A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors.

Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.

The human adenovirus E1B-55K oncoprotein coordinates cell transformation through regulation of DNA-bound host transcription factors.

The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.

A Case Series of Children with Acute Hepatitis and Human Adenovirus Infection.

BACKGROUND: Human adenoviruses typically cause self-limited respiratory, gastrointestinal, and conjunctival infections in healthy children. In late 2021 and early 2022, several previously healthy children were identified with acute hepatitis and human adenovirus viremia. METHODS: We used International Classification of Diseases, 10th Revision, codes to identify all children (<18 years of age) with hepatitis who were admitted to Children's of Alabama hospital between October 1, 2021, and February 28, 2022; those with acute hepatitis who also tested positive for human adenovirus by whole-blood quantitative polymerase chain reaction (PCR) were included in our case series. Demographic, clinical, laboratory, and treatment data were obtained from medical records. Residual blood specimens were sent for diagnostic confirmation and human adenovirus typing. RESULTS: A total of 15 children were identified with acute hepatitis - 6 (40%) who had hepatitis with an identified cause and 9 (60%) who had hepatitis without a known cause. Eight (89%) of the patients with hepatitis of unknown cause tested positive for human adenovirus. These 8 patients plus 1 additional patient referred to this facility for follow-up were included in this case series (median age, 2 years 11 months; age range, 1 year 1 month to 6 years 5 months). Liver biopsies indicated mild-to-moderate active hepatitis in 6 children, some with and some without cholestasis, but did not show evidence of human adenovirus on immunohistochemical examination or electron microscopy. PCR testing of liver tissue for human adenovirus was positive in 3 children (50%). Sequencing of specimens from 5 children showed three distinct human adenovirus type 41 hexon variants. Two children underwent liver transplantation; all the others recovered with supportive care. CONCLUSIONS: Human adenovirus viremia was present in the majority of children with acute hepatitis of unknown cause admitted to Children's of Alabama from October 1, 2021, to February 28, 2022, but whether human adenovirus was causative remains unclear. Sequencing results suggest that if human adenovirus was causative, this was not an outbreak driven by a single strain. (Funded in part by the Centers for Disease Control and Prevention.).

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

Isolation of a recombinant simian adenovirus encoding the human adenovirus G52 hexon suggests a simian origin for human adenovirus G52.

Human adenoviruses (HAdVs) are causative agents of morbidity and mortality throughout the world. These double-stranded DNA viruses are phylogenetically classified into seven different species (A-G). HAdV-G52, originally isolated in 2008 from a patient presenting with gastroenteritis, is the sole human-derived member of species G. Phylogenetic analysis previously suggested that HAdV-G52 may have a simian origin, indicating a potential zoonotic spillover into humans. However, evidence of HAdV-G52 in either human or simian populations has not been reported since. Here, we describe the isolation and in vitro characterization of rhesus (rh)AdV-69, a novel simian AdV with clear evidence of recombination with HAdV-G52, from the stool of a rhesus macaque. Specifically, the rhAdV-69 hexon capsid protein is 100% identical to that of HAdV-G52, whereas the remainder of the genome is most similar to rhAdV-55, sharing 95.36% nucleic acid identity. A second recombination event with an unknown adenovirus (AdV) is evident at the short fiber gene. From the same sample, we also isolated a second, highly related recombinant AdV (rhAdV-68) that harbors a distinct hexon gene but nearly identical backbone compared to rhAdV-69. In vitro, rhAdV-68 and rhAdV-69 demonstrate comparable growth kinetics and tropisms in human cell lines, nonhuman cell lines, and human enteroids. Furthermore, we show that coinfection of highly related AdVs is not unique to this sample since we also isolated coinfecting rhAdVs from two additional rhesus macaque stool samples. Our data collectively contribute to elucidating the origins of HAdV-G52 and provide insights into the frequency of coinfections and subsequent recombination in AdV evolution.IMPORTANCEUnderstanding the host origins of adenoviruses (AdVs) is critical for public health as transmission of viruses from animals to humans can lead to emergent viruses. Recombination between animal and human AdVs can also produce emergent viruses. HAdV-G52 is the only human-derived member of the HAdV G species. It has been suggested that HAdV-G52 has a simian origin. Here, we isolated from a rhesus macaque, a novel rhAdV, rhAdV-69, that encodes a hexon protein that is 100% identical to that of HAdV-G52. This observation suggests that HAdV-G52 may indeed have a simian origin. We also isolated a highly related rhAdV, differing only in the hexon gene, from the same rhesus macaque stool sample as rhAdV-69, illustrating the potential for co-infection of closely related AdVs and recombination at the hexon gene. Furthermore, our study highlights the critical role of whole-genome sequencing in understanding AdV evolution and monitoring the emergence of pathogenic AdVs.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

The adenovirus DNA-binding protein DBP.

Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research.

Development of oncolytic and gene therapy vectors based on adenovirus serotype 4 as an alternative to adenovirus serotype 5.

BACKGROUND: Adenoviral vectors are among the most frequently used vectors for gene therapy and cancer treatment. Most vectors are derived from human adenovirus (Ad) serotype 5 despite limited applicability caused by pre-existing immunity and unfavorable liver tropism, whereas the other more than 100 known human serotypes remain largely unused. Here, we screened a library of human Ad types and identified Ad4 as a promising candidate vector. METHODS: Reporter-gene-expressing viruses representative of the natural human Ad diversity were used to transduce an array of muscle cell lines and two- or three-dimensional tumor cultures. The time-course of transgene expression was monitored by fluorescence or luminescence measurements. To generate replication-deficient Ad4 vector genomes, successive homologous recombination was applied. RESULTS: Ad4, 17 and 50 transduced human cardiomyocytes more efficiently than Ad5, whereas Ad37 was found to be superior in rhabdomyocytes. Despite its moderate transduction efficiency, Ad4 showed efficient and long-lasting gene expression in papillomavirus (HPV) positive tumor organoids. Therefore, we aimed to harness the potential of Ad4 for improved muscle transduction or oncolytic virotherapy of HPV-positive tumors. We deleted the E1 and E3 transcription units to produce first generation Ad vectors for gene therapy. The E1- and E1/E3-deleted vectors were replication-competent in HEK293 cells stably expressing E1 but not in the other cell lines tested. Furthermore, we show that the Ad5 E1 transcription unit can complement the replication of E1-deleted Ad4 vectors. CONCLUSIONS: Our Ad4-based gene therapy vector platform contributes to the development of improved Ad vectors based on non-canonical serotypes for a broad range of applications.

Increased circulation of human adenovirus in 2023: an investigation of the circulating genotypes, upper respiratory viral loads, and hospital admissions in a large academic medical center.

IMPORTANCE: The circulation of human adenoviruses (HAdV) increased in 2023. In this manuscript, we show that HAdV-B3 was predominant in 2023 in a cohort characterized by the Johns Hopkins Hospital System. We also show that HAdV-B3 was associated with an increase in viral loads in respiratory samples and provide a correlation with the clinical presentations and outcomes.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

IMPORTANCE: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

Fragile X-Related Protein FXR1 Controls Human Adenovirus Capsid mRNA Metabolism.

Human adenoviruses (HAdVs) are widespread pathogens causing a variety of diseases. A well-controlled expression of virus capsid mRNAs originating from the major late transcription unit (MLTU) is essential for forming the infectious virus progeny. However, regulation of the MLTU mRNA metabolism has mainly remained enigmatic. In this study, we show that the cellular RNA-binding protein FXR1 controls the stability of the HAdV-5 MLTU mRNAs, as depletion of FXR1 resulted in increased steady-state levels of MLTU mRNAs. Surprisingly, the lack of FXR1 reduced viral capsid protein accumulation and formation of the infectious virus progeny, indicating an opposing function of FXR1 in HAdV-5 infection. Further, the long FXR1 isoform interfered with MLTU mRNA translation, suggesting FXR1 isoform-specific functions in virus-infected cells. We also show that the FXR1 protein interacts with N6-methyladenosine (m6A)-modified MLTU mRNAs, thereby acting as a novel m6A reader protein in HAdV-5 infected cells. Collectively, our study identifies FXR1 as a regulator of MLTU mRNA metabolism in the lytic HAdV-5 life cycle. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, such as the common cold and conjunctivitis. Even though adenoviruses have been studied for more than 6 decades, there are still gaps in understanding how the virus interferes with the host cell to achieve efficient growth. In this study, we identified the cellular RNA-binding protein FXR1 as a factor manipulating the HAdV life cycle. We show that the FXR1 protein specifically interferes with mRNAs encoding essential viral capsid proteins. Since the lack of the FXR1 protein reduces virus growth, we propose that FXR1 can be considered a novel cellular proviral factor needed for efficient HAdV growth. Collectively, our study provides new detailed insights about the HAdV-host interactions, which might be helpful when developing countermeasures against pathogenic adenovirus infections and for improving adenovirus-based therapies.

Inhibition of human adenovirus replication by TRIM35-mediated degradation of E1A.

