Nanomolar concentrations of the photodynamic compound TLD-1433 effectively inactivate numerous human pathogenic viruses.

The anti-viral properties of a small (≈1 kDa), novel Ru(II) photo dynamic compound (PDC), referred to as TLD-1433 (Ruvidar™), are presented. TLD-1433 had previously been demonstrated to exert strong anti-bacterial and anti-cancer properties. We evaluated the capacity of TLD-1433 to inactivate several human pathogenic viruses. TLD-1433 that was not photo-activated was capable of effectively inactivating 50 % of influenza H1N1 virus (ID50) at a concentration of 117 nM. After photo-activation, the ID50 was reduced to <10 nM. The dose of photo-activated TLD-1433 needed to reduce H1N1 infectivity >99 % (ID99) was approximately 170 nM. Similarly, the ID99 of photo-activated TLD-1433 was determined to range from about 20 to 120 nM for other tested enveloped viruses; specifically, a human coronavirus, herpes simplex virus, the poxvirus Vaccinia virus, and Zika virus. TLD-1433 also inactivated two tested non-enveloped viruses; specifically, adenovirus type 5 and mammalian orthoreovirus, but at considerably higher concentrations. Analyses of TLD-1433-treated membranes suggested that lipid peroxidation was a major contributor to enveloped virus inactivation. TLD-1433-mediated virus inactivation was temperature-dependent, with approximately 10-fold more efficient virucidal activity when viruses were treated at 37 °C than when treated at room temperature (∼22 °C). The presence of fetal bovine serum and virus solution turbidity reduced TLD-1433-mediated virucidal efficiency. Immunoblots of TLD-1433-treated human coronavirus indicated the treated spike protein remained particle-associated.

Molecular insights into type I interferon suppression and enhanced pathogenicity by species B human adenoviruses B7 and B14.

Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes.

HBO1/KAT7/MYST2 HAT complex regulates human adenovirus replicative cycle.

Human adenoviruses (HAdV) belong to a small DNA tumor virus family that continues as valuable models in understanding the viral strategies of usurping cell growth regulation. A number of HAdV type 2/5 early viral gene products interact with a variety of cellular proteins to build a conducive environment that promotes viral replication. Here we show that HBO1 (Histone Acetyltransferase Binding to ORC1), a member of the MYST histone acetyltransferase (HAT) complex (also known as KAT7 and MYST2) that acetylates most of the histone H3 lysine 14, is essential for HAdV5 growth. HBO1/MYST2/KAT7 HAT complexes are critical for a variety of cellular processes including control of cell proliferation. In HBO1 downregulated human cells, HAdV5 infection results in reduced expression of E1A and other viral early genes, virus growth is also reduced significantly. Importantly, HBO1 downregulation reduced H3 lysine 14 acetylation at viral promoters during productive infection, likely driving reduced viral gene expression. HBO1 was also associated with viral promoters during infection and co-localized with viral replication centers in the nuclei of infected cells. In transiently transfected cells, overexpression of E1A along with HBO1 stimulated histone acetyltransferase activity of HBO1. E1A also co-immunoprecipitated with HBO1 in transiently transfected cells. In summary, our results demonstrate that HAdV recruits the HBO1 HAT complex to aid in viral replication.

ARGLU1 enhances promoter-proximal pausing of RNA polymerase II and stimulates DNA damage repair.

Arginine and glutamate rich 1 (ARGLU1) is a poorly understood cellular protein with functions in RNA splicing and transcription. Computational prediction suggests that ARGLU1 contains intrinsically disordered regions and lacks any known structural or functional domains. We used adenovirus Early protein 1A (E1A) to probe for critical regulators of important cellular pathways and identified ARGLU1 as a significant player in transcription and the DNA damage response pathway. Transcriptional effects induced by ARGLU1 occur via enhancement of promoter-proximal RNA polymerase II pausing, likely by inhibiting the interaction between JMJD6 and BRD4. When overexpressed, ARGLU1 increases the growth rate of cancer cells, while its knockdown leads to growth arrest. Significantly, overexpression of ARGLU1 increased cancer cell resistance to genotoxic drugs and promoted DNA damage repair. These results identify new roles for ARGLU1 in cancer cell survival and the DNA damage repair pathway, with potential clinical implications for chemotherapy resistance.

