In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection.

Zika virus (ZIKV) infection may lead to severe neurological consequences, including seizures, and early infancy death. However, the involved mechanisms are still largely unknown. TRPC channels play an important role in regulating nervous system excitability and are implicated in seizure development. We investigated whether TRPCs might be involved in the pathogenesis of ZIKV infection. We found that ZIKV infection increases TRPC4 expression in host cells via the interaction between the ZIKV-NS3 protein and CaMKII, enhancing TRPC4-mediated calcium influx. Pharmacological inhibition of CaMKII decreased both pCREB and TRPC4 protein levels, whereas the suppression of either TRPC4 or CaMKII improved the survival rate of ZIKV-infected cells and reduced viral protein production, likely by impeding the replication phase of the viral life cycle. TRPC4 or CaMKII inhibitors also reduced seizures and increased the survival of ZIKV-infected neonatal mice and blocked the spread of ZIKV in brain organoids derived from human-induced pluripotent stem cells. These findings suggest that targeting CaMKII or TRPC4 may offer a promising approach for developing novel anti-ZIKV therapies, capable of preventing ZIKV-associated seizures and death.

SARS-CoV-2 ORF3a Protein as a Therapeutic Target against COVID-19 and Long-Term Post-Infection Effects.

The COVID-19 pandemic caused by SARS-CoV-2 has posed unparalleled challenges due to its rapid transmission, ability to mutate, high mortality and morbidity, and enduring health complications. Vaccines have exhibited effectiveness, but their efficacy diminishes over time while new variants continue to emerge. Antiviral medications offer a viable alternative, but their success has been inconsistent. Therefore, there remains an ongoing need to identify innovative antiviral drugs for treating COVID-19 and its post-infection complications. The ORF3a (open reading frame 3a) protein found in SARS-CoV-2, represents a promising target for antiviral treatment due to its multifaceted role in viral pathogenesis, cytokine storms, disease severity, and mortality. ORF3a contributes significantly to viral pathogenesis by facilitating viral assembly and release, essential processes in the viral life cycle, while also suppressing the body's antiviral responses, thus aiding viral replication. ORF3a also has been implicated in triggering excessive inflammation, characterized by NF-κB-mediated cytokine production, ultimately leading to apoptotic cell death and tissue damage in the lungs, kidneys, and the central nervous system. Additionally, ORF3a triggers the activation of the NLRP3 inflammasome, inciting a cytokine storm, which is a major contributor to the severity of the disease and subsequent mortality. As with the spike protein, ORF3a also undergoes mutations, and certain mutant variants correlate with heightened disease severity in COVID-19. These mutations may influence viral replication and host cellular inflammatory responses. While establishing a direct link between ORF3a and mortality is difficult, its involvement in promoting inflammation and exacerbating disease severity likely contributes to higher mortality rates in severe COVID-19 cases. This review offers a comprehensive and detailed exploration of ORF3a's potential as an innovative antiviral drug target. Additionally, we outline potential strategies for discovering and developing ORF3a inhibitor drugs to counteract its harmful effects, alleviate tissue damage, and reduce the severity of COVID-19 and its lingering complications.

Cell-specific genome-scale metabolic modeling of SARS-CoV-2-infected lung to identify antiviral enzymes.

Computational systems biology plays a key role in the discovery of suitable antiviral targets. We designed a cell-specific, constraint-based modeling technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected lungs. We used the gene sequence of the alpha variant of SARS-CoV-2 to build a viral biomass reaction (VBR). We also used the mass proportion of lipids between the viral biomass and its host cell to estimate the stoichiometric coefficients of viral lipids in the reaction. We then integrated the VBR, the gene expression of the alpha variant of SARS-CoV-2, and the generic human metabolic network Recon3D to reconstruct a cell-specific genome-scale metabolic model. An antiviral target discovery (AVTD) platform was introduced using this model to identify therapeutic drug targets for combating COVID-19. The AVTD platform not only identified antiviral genes for eliminating viral replication but also predicted side effects of treatments. Our computational results revealed that knocking out dihydroorotate dehydrogenase (DHODH) might reduce the synthesis rate of cytidine-5'-triphosphate and uridine-5'-triphosphate, which terminate the viral building blocks of DNA and RNA for SARS-CoV-2 replication. Our results also indicated that DHODH is a promising antiviral target that causes minor side effects, which is consistent with the results of recent reports. Moreover, we discovered that the genes that participate in the de novo biosynthesis of glycerophospholipids and ceramides become unidentifiable if the VBR does not involve the stoichiometry of lipids.

