dsRNA-induced condensation of antiviral proteins modulates PKR activity.

Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.

Biochemical strategies of E3 ubiquitin ligases target viruses in critical diseases.

Viruses are known to cause various diseases in human and also infect other species such as animal plants, fungi, and bacteria. Replication of viruses depends upon their interaction with hosts. Human cells are prone to such unwanted viral infections. Disintegration and reconstitution require host machinery and various macromolecules like DNA, RNA, and proteins are invaded by viral particles. E3 ubiquitin ligases are known for their specific function, that is, recognition of their respective substrates for intracellular degradation. Still, we do not understand how ubiquitin proteasome system-based enzymes E3 ubiquitin ligases do their functional interaction with different viruses. Whether E3 ubiquitin ligases help in the elimination of viral components or viruses utilize their molecular capabilities in their intracellular propagation is not clear. The first time our current article comprehends fundamental concepts and new insights on the different viruses and their interaction with various E3 Ubiquitin Ligases. In this review, we highlight the molecular pathomechanism of viruses linked with E3 Ubiquitin Ligases dependent mechanisms. An enhanced understanding of E3 Ubiquitin Ligase-mediated removal of viral proteins may open new therapeutic strategies against viral infections.

The PCSK9 discovery, an inactive protease with varied functions in hypercholesterolemia, viral infections, and cancer.

In 2003, the sequences of mammalian proprotein convertase subtilisin/kexin type 9 (PCSK9) were reported. Radiolabeling pulse-chase analyses demonstrated that PCSK9 was synthesized as a precursor (proPCSK9) that undergoes autocatalytic cleavage in the endoplasmic reticulum into PCSK9, which is then secreted as an inactive enzyme in complex with its inhibitory prodomain. Its high mRNA expression in liver hepatocytes and its gene localization on chromosome 1p32, a third locus associated with familial hypercholesterolemia, other than LDLR or APOB, led us to identify three patient families expressing the PCSK9 variants S127R or F216L. Although Pcsk9 and Ldlr were downregulated in mice that were fed a cholesterol-rich diet, PCSK9 overexpression led to the degradation of the LDLR. This led to the demonstration that gain-of-function and loss-of-function variations in PCSK9 modulate its bioactivity, whereby PCSK9 binds the LDLR in a nonenzymatic fashion to induce its degradation in endosomes/lysosomes. PCSK9 was also shown to play major roles in targeting other receptors for degradation, thereby regulating various processes, including hypercholesterolemia and associated atherosclerosis, vascular inflammation, viral infections, and immune checkpoint regulation in cancer. Injectable PCSK9 monoclonal antibody or siRNA is currently used in clinics worldwide to treat hypercholesterolemia and could be combined with current therapies in cancer/metastasis. In this review, we present the critical information that led to the discovery of PCSK9 and its implication in LDL-C metabolism. We further analyze the underlying functional mechanism(s) in the regulation of LDL-C, as well as the evolving novel roles of PCSK9 in both health and disease states.

The Heparanase Regulatory Network in Health and Disease.

The extracellular matrix (ECM) is a structural framework that has many important physiological functions which include maintaining tissue structure and integrity, serving as a barrier to invading pathogens, and acting as a reservoir for bioactive molecules. This cellular scaffold is made up of various types of macromolecules including heparan sulfate proteoglycans (HSPGs). HSPGs comprise a protein core linked to the complex glycosaminoglycan heparan sulfate (HS), the remodeling of which is important for many physiological processes such as wound healing as well as pathological processes including cancer metastasis. Turnover of HS is tightly regulated by a single enzyme capable of cleaving HS side chains: heparanase. Heparanase upregulation has been identified in many inflammatory diseases including atherosclerosis, fibrosis, and cancer, where it has been shown to play multiple roles in processes such as epithelial-mesenchymal transition, angiogenesis, and cancer metastasis. Heparanase expression and activity are tightly regulated. Understanding the regulation of heparanase and its downstream targets is attractive for the development of treatments for these diseases. This review provides a comprehensive overview of the regulators of heparanase as well as the enzyme's downstream gene and protein targets, and implications for the development of new therapeutic strategies.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8+ T cells at the expense of central memory.

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.

PDIA3: Structure, functions and its potential role in viral infections.

