Deamidation, a type of post-translational modification commonly considered a hallmark of protein "aging" and function decay, is increasingly recognized for its pivotal role in regulating biological processes and viral infection. Our previous study has demonstrated that the deamidation of replication and transcription activator (RTA), a master regulator of ubiquitous and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), mediated by phosphoribosylformylglycinamidine synthetase (PFAS), hinders its nuclear import and transcriptional activity. Here we report that the viral glutamine amidotransferase (vGAT) pseudo-enzyme was exploited to facilitate KSHV lytic infection by inhibiting RTA deamidation. To be more specific, vGAT interacted with both RTA and cellular PFAS, and inhibited PFAS-mediated RTA deamidation, thus facilitating RTA nuclear localization and suppressing nuclear factor-kappa B (NF-κB) signaling activation, as well as augmenting RTA-mediated transcriptional activation of viral open reading frames (ORFs). In addition, vGAT appeared to regulate the deamidation process of several viral ORFs of KSHV. Collectively, these findings unveil that a viral pseudo-enzyme was exploited to enhance viral infection via deamidation regulation.

Metabolic enzymes are central players for cell metabolism and cell proliferation. These enzymes perform distinct functions in various cellular processes, such as cell metabolism and immune defense. Because viral infections inevitably trigger host immune activation, viruses have evolved diverse strategies to blunt or exploit the host immune response to enable viral replication. Meanwhile, viruses hijack key cellular metabolic enzymes to reprogram metabolism, which generates the necessary biomolecules for viral replication. An emerging theme arising from the metabolic studies of viral infection is that metabolic enzymes are key players of immune response and, conversely, immune components regulate cellular metabolism, revealing unexpected communication between these two fundamental processes that are otherwise disjointed. This review aims to summarize our present comprehension of the involvement of metabolic enzymes in viral infections and host immunity and to provide insights for potential antiviral therapy targeting metabolic enzymes.

Pseudokinases regulate diverse cellular processes associated with normal cellular functions and disease. They are defined bioinformatically based on the absence of one or more catalytic residues that are required for canonical protein kinase functions. The ability to define pseudokinases based on primary sequence comparison has enabled the systematic mapping and cataloging of pseudokinase orthologs across the tree of life. While these sequences contain critical information regarding pseudokinase evolution and functional specialization, extracting this information and generating testable hypotheses based on integrative mining of sequence and structural data requires specialized computational tools and resources. In this chapter, we review recent advances in the development and application of open-source tools and resources for pseudokinase research. Specifically, we describe the application of an interactive data analytics framework, KinView, for visualizing the patterns of conservation and variation in the catalytic domain motifs of pseudokinases and evolutionarily related canonical kinases using a consistent set of curated alignments organized based on the widely used kinome evolutionary hierarchy. We also demonstrate the application of an integrated Protein Kinase Ontology (ProKinO) and an interactive viewer, ProtVista, for mapping and analyzing primary sequence motifs and annotations in the context of 3D structures and AlphaFold2 models. We provide examples and protocols for generating testable hypotheses on pseudokinase functions both for bench biologists and advanced users.

This Special Issue comprises twelve authoritative reviews that highlight an understudied but rapidly developing area of biology: catalytically inactive enzyme homologs. These pseudoenzymes, sometimes called 'dead enzymes', are found within most enzyme families and generally arose via gene duplication events. Dead enzymes have lost their enzymatic capacity, often via the evolutionary loss of key catalytic residues. However, as this Special Issue highlights, pseudoenzymes are far from being functionally 'dead'. In fact, they fulfill a range of critical biochemical roles, frequently appearing more versatile as biochemical regulators than their catalytic cousins. The functions of dead enzymes from diverse enzyme families often follow recurring themes, including allosteric regulation of their catalytically active counterparts, acting as signaling scaffolds, or as inhibitors that recognize and sequester the substrates of their catalytic homologs. As well as highlighting the breadth and depth of dead enzyme biology, this Special Issue emphasizes the power of pseudoenzymes as key biochemical regulators in health and disease and potentially as more tractable drug targets than some enzymes themselves. We hope you find these reviews enlivening, and we thank the authors for these excellent contributions.

The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulates a shared host pathway governing viral DNA replication and antiviral defense. While the molecular mechanisms involved are incompletely understood, B1 and B12 seem to intersect with signaling processes mediated by their cellular homologs termed the vaccinia-related kinases (VRKs). In this study, we expand upon our previous characterization of the B1-B12 signaling axis to gain insights into B12 function. We begin our studies by demonstrating that modulation of B12 repressive activity is a conserved function of B1 orthologs from divergent poxviruses. Next, we characterize the protein interactome of B12 using multiple cell lines and expression systems and discover that the cellular kinase VRK1 is a highly enriched B12 interactor. Using complementary VRK1 knockdown and overexpression assays, we first demonstrate that VRK1 is required for the rescue of a B1-deleted virus upon mutation of B12. Second, we find that VRK1 overexpression is sufficient to overcome repressive B12 activity during B1-deleted virus replication. Interestingly, we also evince that B12 interferes with the ability of VRK1 to phosphoinactivate the host defense protein BAF. Thus, B12 restricts vaccinia virus DNA accumulation in part by repressing the ability of VRK1 to inactivate BAF. Finally, these data establish that a B12-VRK1-BAF signaling axis forms during vaccinia virus infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms that poxviruses use to hijack homologous cellular signaling pathways during infection.IMPORTANCE Viruses from diverse families encode both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology. Studies of poxvirus-encoded genes have largely concentrated on positive regulators of their replication; however, examples of fitness gains attributed to poxvirus gene loss suggests that negative regulators of poxvirus replication also impact infection dynamics. This study focuses on the vaccinia B12 pseudokinase, a protein capable of inhibiting vaccinia DNA replication. Here, we elucidate the mechanisms by which B12 inhibits vaccinia DNA replication, demonstrating that B12 activates the antiviral protein BAF by inhibiting the activity of VRK1, a cellular modulator of BAF. Combined with previous data, these studies provide evidence that poxviruses govern their replication by employing both positive and negative regulators of viral replication.

The FEBS Journal, a leading multidisciplinary journal in the life sciences, continues to grow in visibility and impact. Here, the Editor-in-Chief Seamus Martin discusses developments at the journal over the past year and the impact of the COVID-19 crisis on research activities.