A Single Amino Acid Switch in the Adenoviral DNA Binding Protein Abrogates Replication Center Formation and Productive Viral Infection.

Adenoviruses are very efficient high-capacity vaccine vectors and are common gene delivery systems. Despite their extensive use in preclinical models and clinical trials over the past decades, adenoviral vectors still require optimization. To achieve that, more thorough characterizations of adenoviral genes and gene products, as well as pathogen-host interactions, are indispensable. The adenoviral DNA binding protein (DBP) is a key regulatory protein involved in various cellular and viral processes. Here, we show that single amino acid exchange mutations in human adenovirus C5 (HAdV-C5) DBP strongly influence adenoviral replication by altering interaction with the cellular ubiquitination machinery. Specifically, phenotypic analyses of DBP mutants demonstrate that single amino acid substitutions can regulate interactions with the cellular USP7 deubiquitinase, impede viral DNA synthesis, and completely abolish viral late protein expression and progeny production. Importantly, cells infected with the DBP mutant UBM5 consistently lack DBP-positive replication centers (RCs), which are usually formed during the transition from the early to the late phase of infection. Our findings demonstrate that DBP regulates a key step at the onset of the late phase of infection and that this activity is unambiguously linked to the formation and integrity of viral RCs. These data provide the experimental basis for future work that targets DBP and its interference with the formation of viral RCs during productive infection. Consequently, this work will have immediate impact on DNA virus and adenovirus research in general and, potentially, also on safety optimization of existing and development of novel adenoviral vectors and anti-adenoviral compounds. IMPORTANCE To further understand the biology of human adenoviruses (HAdVs) and to optimize HAdVs for use in prophylactic and therapeutic therapies, a thorough understanding of key viral proteins is paramount. As one of the essential HAdV proteins, the DNA binding protein DBP plays important roles in various steps of the viral replication cycle. In this work, we aimed at deciphering the role of single amino acid exchange mutations in the HAdV-C5 DBP on interaction with the cellular deubiquitinase USP7 and regulation of viral replication. We identify interaction with USP7, viral replication center formation, and viral progeny production as potently regulated steps of the viral life cycle that are affected by these few and distinct mutations in DBP.

KIR3DS1 directs NK cell-mediated protection against human adenovirus infections.

Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving non­KIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.

A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation.

Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

The human adenovirus type 5 E1B 55-kilodalton protein is phosphorylated by protein kinase CK2.

The human adenovirus type 5 (HAdV5) early region 1B 55-kDa protein (E1B-55K) is a multifunctional phosphoprotein playing several critical roles during adenoviral productive infection, e.g., degradation of host cell proteins, viral late mRNA export, and inhibition of p53-mediated transcription. Many of these functions are apparently regulated at least in part by the phosphorylation of E1B-55K occurring at a stretch of amino acids resembling a potential CK2 consensus phosphorylation motif. We therefore investigated the potential role of CK2 phosphorylation upon E1B-55K during adenoviral infection. A phosphonegative E1B-55K mutant showed severely reduced virus progeny production, although viral early, late, and structural protein levels and viral DNA replication were not obviously affected. Binding studies revealed an interaction between the CK2α catalytic subunit and wild-type E1B-55K, which is severely impaired in the phosphonegative E1B mutant. In addition, in situ the α-catalytic subunit is redistributed into ring-like structures surrounding E1B-55K nuclear areas and distinct cytoplasmic accumulations, where a significant amount of CK2α colocalizes with E1B-55K. Furthermore, in in vitro phosphorylation assays, wild-type E1B-55K glutathione S-transferase fusion proteins were readily phosphorylated by the CK2α subunit but inefficiently phosphorylated by the CK2 holoenzyme. Addition of the CK2-specific inhibitors TBB (4,5,6,7-tetrabromobenzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole) to infected cells confirmed that CK2α binding to E1B-55K is necessary for efficient phosphorylation of E1B-55K. In summary, our data show that CK2α interacts with and phosphorylates HAdV5 E1B-55K at residues S490/491 and T495 and that these posttranslational modifications are essential for E1B-55K lytic functions.