The catalysis of disulphide (SS) bonds is the most important characteristic of protein disulphide isomerase (PDI) family. Catalysis occurs in the endoplasmic reticulum, which contains many proteins, most of which are secretory in nature and that have at least one s-s bond. Protein disulphide isomerase A3 (PDIA3) is a member of the PDI family that acts as a chaperone. PDIA3 is highly expressed in response to cellular stress, and also intercept the apoptotic cellular death related to endoplasmic reticulum (ER) stress, and protein misfolding. PDIA3 expression is elevated in almost 70% of cancers and its expression has been linked with overall low cell invasiveness, survival and metastasis. Viral diseases present a significant public health threat. The presence of PDIA3 on the cell surface helps different viruses to enter the cells and also helps in replication. Therefore, inhibitors of PDIA3 have great potential to interfere with viral infections. In this review, we summarize what is known about the basic structure, functions and role of PDIA3 in viral infections. The review will inspire studies of pathogenic mechanisms and drug targeting to counter viral diseases.

Kallikreins emerge as new regulators of viral infections.

Kallikrein-related peptidases (KLKs) or kallikreins have been linked to diverse (patho) physiological processes, such as the epidermal desquamation and inflammation, seminal clot liquefaction, neurodegeneration, and cancer. Recent mounting evidence suggests that KLKs also represent important regulators of viral infections. It is well-established that certain enveloped viruses, including influenza and coronaviruses, require proteolytic processing of their hemagglutinin or spike proteins, respectively, to infect host cells. Similarly, the capsid protein of the non-enveloped papillomavirus L1 should be proteolytically cleaved for viral uncoating. Consequently, extracellular or membrane-bound proteases of the host cells are instrumental for viral infections and represent potential targets for drug development. Here, we summarize how extracellular proteolysis mediated by the kallikreins is implicated in the process of influenza (and potentially coronavirus and papillomavirus) entry into host cells. Besides direct proteolytic activation of viruses, KLK5 and 12 promote viral entry indirectly through proteolytic cascade events, like the activation of thrombolytic enzymes that also can process hemagglutinin, while additional functions of KLKs in infection cannot be excluded. In the light of recent evidence, KLKs represent potential host targets for the development of new antivirals. Humanized animal models to validate their key functions in viral infections will be valuable.

How can a traffic light properly work if it is always green? The paradox of CK2 signaling.

CK2 is a constitutively active protein kinase that assuring a constant level of phosphorylation to its numerous substrates supports many of the most important biological functions. Nevertheless, its activity has to be controlled and adjusted in order to cope with the varying needs of a cell, and several examples of a fine-tune regulation of its activity have been described. More importantly, aberrant regulation of this enzyme may have pathological consequences, e.g. in cancer, chronic inflammation, neurodegeneration, and viral infection. Our review aims at summarizing our current knowledge about CK2 regulation. In the first part, we have considered the most important stimuli shown to affect protein kinase CK2 activity/expression. In the second part, we focus on the molecular mechanisms by which CK2 can be regulated, discussing controversial aspects and future perspectives.

Role of Virally-Encoded Deubiquitinating Enzymes in Regulation of the Virus Life Cycle.

The ubiquitin (Ub) proteasome system (UPS) plays a pivotal role in regulation of numerous cellular processes, including innate and adaptive immune responses that are essential for restriction of the virus life cycle in the infected cells. Deubiquitination by the deubiquitinating enzyme, deubiquitinase (DUB), is a reversible molecular process to remove Ub or Ub chains from the target proteins. Deubiquitination is an integral strategy within the UPS in regulating survival and proliferation of the infecting virus and the virus-invaded cells. Many viruses in the infected cells are reported to encode viral DUB, and these vial DUBs actively disrupt cellular Ub-dependent processes to suppress host antiviral immune response, enhancing virus replication and thus proliferation. This review surveys the types of DUBs encoded by different viruses and their molecular processes for how the infecting viruses take advantage of the DUB system to evade the host immune response and expedite their replication.

ABCE1 Regulates RNase L-Induced Autophagy during Viral Infections.

Host response to a viral infection includes the production of type I interferon (IFN) and the induction of interferon-stimulated genes that have broad antiviral effects. One of the key antiviral effectors is the IFN-inducible oligoadenylate synthetase/ribonuclease L (OAS/RNase L) pathway, which is activated by double-stranded RNA to synthesize unique oligoadenylates, 2-5A, to activate RNase L. RNase L exerts an antiviral effect by cleaving diverse RNA substrates, limiting viral replication; many viruses have evolved mechanisms to counteract the OAS/RNase L pathway. Here, we show that the ATP-binding cassette E1 (ABCE1) transporter, identified as an inhibitor of RNase L, regulates RNase L activity and RNase L-induced autophagy during viral infections. ABCE1 knockdown cells show increased RNase L activity when activated by 2-5A. Compared to parental cells, the autophagy-inducing activity of RNase L in ABCE1-depleted cells is enhanced with early onset. RNase L activation in ABCE1-depleted cells inhibits cellular proliferation and sensitizes cells to apoptosis. Increased activity of caspase-3 causes premature cleavage of autophagy protein, Beclin-1, promoting a switch from autophagy to apoptosis. ABCE1 regulates autophagy during EMCV infection, and enhanced autophagy in ABCE1 knockdown cells promotes EMCV replication. We identify ABCE1 as a host protein that inhibits the OAS/RNase L pathway by regulating RNase L activity, potentially affecting antiviral effects.

