Adenoviruses induce an extensive reorganization of the host cell nucleus during replication. Such a process results in the assembly of viral and cellular macromolecules into nuclear structures called adenovirus replication compartments (AdRCs), which function as platforms for viral DNA replication and gene expression. AdRCs co-opt host proteins and cellular pathways that restrict viral replication, suggesting that the mechanisms that control AdRC formation and function are essential for viral replication and lay at the basis of virus-host interactions. Here, we review the hallmarks of AdRCs and recent progress in our understanding of the formation, composition, and function of AdRCs. Furthermore, we discuss how AdRCs facilitate the interplay between viral and cellular machineries and hijack cellular functions to promote viral genome replication and expression.

Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. IMPORTANCERC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that the synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle.

The adenovirus E1B 55K (E1B) protein plays major roles in productive adenoviral infection and cellular transformation. Interest in E1B increased because of the potential of adenoviruses as therapeutic vectors, and the E1B gene is commonly deleted from adenovirus vectors for anticancer therapy. E1B activities are spatiotemporally regulated through SUMOylation and phosphorylation, and through interactions with multiple partners that occur presumably at different intracellular sites and times postinfection. E1B is implicated in the formation of viral replication compartments and regulates viral genome replication and transcription, transcriptional repression, degradation of cellular proteins, and several intranuclear steps of viral late mRNA biogenesis. Here, we review advances in our understanding of E1B during productive adenovirus replication and discuss fundamental aspects that remain unresolved.The adenovirus E1B 55K protein (called E1B throughout this review) plays key roles during productive viral replication and contributes to oncogenic transformation. The E1B gene is usually deleted or modified in oncolytic adenoviruses because such viruses preferentially replicate in and kill cancer cells (reviewed in [1,2]). Although structural data for the 496 amino acid (aa) polypeptide are scarce, some structural or functional motifs are known or have been proposed, and the E1B protein may contain intrinsically disordered regions (IDR). E1B activities are regulated by posttranslational modifications (PTMs) that impact on both intracellular localization and the interactions that E1B establishes with viral and cellular proteins. E1B acts as a small ubiquitin-like modifier (SUMO)-1 ligase for p53 and participates in the polyubiquitylation-induced degradation of cellular targets that would otherwise interfere with viral replication. E1B also promotes viral replication through repression of interferon (IFN)-stimulated genes (ISG) and p53 regulation. Of note, efficient viral DNA replication depends on the formation of adenoviral replication compartments (RCs), another process in which E1B is involved. Furthermore, E1B interacts with viral RNA and this interaction optimizes production and splicing of viral late mRNAs. Yet, many aspects of the biology of E1B remain to be elucidated, including the protein's threedimensional structure and the molecular mechanisms whereby E1B regulates viral genome replication and gene expression. Most of the knowledge of E1B comes from the study of the human serotypes 2 and 5 from species C; however, sequences and functional features from other adenovirus species have also been described. A very complete review of the E1B protein Abbreviations CRM1, chromosome region maintenance 1 protein homolog; CDK, cyclin-dependent kinase; E1B-AP5, E1B-associated protein 5; eIF4E, eukaryotic translation initiation factor 4E; hnRNP, heteronuclear ribonucleoprotein; LH3, hexon-interlacing protein LH3; I-TASSER, iterative threading ASSEmbly refinement; Mre11, meiotic recombination 11; Nxf1/Tap, nuclear RNA expo...

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