Human adenovirus (HAdV) is ubiquitous in the human population, constituting a significant burden of global respiratory diseases. Children and individuals with low immunity are at risk of developing severe infections without approved antiviral treatment for HAdV. Our study demonstrated that TRIM35 inhibited HAdV-C5 early gene transcription, early protein expression, genome replication, and infectious virus progeny production. Furthermore, TRIM35 was found to inhibit HAdV replication by attenuating E1A expression. Mechanistically, TRIM35 interacts with and degrades E1A by promoting its K48-linked ubiquitination. Additionally, K253 and K285 are the key sites necessary for TRIM35 degradation. Moreover, an oncolytic adenovirus carrying shTRIM35 was constructed and observed to exhibit improved oncolysis in vivo, providing new ideas for clinical tumor treatment. Our results expand the broad antiviral activity of TRIM35 and mechanically support its application as a HAdV replication inhibitor. IMPORTANCE E1A is an essential human adenovirus (HAdV) protein responsible for the early replication of adenovirus while interacting with multiple host proteins. Understanding the interaction between HAdV E1A and TRIM35 helps identify effective antiviral therapeutic targets. The viral E1A protein is a crucial activator and regulator of viral transcription during the early infection stages. We first reported that TRIM35 interacts with E1A to resist adenovirus infection. Our study demonstrated that TRIM35 targets E1A to resist adenovirus, indicating the applicability of targeting virus-dependent host factors as a suitable antiviral strategy.

Molecular Mechanism of Adenovirus Late Protein L4-100K Chaperones the Trimerization of Hexon.

Assembly of the adenovirus capsid protein hexon depends on the assistance of the molecular chaperone L4-100K. However, the chaperone mechanisms remain unclear. In this study, we found that L4-100K was involved in the hexon translation process and could prevent hexon degradation by the proteasome in cotransfected human cells. Two nonadjacent domains, 84-133 and 656-697, at the N-terminal and C-terminal regions of human adenovirus type 5 L4-100K, respectively, were found to be crucial and cooperatively responsible for hexon trimer expression and assembly. These two chaperone-related domains were conserved in the sequence of L4-100K and in the function of hexon assembly across different adenovirus serotypes. Different degrees of cross-activity of hexon trimerization with different serotypes were detected in subgroups B, C, and D, which were proven to be controlled by the interaction between the C-terminal chaperone-related domain of L4-100K and hypervariable regions (HVR) of hexon. Additionally, HVR-chimeric hexon mutants were successfully assembled with the assistance of the 1-697 mutant. Structural analysis of 656-697 by nuclear magnetic resonance and structural prediction of L4-100K using Robetta showed that the two conserved domains are mainly composed of α-helices and are located on the surface of the highly folded core region. Our research provides a more complete understanding of hexon assembly and guidance for the development of hexon-chimeric adenovirus vectors that will be safer, smarter, and more efficient. IMPORTANCE Adenovirus vectors have been widely used in clinical trials of vaccines and gene therapy, although some deficiencies remain. Chimeric modification of the hexon was expected to improve the potency of preexisting immune evasion and targeting, but in many cases, viral packaging is prevented by the inability of the chimeric hexon to assemble correctly. So far, few studies have examined the mechanisms of hexon trimer assembly. Here, we show how the chaperone protein L4-100K contributes to the assembly of the adenovirus capsid protein hexon, and these data will provide a guide for novel adenovirus vector design and development, as we desired.

Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis.

IMPORTANCE: The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there's still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.

Enforced Dimerization of CD45 by the Adenovirus E3/49K Protein Inhibits T Cell Receptor Signaling.

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses, HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D HAdVs. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45, causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both secreted sec49K as well as membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer. IMPORTANCE The battle between viruses and their hosts is an ongoing arms race. Whereas the host tries to detect and eliminate the virus, the latter counteracts such antiviral measures to replicate and spread. Adenoviruses have evolved various mechanisms to evade the human immune response. The E3/49K protein of species D adenoviruses mediates the inhibition of immune cell function via binding to the protein tyrosine phosphatase CD45. Here, we show that E3/49K triggers the dimerization of CD45 and thereby inhibits its phosphatase activity. Intriguingly, the membrane-anchored E3/49K seems to be designed like a "molecular fishing rod" with the two CD45 binding domains of E3/49K as baits positioned at the end of an extended disordered region reminiscent of a fishing line. The adenoviral strategy to inhibit CD45 activity by forced dimerization may be used for therapeutic intervention in autoimmune diseases or to prevent graft rejection after transplantation.

Isolation of novel simian adenoviruses from macaques for development of a vector for human gene therapy and vaccines.

IMPORTANCE: Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells.

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.