Cyclin-dependent Kinases as Emerging Targets for Developing Novel Antiviral Therapeutics.

Besides its prominent role in cell proliferation, cyclin-dependent kinases (CDKs) are key players in viral infections as both DNA and RNA viruses modify CDK function to favor viral replication. Recently, a number of specific pharmacological CDK inhibitors have been developed and approved for cancer treatment. The repurposing of these specific CDK inhibitors for the treatment of viral infections may represent a novel effective therapeutic strategy to combat old and emergent viruses. In this review, we describe the role, mechanisms of action, and potential of CDKs as antiviral drug targets. We also discuss the current clinical state of novel specific CDK inhibitors, focusing on their putative use as antivirals, especially against new emerging viruses.

The emerging roles of the MARCH ligases in antiviral innate immunity.

Membrane-associated RING (really interesting new gene)-cysteine-histidine (CH) (MARCH) ubiquitin ligases belong to a RING finger domain E3 ligases family. So far, eleven members have been found in the MARCH family, which are MARCH 1 to 11. The members of the MARCH family are widely distributed and involve in a variety of cellular functions, including regulation of the immune system, transmembrane transport of proteins, protein stability, endoplasmic reticulum-related degradation, and endosome protein transport. Several seminal studies over the past decade have delineated that MARCH affects viral replication through various mechanisms by regulating the activity of signaling molecules and their expression in the antiviral innate immune responses. Here, we summarize the complex roles of MARCH ligases in the antiviral innate immune signaling pathway and its impact on viral replication in host immune defense systems. A better understanding of this interplay's molecular mechanisms is important concerning the development of new therapeutics targeting viral infections.

When PARPs Meet Antiviral Innate Immunity.

The poly(ADP-ribose) polymerases (PARPs) family contains 17 members in humans, sharing a PARP domain to transfer ADP-ribose groups to target proteins to trigger ADP-ribosylation. The roles of PARPs have evolved from DNA damage repair to diverse biological processes, such as gene transcription, cellular stress response, etc. Recently, seminal studies have demonstrated the critical roles of PAPRs in antiviral innate immunity. PARPs catalyze ADP-ribosylation, a fundamental post-translational modification, using NAD+ as a substrate. ADP-ribosylation can occur either as mono- or poly-(ADP-ribosyl)ation, which is initially linked to DNA damage repair, as exemplified by PARP1. Recent advances in host antiviral immunity demonstrated that several PARPs, such as PARP9, 11, 12, 13, 14, etc., have broad-spectrum antiviral activities that are independent of their ADP-ribosylation.

Drugging the Phosphoinositide 3-Kinase (PI3K) and Phosphatidylinositol 4-Kinase (PI4K) Family of Enzymes for Treatment of Cancer, Immune Disorders, and Viral/Parasitic Infections.

The lipid kinases that generate the lipid signalling phosphoinositides have been established as fundamental signalling enzymes that control numerous aspects of how cells respond to their extracellular environment. In addition, they play critical roles in regulating membrane trafficking and lipid transport within the cell. The class I phosphoinositide kinases which generate the critical lipid signal PIP3 are hyperactivated in numerous human pathologies including cancer, overgrowth syndromes, and primary immunodeficiencies. The type III phosphatidylinositol 4-kinase beta isoform (PI4KB), which are evolutionarily similar to the class I PI3Ks, have been found to be essential host factors mediating the replication of numerous devastating pathogenic viruses. Finally, targeting the parasite variant of PI4KB has been established as one of the most promising strategies for the development of anti-malarial and anti-cryptosporidium strategies. Therefore, the development of targeted isoform selective inhibitors for these enzymes are of paramount importance. The first generation of PI3K inhibitors have recently been clinically approved for a number of different cancers, highlighting their therapeutic value. This review will examine the history of the class I PI3Ks, and the type III PI4Ks, their relevance to human disease, and the structural basis for their regulation and inhibition by potent and selective inhibitors.

Expression, purification and crystallization of CLK1 kinase - A potential target for antiviral therapy.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

SnapShot: Enveloped Virus Entry.

In order to initiate successful infection, viruses have to transmit and deliver their genome from one host cell or organism to another. To achieve this, enveloped viruses must first fuse their membrane with those of the target host cell. Here, we describe the sequence of events leading to the entry of representative enveloped viruses, highlighting the strategies they use to gain access to the host cell cytosol.

