E1B-55K Is a Phosphorylation-Dependent Transcriptional and Posttranscriptional Regulator of Viral Gene Expression in Human Adenovirus C5 Infection.

The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. IMPORTANCE Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.

Automatic detection and measurement of viral replication compartments by ellipse adjustment.

Viruses employ a variety of strategies to hijack cellular activities through the orchestrated recruitment of macromolecules to specific virus-induced cellular micro-environments. Adenoviruses (Ad) and other DNA viruses induce extensive reorganization of the cell nucleus and formation of nuclear Replication Compartments (RCs), where the viral genome is replicated and expressed. In this work an automatic algorithm designed for detection and segmentation of RCs using ellipses is presented. Unlike algorithms available in the literature, this approach is deterministic, automatic, and can adjust multiple RCs using ellipses. The proposed algorithm is non iterative, computationally efficient and is invariant to affine transformations. The method was validated over both synthetic images and more than 400 real images of Ad-infected cells at various timepoints of the viral replication cycle obtaining relevant information about the biogenesis of adenoviral RCs. As proof of concept the algorithm was then used to quantitatively compare RCs in cells infected with the adenovirus wild type or an adenovirus mutant that is null for expression of a viral protein that is known to affect activities associated with RCs that result in deficient viral progeny production.