as pVII(Δ24) (14)] organizes the viral genome into a nucleosome-like structure (15)(16)(17), protects viral DNA from the cellular DNA damage response (18), and also can act as a protein regulating transcription (19,20).…”

The Kinase Activity of Ataxia-Telangiectasia Mutated Interferes with Adenovirus E4 Mutant DNA Replication

“…Mre11 and mediator of DNA damage checkpoint protein 1 (Mdc1) are redistributed to early foci that appear prior to the onset of viral DNA replication (4,5). Phosphorylated ATM (pATM) is found in foci that contain input E4 mutant DNA (15). In E4 mutant-infected cells, Mre11 and pATM are present in viral replication centers that contain the 72-kDa DNA binding protein produced from E2 (E2-72kDa) (9,10).…”

Differential Activation of Cellular DNA Damage Responses by Replication-Defective and Replication-Competent Adenovirus Mutants

“…During the process of early transcription the genome undergoes chromatin remodeling and pVII is replaced by acetylated histones (26). Karen and Hearing (24) have studied the formation of foci containing phosphorylated ATM (pATM) in E4 mutant infections, and find that pATM colocalizes with Ad genomes that no longer contain the viral core DNA-binding protein pVII. Furthermore, pATM foci are reduced in cells treated with a transcription inhibitor and after infection by Ad E1 mutants that fail to activate any of the viral genome's transcription units.…”

“…Since recent results from Karen and Hearing (24) suggest that transcription-mediated remodeling of the viral chromatin is needed to activate phosphorylated ATM focus formation, we next sought to determine whether active transcription was required for Mdc1 focus formation in infected cells. HeLa cells were infected with Ad5, AdRSV␤gal, or H5dl1007 and immediately treated with ␣-amanitin to inhibit transcription from the incoming genomes.…”