Adenovirus 5 Vectors Expressing SARS-CoV-2 Proteins.

SARS-CoV-2 coronavirus is a recently identified novel coronavirus that is the causative agent of the COVID-19 pandemic that began in 2020. An intense research effort has been undertaken by the research community in order to better understand the molecular etiology of this virus and its mechanisms of host cell subjugation and immune system evasion. To facilitate further research into the SARS-CoV-2 coronavirus we have generated adenovirus 5-based viral vectors that express SARS-CoV-2 proteins-S, N, E, NSP7, NSP8, and NSP12 as hemagglutinin (HA)-tagged and untagged variants. We have also engineered two additional viruses that express the S protein receptor binding domain and a fusion of the receptor binding domain to the N protein. We show that these vectors are expressed in several different cell lines by Western blotting and real-time quantitative reverse transcriptase (qRT-PCR), we evaluate the subcellular localization of these viral proteins, and we show that these coronavirus proteins bind to a variety of cellular targets. The flexibility of adenovirus vectors allows them to be used in a variety of cell models and, importantly, in animal models as well. IMPORTANCE The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has brought untold personal and economic suffering to the world. Intense research has made tremendous progress in understanding how this virus works, yet much research remains to be done as new variants and continued evolution of the virus keep shifting the rules of engagement on the pandemic battlefield. Therefore, wide availability of resources and reagents to study SARS-CoV-2 is essential in overcoming the pandemic and for the prevention of future outbreaks. Our viral vectors provide additional tools for researchers to use in order to better understand the molecular biology of virus-host interactions and other aspects of SARS-CoV-2.

Differential Splicing of Human Adenovirus 5 E1A RNA Expressed in cis versus in trans.

Human adenovirus (HAdV) is used extensively as a vector for gene delivery for a variety of purposes, including gene therapy and vaccine development. Most adenoviral vectors used for these approaches have a deletion of early region 1 (E1), which is complemented by the cell line. Most commonly, these are 293 cells for HAdV serotype 2 or 5. The 293 cells have the left end of HAdV5 integrated into chromosome 19 and express the E1 genes and protein IX. We observed that viruses with the E1 region deleted often grow less well on 293 cells than E1 wild-type viruses. Therefore, we investigated whether this poor growth is caused by splicing differences between the E1A RNA provided by the cell line (in trans) and the E1A RNA provided by the infecting viral genome (in cis). We observed that E1A RNA that was expressed from the genomes of 293 cells was spliced differently during infection with an E1A-deleted dl312 virus than E1A RNA from the same cells infected with dl309 or wt300. Importantly, 293 cells were not able to fully complement the late E1A transcripts, specifically 11S, 10S, and 9S RNA, which express the E1A217R, E1A171R, and E1A55R isoforms, respectively. We observed that these splicing differences likely arise due to different subnuclear localizations of E1A RNA. E1A RNA expressed from the viral genome was localized to viral replication centers, while E1A RNA expressed from the cell's genome was not. This loss of the late E1A mRNAs and their associated proteins impacts viral growth, gene expression, and protein levels. Complementation of the late E1A mRNAs in 293 cells restored some of the growth defect observed with dl312 and resulted in higher virus growth.IMPORTANCE Human adenovirus has become an important tool for medicine and research, and 293 cells and various similar cell lines are used extensively for virus production in situations where high viral yields are important. Such complementing cell lines are used for the production of viral vectors and vaccines, which often have deletions and replacements in various viral genes. Deletions in essential genes, such as E1, are often complemented by the cell line that is used for virus propagation in trans Here, we show that even complete genetic complementation of a viral gene does not result in full protein complementation, a defect that compromises virus growth. This is particularly important when high viral yields are crucial, as in virus production for vaccine development or gene therapy.

Differential Effects of Human Adenovirus E1A Protein Isoforms on Aerobic Glycolysis in A549 Human Lung Epithelial Cells.