Inhibitors of DNA Glycosylases as Prospective Drugs.

DNA glycosylases are enzymes that initiate the base excision repair pathway, a major biochemical process that protects the genomes of all living organisms from intrinsically and environmentally inflicted damage. Recently, base excision repair inhibition proved to be a viable strategy for the therapy of tumors that have lost alternative repair pathways, such as BRCA-deficient cancers sensitive to poly(ADP-ribose)polymerase inhibition. However, drugs targeting DNA glycosylases are still in development and so far have not advanced to clinical trials. In this review, we cover the attempts to validate DNA glycosylases as suitable targets for inhibition in the pharmacological treatment of cancer, neurodegenerative diseases, chronic inflammation, bacterial and viral infections. We discuss the glycosylase inhibitors described so far and survey the advances in the assays for DNA glycosylase reactions that may be used to screen pharmacological libraries for new active compounds.

Heparanase, Heparan Sulfate and Viral Infection.

The story of heparanase (HPSE) in viral infection has roots in the longstanding connection between heparan sulfate (HS) and a large number of viruses. As a major viral attachment and entry receptor present on the cell surface, HS serves as the first point of contact between a virus particle and its target host cell. Likewise, direct regulation of HS levels on the cell surface by HPSE enzymatic activity dictates the extent of virus release after replication has occurred. Additionally, virus-induced HPSE activation and nuclear translocation results in higher expression of pro-inflammatory factors and delayed wound healing leading to worsened disease. In this chapter, using herpes simplex virus (HSV) as a prototype virus we provide a brief synopsis of important stages in viral infection, describe how these processes are governed by HS and HPSE, and discuss the recent discoveries that designate HPSE as a major host virulence factor and driver of pathogenesis for several different viruses.

Inhibition of topoisomerase IIA (Top2α) induces telomeric DNA damage and T cell dysfunction during chronic viral infection.

T cells play a critical role in controlling viral infection; however, the mechanisms regulating their responses remain incompletely understood. Here, we investigated the role of topoisomerase IIA (Top2α, an enzyme that is essential in resolving entangled DNA strands during replication) in telomeric DNA damage and T cell dysfunction during viral infection. We demonstrated that T cells derived from patients with chronic viral (HBV, HCV, and HIV) infection had lower Top2α protein levels and enzymatic activity, along with an accumulation of the Top2α cleavage complex (Top2cc) in genomic DNA. In addition, T cells from virally infected subjects with lower Top2α levels were vulnerable to Top2α inhibitor-induced cell apoptosis, indicating an important role for Top2α in preventing DNA topological disruption and cell death. Using Top2α inhibitor (ICRF193 or Etoposide)-treated primary T cells as a model, we demonstrated that disrupting the DNA topology promoted DNA damage and T cell apoptosis via Top2cc accumulation that is associated with protein-DNA breaks (PDB) at genomic DNA. Disruption of the DNA topology was likely due to diminished expression of tyrosyl-DNA phosphodiesterase 2 (TDP2), which was inhibited in T cells in vitro by Top2α inhibitor and in vivo by chronic viral infection. These results suggest that immune-evasive viruses (HBV, HCV, and HIV) can disrupt T cell DNA topology as a mechanism of dysregulating host immunity and establishing chronic infection. Thus, restoring the DNA topologic machinery may serve as a novel strategy to protect T cells from unwanted DNA damage and to maintain immune competence.

DEAD-Box Helicases: Sensors, Regulators, and Effectors for Antiviral Defense.

DEAD-box helicases are a large family of conserved RNA-binding proteins that belong to the broader group of cellular DExD/H helicases. Members of the DEAD-box helicase family have roles throughout cellular RNA metabolism from biogenesis to decay. Moreover, there is emerging evidence that cellular RNA helicases, including DEAD-box helicases, play roles in the recognition of foreign nucleic acids and the modulation of viral infection. As intracellular parasites, viruses must evade detection by innate immune sensing mechanisms and degradation by cellular machinery while also manipulating host cell processes to facilitate replication. The ability of DEAD-box helicases to recognize RNA in a sequence-independent manner, as well as the breadth of cellular functions carried out by members of this family, lead them to influence innate recognition and viral infections in multiple ways. Indeed, DEAD-box helicases have been shown to contribute to intracellular immune sensing, act as antiviral effectors, and even to be coopted by viruses to promote their replication. However, our understanding of the mechanisms underlying these interactions, as well as the cellular roles of DEAD-box helicases themselves, is limited in many cases. We will discuss the diverse roles that members of the DEAD-box helicase family play during viral infections.