miRNAs in Herpesvirus Infection: Powerful Regulators in Small Packages.

microRNAs are a class of small, single-stranded, noncoding RNAs that regulate gene expression. They can be significantly dysregulated upon exposure to any infection, serving as important biomarkers and therapeutic targets. Numerous human DNA viruses, along with several herpesviruses, have been found to encode and express functional viral microRNAs known as vmiRNAs, which can play a vital role in host-pathogen interactions by controlling the viral life cycle and altering host biological pathways. Viruses have also adopted a variety of strategies to prevent being targeted by cellular miRNAs. Cellular miRNAs can act as anti- or proviral components, and their dysregulation occurs during a wide range of infections, including herpesvirus infection. This demonstrates the significance of miRNAs in host herpesvirus infection. The current state of knowledge regarding microRNAs and their role in the different stages of herpes virus infection are discussed in this review. It also delineates the therapeutic and biomarker potential of these microRNAs in future research directions.

Development and therapeutic potential of adaptor-associated kinase 1 inhibitors in human multifaceted diseases.

Adaptor-Associated Kinase 1 (AAK1), a Ser/Thr protein kinase, responsible for regulating clathrin-mediated endocytosis, is ubiquitous in the central nervous system (CNS). AAK1 plays an important role in neuropathic pain and a variety of other human diseases, including viral invasion, Alzheimer's disease, Parkinson's syndrome, etc. Therefore, targeting AAK1 is a promising therapeutic strategy. However, although small molecule AAK1 inhibitors have been vigorously developed, only BMS-986176/LX-9211 has entered clinical trials. Simultaneously, new small molecule inhibitors, including BMS-911172 and LP-935509, exhibited excellent druggability. This review elaborates on the structure, biological function, and disease relevance of AAK1. We emphatically analyze the structure-activity relationships (SARs) of small molecule AAK1 inhibitors based on different binding modalities and discuss prospective strategies to provide insights into novel AAK1 therapeutic agents for clinical practice.

Conserved requirement of autophagy-related effectors during coronavirus replication.

The recurrence of zoonotic transmission events highlights the need for novel treatment strategies against emerging coronaviruses (CoVs), namely SARS-CoV, MERS-CoV and most notably SARS-CoV-2. Our recently performed genome-wide CRISPR knockout screen revealed a list of conserved pan-coronavirus as well as MERS-CoV or HCoV-229E-specific host dependency factors (HDF) essential during the viral life cycle. Intriguingly, we identified the macroautophagy/autophagy pathway-regulating immunophilins FKBP8, TMEM41B, and MINAR1 as conserved MERS-CoV, HCoV-229E, SARS-CoV, and SARS-CoV-2 host factors, which further constitute potential targets for therapeutic intervention by clinically approved drugs.

'Shared-Hook' and 'Changed-Hook' Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis.

Herpesviruses replicate their genomes and assemble their capsids in the host cell nucleus. To progress towards morphogenesis in the cytoplasm, herpesviruses evolved the strategy of nuclear egress as a highly regulated process of nucleo-cytoplasmic capsid transition. The process is conserved among α-, ß- and γ-herpesviruses and involves the formation of a core and multicomponent nuclear egress complex (NEC). Core NEC is assembled by the interaction between the nucleoplasmic hook protein, i.e., pUL53 (human cytomegalovirus, HCMV), and the integral membrane-associated groove protein, i.e., pUL50. Our study aimed at the question of whether a panherpesviral NEC scaffold may enable hook-into-groove interaction across herpesviral subfamilies. For this purpose, NEC constructs were generated for members of all three subfamilies and analyzed for multi-ligand interaction using a yeast two-hybrid (Y2H) approach with randomized pUL53 mutagenesis libraries. The screening identified ten library clones displaying cross-viral shared hook-into-groove interaction. Interestingly, a slightly modified Y2H screening strategy provided thirteen further changed-hook pUL53 clones having lost parental pUL50 interaction but gained homolog interaction. In addition, we designed a sequence-predicted hybrid construct based on HCMV and Epstein-Barr virus (EBV) core NEC proteins and identified a cross-viral interaction phenotype. Confirmation was provided by applying protein-protein interaction analyses in human cells, such as coimmunoprecipitation settings, confocal nuclear rim colocalization assays, and HCMV ΔUL53 infection experiments with pUL53-complementing cells. Combined, the study provided the first examples of cross-viral NEC interaction patterns and revealed a higher yield of human cell-confirmed binding clones using a library exchange rate of 3.4 than 2.7. Thus, the study provides improved insights into herpesviral NEC protein binding specificities of core NEC formation. This novel information might be exploited to gain a potential target scaffold for the development of broadly acting NEC-directed inhibitory small molecules.

