Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR.

Human adenovirus infects terminally differentiated cells and to replicate it must induce S-phase. The chief architects that drive adenovirus-infected cells into S-phase are the E1A proteins, with 5 different isoforms expressed during infection. E1A remodels the infected cell by associating with cellular factors and modulating their activity. The C-terminus of E1A is known to bind to only a handful of proteins. We have identified a novel E1A C-terminus binding protein, Ku70 (XRCC6), which was found to bind directly within the CR4 of E1A from human adenovirus type 5. Depletion of Ku70 reduced virus growth, possibly by activating the DNA damage response pathway. Ku70 was found to localize to viral replication centers and associate with the viral genome. Ku70 was also recruited to cellular cell cycle regulated promoters following viral infection. Our study has identified, for the first time, Ku70 as a novel E1A-binding protein which affects virus life cycle.

Adenovirus Early 1A proteins (E1A) are crucial for initiation of the viral life cycle after infection. The E1A gene is encoded at the left end of the viral genome and consists of two exons, the first encoding 185 amino acids in the 289 residues adenovirus 5 E1A, while the second exon encodes 104 residues. The second exon-encoded region of E1A is conserved across all E1A isoforms except for the 55 residues protein, which has a unique C-terminus due to a frame shift following splicing into the second exon. This region of E1A contributes to a variety of processes including the regulation of viral and cellular gene expression, immortalization and transformation. Here we evaluated the contributions that different regions of the second exon of E1A make to the viral life cycle using deletion mutants. The region of E1A encoded by the second exon was found to be important for overall virus growth, induction of viral and cellular gene expression, viral genome replication and deregulation of the cell cycle. Efficient viral replication was found to require exon 2 and the nuclear localization signal, as loss of either resulted in severe growth deficiency. Induction of cellular DNA synthesis was also deficient with any deletion of E1A within the C-terminus even if these deletions were outside of conserved region 4. Overall, our study provides the first comprehensive insight into the contributions of the C-terminus of E1A to the replicative fitness of human adenovirus 5 in arrested lung fibroblasts.

Far-upstream element (FUSE) binding protein 1 (FUBP1) was originally identified as a regulator of the oncogene via binding to the FUSE within the promoter and activating the expression of the gene. Recent studies have identified FUBP1 as a regulator of transcription, translation, and splicing via its DNA and RNA binding activities. Here we report the identification of FUBP1 as a novel binding partner of E1A. FUBP1 binds directly to E1A via the N terminus (residues 1 to 82) and conserved region 3 (residues 139 to 204) of adenovirus 5 E1A. The depletion of FUBP1 via short interfering RNAs (siRNA) reduces virus growth and drives the upregulation of the cellular stress response by activating the expression of p53-regulated genes. During infection, FUBP1 is relocalized within the nucleus, and it is recruited to viral promoters together with E1A while at the same time being lost from the FUSE upstream of the promoter. The depletion of FUBP1 affects viral and cellular gene expression. Importantly, in FUBP1-depleted cells, p53-responsive genes are upregulated, p53 occupancy on target promoters is enhanced, and histone H3 lysine 9 is hyperacetylated. This is likely due to the loss of the FUBP1-mediated suppression of p53 DNA binding. We also observed that E1A stabilizes the FUBP1-p53 complex, preventing p53 promoter binding. Together, our results identify, for the first time, FUBP1 as a novel E1A binding protein that participates in aspects of viral replication and is involved in the E1A-mediated suppression of p53 function. Viral infection triggers innate cellular defense mechanisms that have evolved to block virus replication. To overcome this, viruses have counterevolved mechanisms that ensure that cellular defenses are either disarmed or not activated to guarantee successful replication. One of the key regulators of cellular stress is the tumor suppressor p53 that responds to a variety of cellular stress stimuli and safeguards the integrity of the genome. During infection, many viruses target the p53 pathway in order to deactivate it. Here we report that human adenovirus 5 coopts the cellular protein FUBP1 to prevent the activation of the p53 stress response pathway that would block viral replication. This finding adds to our understanding of p53 deactivation by adenovirus and highlights its importance in infection and innate immunity.

Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression. IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.