Viruses alter a multitude of host-cell processes to create a more optimal environment for viral replication. This includes altering metabolism to provide adequate substrates and energy required for replication. Typically, viral infections induce a metabolic phenotype resembling the Warburg effect, with an upregulation of glycolysis and a concurrent decrease in cellular respiration. Human adenovirus (HAdV) has been observed to induce the Warburg effect, which can be partially attributed to the adenovirus protein early region 4, open reading frame 1 (E4orf1). E4orf1 regulates a multitude of host-cell processes to benefit viral replication and can influence cellular metabolism through the transcription factor avian myelocytomatosis viral oncogene homolog (MYC). However, E4orf1 does not explain the full extent of Warburg-like HAdV metabolic reprogramming, especially the accompanying decrease in cellular respiration. The HAdV protein early region 1A (E1A) also modulates the function of the infected cell to promote viral replication. E1A can interact with a wide variety of host-cell proteins, some of which have been shown to interact with metabolic enzymes independently of an interaction with E1A. To determine if the HAdV E1A proteins are responsible for reprogramming cell metabolism, we measured the extracellular acidification rate and oxygen consumption rate of A549 human lung epithelial cells with constitutive endogenous expression of either of the two major E1A isoforms. This was followed by the characterization of transcript levels for genes involved in glycolysis and cellular respiration, and related metabolic pathways. Cells expressing the 13S encoded E1A isoform had drastically increased baseline glycolysis and lower maximal cellular respiration than cells expressing the 12S encoded E1A isoform. Cells expressing the 13S encoded E1A isoform exhibited upregulated expression of glycolysis genes and downregulated expression of cellular respiration genes. However, tricarboxylic acid cycle genes were upregulated, resembling anaplerotic metabolism employed by certain cancers. Upregulation of glycolysis and tricarboxylic acid cycle genes was also apparent in IMR-90 human primary lung fibroblast cells infected with a HAdV-5 mutant virus that expressed the 13S, but not the 12S encoded E1A isoform. In conclusion, it appears that the two major isoforms of E1A differentially influence cellular glycolysis and oxidative phosphorylation and this is at least partially due to the altered regulation of mRNA expression for the genes in these pathways.

Characterization of Adenovirus 5 E1A Exon 1 Deletion Mutants in the Viral Replicative Cycle.

Human adenovirus infection is driven by Early region 1A (E1A) proteins, which are the first proteins expressed following the delivery of the viral genome to the cellular nucleus. E1A is responsible for reprogramming the infected cell to support virus replication alongside the activation of expression of all viral transcriptional units during the course of the infection. Although E1A has been extensively studied, most of these studies have focused on understanding the conserved region functions outside of a full infection. Here, we investigated the effects of small deletions in E1A exon 1 on the viral replicative cycle. Almost all deletions were found to have a negative impact on viral replication with the exception of one deletion found in the mutant dl1106, which replicated better than the wild-type E1A expressing dl309. In addition to growth, we assessed the virus mutants for genome replication, induction of the cytopathic effect, gene and protein expression, sub-cellular localization of E1A mutant proteins, induction of cellular S-phase, and activation of S-phase specific cellular genes. Importantly, our study found that virus replication is likely limited by host-specific factors, rather than specific viral aspects such as the ability to replicate genomes or express late proteins, after a certain level of these has been expressed. Furthermore, we show that mutants outside of the conserved regions have significant influence on viral fitness. Overall, our study is the first comprehensive evaluation of the dl1100 series of exon 1 E1A deletion mutants in viral fitness and provides important insights into the contribution that E1A makes to viral replication in normal human cells.

Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization.

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.

Temporal dynamics of adenovirus 5 gene expression in normal human cells.

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.

Adenovirus 5 E1A-Mediated Suppression of p53 via FUBP1.

Far-upstream element (FUSE) binding protein 1 (FUBP1) was originally identified as a regulator of the oncogene c-Myc via binding to the FUSE within the c-Myc 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 c-Myc 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.IMPORTANCE 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.

The Influence of E1A C-Terminus on Adenovirus Replicative 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.

Suppression of Type I Interferon Signaling by E1A via RuvBL1/Pontin.