DDX5: an expectable treater for viral infection- a literature review.

Background and Objective: DEAD-box protein (DDX)5 plays important roles in multiple aspects of cellular processes that require modulating RNA structure. Alongside the canonical role of DDX5 in RNA metabolism, many reports have shown that DDX5 influences viral infection by directly interacting with viral proteins. However, the functional role of DDX5 in virus-associated cancers, as well as the identity of DDX5 in virus infection-associated signaling pathways, has remained largely unexplained. Here, we further explore the precise functions of DDX5 and its potential targets for antiviral treatment. Methods: We searched the PubMed and PMC databases to identify studies on role of DDXs, especially DDX5, during various viral infection published up to May 2022. Key Content and Findings: DDX5 functions as both a viral infection helper and inhibitor, which depends on virus type. DDXs proteins have been identified to play roles on multiple aspects covering RNA metabolism and function. Conclusions: DDX5 influences viral pathogenesis by participating in viral replication and multiple viral infection-related signaling pathways, it also plays a double-edge sword role under different viral infection conditions. Deep investigation into the mechanism of DDX5 modulating immune response in host cells revealed that it holds highly potential usage for future antiviral therapy. We reviewed current studies to provide a comprehensive update of the role of DDX5 in viral infection.

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors.

The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.

Channel catfish virus entry into host cells via clathrin-mediated endocytosis.

Channel catfish virus (CCV), an important member of the family Alloherpesviridae, causes a lethal infection in channel catfish. As with most animal viruses, the initial step of infection by CCV is entry into host cells, which is also a promising antiviral target for CCV disease. This study investigated the mechanism of host cell invasion by CCV using a series of biochemical inhibitor assays in channel catfish cells. CCV infection in host cells was does-dependently inhibited when cells were treated with endosomal acidification inhibitors (5 µM chloroquine, 50 nM bafilomycin A1, and 1 mM ammonium chloride) and hypertonic medium (50 mM sucrose) , which suggests that CCV invades host cells in a manner dependent on low-pH and the endocytic pathway. Moreover, when the cells were pretreated with inhibitors of clathrin-mediated endocytosis, including chlorpromazine (2 µM) and dynasore (50 µM), the CCV infection in the host cells was strongly inhibited. In contrast, the destruction of cellular cholesterol by methyl-ß-cyclodextrin and nystatin and inhibition of macropinocytosis had no effect on viral entry. Altogether, these findings indicate that CCV infects host cells via clathrin-mediated endocytosis in a low-pH-dependent manner, suggesting that this CCV entry pathway offers an antiviral target against CCV disease.

p38 activation and viral infection.

Viruses completely rely on the energy and metabolic systems of host cells for life activities. Viral infections usually lead to cytopathic effects and host diseases. To date, there are still no specific clinical vaccines or drugs against most viral infections. Therefore, understanding the molecular and cellular mechanisms of viral infections is of great significance to prevent and treat viral diseases. A variety of viral infections are related to the p38 MAPK signalling pathway, and p38 is an important host factor in virus-infected cells. Here, we introduce the different signalling pathways of p38 activation and then summarise how different viruses induce p38 phosphorylation. Finally, we provide a general summary of the effect of p38 activation on virus replication. Our review provides integrated data on p38 activation and viral infections and describes the potential application of targeting p38 as an antiviral strategy.

PI3K/AKT/p53 pathway inhibits infectious spleen and kidney necrosis virus infection by regulating autophagy and immune responses.

