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...

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.Adenovirus (Ad) has a linear, double-stranded DNA genome with inverted terminal repeats (ITRs) at each extremity that contain the origins of replication. Several viral proteins have been found to be key components in supporting viral DNA replication, and of particular importance here, this includes the E4-ORF3 and E4-ORF6 products. With mutant viruses that lack E4-ORF3 and E4-ORF6, early viral transcription and gene expression are normal; however, there is a significant delay and reduction in viral DNA replication, and virus yield is reduced by ϳ1,000-fold (20, 49). Either the E4-ORF3 or E4-ORF6 protein is individually sufficient to complement the DNA replication defect and virus growth (7, 24); thus, these proteins are considered functionally redundant.An important function of these two Ad E4 proteins is the inhibition of the Mre11-Rad50-Nbs1 (MRN) complex (30, 50). The MRN complex is involved in double-strand break repair (DSBR) and is regarded as the sensor of double-strand breaks (DSBs) (9, 12, 46). When a DSB occurs in the cellular genome due to a multitude of causes, ranging from ionizing radiation (33) to VDJ recombination (10), the MRN complex recognizes the lesion and recruits the protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) to the site of the break to initiate the process of nonhomologous end joining (NHEJ) (46). These two kinases...

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world.

bDecades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.