Suppression of interferon signaling is of paramount importance to a virus. Interferon signaling significantly reduces or halts the ability of a virus to replicate; therefore, viruses have evolved sophisticated mechanisms that suppress activation of the interferon pathway or responsiveness of the infected cell to interferon. Adenovirus has multiple modes of inhibiting the cellular response to interferon. Here, we report that E1A, previously shown to regulate interferon signaling in multiple ways, inhibits interferon-stimulated gene expression by modulating RuvBL1 function. RuvBL1 was previously shown to affect type I interferon signaling. E1A binds to RuvBL1 and is recruited to RuvBL1-regulated promoters in an interferon-dependent manner, preventing their activation. Depletion of RuvBL1 impairs adenovirus growth but does not appear to significantly affect viral protein expression. Although RuvBL1 has been shown to play a role in cell growth, its depletion had no effect on the ability of the virus to replicate its genome or to drive cells into S phase. E1A was found to bind to RuvBL1 via the C terminus of E1A, and this interaction was important for suppression of interferon-stimulated gene transcriptional activation and recruitment of E1A to interferon-regulated promoters. Here, we report the identification of RuvBL1 as a new target for adenovirus in its quest to suppress the interferon response.IMPORTANCE For most viruses, suppression of the interferon signaling pathway is crucial to ensure a successful replicative cycle. Human adenovirus has evolved several different mechanisms that prevent activation of interferon or the ability of the cell to respond to interferon. The viral immediate-early gene E1A was previously shown to affect interferon signaling in several different ways. Here, we report a novel mechanism reliant on RuvBL1 that E1A uses to prevent activation of interferon-stimulated gene expression following infection or interferon treatment. This adds to the growing knowledge of how viruses are able to inhibit interferon and identifies a novel target used by adenovirus for modulation of the cellular interferon pathway.

The interaction of adenovirus E1A with the mammalian protein Ku70/XRCC6.

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.

A naturally occurring transcript variant of MARCO reveals the SRCR domain is critical for function.

Macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor (cA-SR) that recognizes and phagocytoses a wide variety of pathogens. Most cA-SRs that contain a C-terminal scavenger receptor cysteine-rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signaling is less clear. The discovery of a naturally occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signaling and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant-negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-like receptor 2 (TLR2)-mediated pro-inflammatory signaling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, whereas MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signaling and MARCO-mediated cellular adhesion.

The Dual Nature of Nek9 in Adenovirus Replication.

UNLABELLED: To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression. IMPORTANCE: In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks to replication. We show that the virus uses a cellular protein, Nek9, in order to block activation of p53-regulated gene GADD45A, which is an important player in stress response and p53-mediated cell cycle arrest. Importantly, our study also identifies Nek9 as a transcriptional repressor.

Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells.

Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts.

Adenovirus E1A targets the DREF nuclear factor to regulate virus gene expression, DNA replication, and growth.

UNLABELLED: The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication. IMPORTANCE: This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to viral promoters during infection together with E1A. These results suggest that DREF contributes to activation of viral gene expression. However, like several other PML-associated proteins, DREF also appears to function as a growth restriction factor for adenovirus infection.

The adenovirus 55 residue E1A protein is a transcriptional activator and binds the unliganded thyroid hormone receptor.

The early region 1A (E1A) of human adenovirus types 2 and 5 is differentially spliced to yield five distinct mRNAs that encode different proteins. The smallest E1A RNA transcript encodes a 55 residue (55R) protein that shares only 28 amino acid residues with the other E1A proteins. Even though it is the most abundant E1A transcript at late times post-infection, little is known about the functions of this E1A isoform. In this study, we show that the E1A 55R protein interacts with, and modulates the activity of the unliganded thyroid hormone receptor (TR). We demonstrate that E1A 55R contains a signature motif known as the CoRNR box that confers interaction with the unliganded TR; this motif was originally identified in cellular corepressors. Using a system reconstituted in the yeast Saccharomyces cerevisiae, which lack endogenous TR and TR coregulators, we show that E1A 55R nonetheless differs from cellular corepressors as it functions as a strong co-activator of TR-dependent transcription and that it possesses an intrinsic transcriptional activation domain. These data indicate that the E1A 55R protein functions as a transcriptional regulator.

Cellular GCN5 is a novel regulator of human adenovirus E1A-conserved region 3 transactivation.

The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (hat/hat) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.