The PI3K/AKT/p53 signaling pathway is activated by various types of cellular stimuli or pathogenic infection, and then regulates fundamental cellular functions to combat these stimulations. Here, we studied the meaningful roles of PI3K/AKT/p53 in regulating cellular machine such as autophagy, immune responses, as well as antiviral activity in Chinese perch brain (CPB) cells infected by infectious spleen and kidney necrosis virus (ISKNV), which is an agent caused devastating losses in mandarin fish (Siniperca chuatsi) industry. We found that ISKNV infection induced up-regulation of host PI3K/AKT/p53 axis, but inhibited autophagy in CPB cells. Interestingly, activation of PI3K/AKT/p53 axis factors trough agonists or overexpression dramatically decreased host autophagy level, inhibited ISKNV replication, and elevated the expression of immune-related genes in CPB cells. In contrast, suppression of PI3K/AKT/p53 pathway by inhibitors or small interfering RNA (siRNA)-mediated gene silence increased the autophagy and ISKNV replication, but down-regulated immune responses in CPB cells. All these results indicate that PI3K/AKT/p53 pathway plays an important role in anti-ISKNV infection and can be used as a new target for controlling ISKNV disease.

NS1 protein as a novel anti-influenza target: Map-and-mutate antiviral rationale reveals new putative druggable hot spots with an important role on viral replication.

Influenza NS1 is a promising anti-influenza target, considering its conserved and druggable structure, and key function in influenza replication and pathogenesis. Notwithstanding, target identification and validation, strengthened by experimental data, are lacking. Here, we further explored our previously designed structure-based antiviral rationale directed to highly conserved druggable NS1 regions across a broad spectrum of influenza A viruses. We aimed to identify NS1-mutated viruses exhibiting a reduced growth phenotype and/or an altered cell apoptosis profile. We found that NS1 mutations Y171A, K175A (consensus druggable pocket 1), W102A (consensus druggable pocket 3), Q121A and G184P (multiple consensus druggable pockets) - located at hot spots amenable for pharmacological modulation - significantly impaired A(H1N1)pdm09 virus replication, in vitro. This is the first time that NS1-K175A, -W102A, and -Q121A mutations are characterized. Our map-and-mutate strategy provides the basis to establish the NS1 as a promising target using a rationale with a higher resilience to resistance development.

Genome-scale metabolic modeling reveals SARS-CoV-2-induced metabolic changes and antiviral targets.

Tremendous progress has been made to control the COVID-19 pandemic caused by the SARS-CoV-2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS-CoV-2 infection using genome-scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS-CoV-2 infection. We next applied the GEM-based metabolic transformation algorithm to predict anti-SARS-CoV-2 targets that counteract the virus-induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco-2 cells. Further generating and analyzing RNA-sequencing data of remdesivir-treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti-SARS-CoV-2 drug. Our study provides clinical data-supported candidate anti-SARS-CoV-2 targets for future evaluation, demonstrating host metabolism targeting as a promising antiviral strategy.

RNA Helicase DDX3: A Double-Edged Sword for Viral Replication and Immune Signaling.

DDX3 is a cellular ATP-dependent RNA helicase involved in different aspects of RNA metabolism ranging from transcription to translation and therefore, DDX3 participates in the regulation of key cellular processes including cell cycle progression, apoptosis, cancer and the antiviral immune response leading to type-I interferon production. DDX3 has also been described as an essential cellular factor for the replication of different viruses, including important human threats such HIV-1 or HCV, and different small molecules targeting DDX3 activity have been developed. Indeed, increasing evidence suggests that DDX3 can be considered not only a promising but also a viable target for anticancer and antiviral treatments. In this review, we summarize distinct functional aspects of DDX3 focusing on its participation as a double-edged sword in the host immune response and in the replication cycle of different viruses.

Matrix metalloproteinase 14 regulates HSV-1 infection in neuroblastoma cells.

Growing evidence supports that chronic or latent infection of the central nervous system might be implicated in Alzheimer's disease (AD). Among them, Herpes simplex virus type 1 (HSV-1) has emerged as a major factor in the etiology of the disease. Our group is devoted to the study of the relationship among HSV-1, oxidative stress (OS) and neurodegeneration. We have found that HSV-1 induces the main neuropathological hallmarks of AD, including the accumulation of intracellular amyloid beta (Aß), hyperphosphorylated tau protein and autophagic vesicles, that OS exacerbates these effects, and that matrix metalloproteinase 14 (MMP-14) participates in the alterations induced by OS. In this work, we focused on the role of MMP-14 in the degenerative markers raised by HSV-1 infection. Interestingly, we found that MMP-14 blockage is a potent inhibitor of HSV-1 infection efficiency, that also reduces the degeneration markers, accumulation of Aß and hyperphosphorylated tau, induced by the virus. Our results point to MMP-14 as a potent antiviral target to control HSV-1 infection and its associated neurodegenerative effects.

Anti-Classical Swine Fever Virus Strategies.

Classical swine fever (CSF), caused by CSF virus (CSFV), is a highly contagious swine disease with high morbidity and mortality, which has caused significant economic losses to the pig industry worldwide. Biosecurity measures and vaccination are the main methods for prevention and control of CSF since no specific drug is available for the effective treatment of CSF. Although a series of biosecurity and vaccination strategies have been developed to curb the outbreak events, it is still difficult to eliminate CSF in CSF-endemic and re-emerging areas. Thus, in addition to implementing enhanced biosecurity measures and exploring more effective CSF vaccines, other strategies are also needed for effectively controlling CSF. Currently, more and more research about anti-CSFV strategies was carried out by scientists, because of the great prospects and value of anti-CSFV strategies in the prevention and control of CSF. Additionally, studies on anti-CSFV strategies could be used as a reference for other viruses in the Flaviviridae family, such as hepatitis C virus, dengue virus, and Zika virus. In this review, we aim to summarize the research on anti-CSFV strategies. In detail, host proteins affecting CSFV replication, drug candidates with anti-CSFV effects, and RNA interference (RNAi) targeting CSFV viral genes were mentioned and the possible mechanisms related to anti-CSFV effects were also summarized.

Comprehensive Analysis of G-Quadruplexes in African Swine Fever Virus Genome Reveals Potential Antiviral Targets by G-Quadruplex Stabilizers.

African Swine Fever Virus (ASFV), a lethal hemorrhagic fever of the swine, poses a major threat to the world's swine population and has so far resulted in devastating socio-economic consequences. The situation is further compounded by the lack of an approved vaccine or antiviral drug. Herein, we investigated a novel anti-ASFV approach by targeting G-Quadruplexes (G4s) in the viral genome. Bioinformatics analysis of putative G-quadruplex-forming sequences (PQSs) in the genome of ASFV BA71V strain revealed 317 PQSs on the forward strand and 322 PQSs on the reverse strand of the viral genome, translating to a density of 3.82 PQSs/kb covering 9.52% of the entire genome, which means that 85% of genes in the ASFV genome have at least 1 PQS on either strand. Biochemical characterization showed that 8 out of 13 conserved PQSs could form stable G4s in the presence of K+, and 4 of them could be stabilized by G4 ligands, N-Methyl Mesoporphyrin (NMM), and pyridostatin (PDS) in vitro. An enhanced green fluorescent protein (EGFP)-based reporter system revealed that the expression of two G4-containing genes, i.e., P1192R and D117L, could be significantly suppressed by NMM and PDS in 293T cells. In addition, a virus infection model showed that NMM could inhibit the replication of ASFV in Porcine Alveolar Macrophages (PAM) cells with an EC50 value of 1.16 µM. Altogether, the present study showed that functional PQSs existent in the promoters, CDS, 3' and 5' UTRs of the ASFV genome could be stabilized by G4 ligands, such as NMM and PDS, and could serve as potential targets for antivirals.

Selective regulation in ribosome biogenesis and protein production for efficient viral translation.

As intracellular parasites, viruses depend heavily on host cell structures and their functions to complete their life cycle and produce new viral particles. Viruses utilize or modulate cellular translational machinery to achieve efficient replication; the role of ribosome biogenesis and protein synthesis in viral replication particularly highlights the importance of the ribosome quantity and/or quality in controlling viral protein synthesis. Recently reported studies have demonstrated that ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs) act as multifaceted regulators in selective translation of viral transcripts. Here we summarize the recent literature on RBFs and RPs and their association with subcellular redistribution, post-translational modification, enzyme catalysis, and direct interaction with viral proteins. The advances described in this literature establish a rationale for targeting ribosome production and function in the design of the next generation of antiviral agents.

SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA Helicase Activities That Can Be Inhibited by Bismuth Salts.

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.