Porcine epidemic diarrhoea virus (PEDV) is a highly pathogenic porcine enteric coronavirus that causes severe watery diarrhoea and mortality in piglets. The nucleocapsid protein (N) is the most abundant viral protein and is highly phosphorylated, with the phosphorylation level directly affecting infection and proliferation. Here, we characterized the phosphorylation level of the N protein and found that its SR (Ser and Arg) motif was highly phosphorylated. The phosphorylation level significantly decreased after mutation of threonine (Thr) to serine (Ser). Through screening, it was determined that GSK3α/β plays a major role in phosphorylating the SR motif. Using GSK3α/β inhibitors or directly knocking out the GSK3α/β gene significantly inhibit PEDV proliferation. Finally, we used yeast recombination technology to develop a reverse genetics system for assessing PEDV and confirmed that no differences existed between the wild-type strain and the rescued virus. Using this platform, we generated a PEDV N protein SR motif mutant strain. We found that, compared to the wild-type strain, the proliferation of the mutant strain was significantly weakened, confirming that the phosphorylation of the SR motif is crucial for PEDV proliferation. In summary, we verified the phosphorylation sites of the PEDV N protein and the associated protein kinases, providing new insights into the development of relevant therapeutic strategies.

Replication of many viruses depends on phosphorylation of viral proteins by host protein kinases and subsequent recruitment of host protein partners. The nucleoprotein (N) of SARS-CoV-2 is heavily phosphorylated and recruits human phosphopeptide-binding 14-3-3 proteins early in infection, which is reversed prior to nucleocapsid assembly in new virions. Among the multiple phosphosites of N, which are particularly dense in the serine/arginine-rich interdomain region, phospho-Thr205 is highly relevant for 14-3-3 recruitment by SARS-CoV-2 N. The context of this site is mutated in most SARS-CoV-2 variants of concern. Among mutations that increase infectious virus titers, the S202R mutation (B.1.526 Iota) causes a striking replication boost (∼166-fold), although its molecular consequences have remained unclear. Here, we show that the S202R-mutated N phosphopeptide exhibits a 5-fold higher affinity for human 14-3-3ζ than the Wuhan variant and we rationalize this effect by solving a high-resolution crystal structure of the complex. The structure revealed an enhanced 14-3-3/N interface contributed by the Arg202 side chain that, in contrast to Ser202, formed multiple stabilizing contacts with 14-3-3, including water-mediated H-bonds and guanidinium pi-pi stacking. These findings provide a compelling link between the replicative fitness of SARS-CoV-2 and the N protein's affinity for host 14-3-3 proteins.

The Global pandemic of coronavirus disease 2019 was initiated by the emergence of severe acute respiratory syndrome coronavirus 2. In addition to conventional pulmonary lesions, a range of neurological injury symptoms have been identified in clinical practice, but the aetiology of neurological disorders linked to SARS-CoV-2 infection remains poorly understood. Syrian hamsters, which are highly susceptible to SARS-CoV-2 infection, exhibit a disease phenotype similar to that observed in human COVID-19 patients. In this study, a hamster model of COVID-19 infection was used to analyze molecular changes in different tissues at various time points post infection with distinct strains using proteomic and phosphoproteomic approaches. Multi-omics analysis showed that SARS-COV-2 infection triggers sustained downregulation of the abundance and phosphorylation levels of neuronal and synapse-associated proteins in the brain, suggesting that neuronal damage persists even during the recovery period. Additionally, infections with SARS-CoV-2 may contribute to the onset of long-term symptoms of COVID-19 by impacting energy metabolism, neurotransmitter release, and synaptic transmission pathways. This study provides a comprehensive molecular profile of hamsters infected with different SARS-CoV-2 strains in different tissues, offering foundational insights into the pathogenic mechanisms of COVID-19.

COVID-19, caused by the SARS-CoV-2, poses significant global health challenges. A key player in its pathogenesis is the nucleocapsid protein (NP), which is crucial for viral replication and assembly. While NPs from other coronaviruses, such as SARS-CoV and MERS-CoV, are known to increase inflammation and cause acute lung injury, the specific effects of the SARS-CoV-2 NP on host cells remain largely unexplored. Recent findings suggest that the NP acts as a pathogen-associated molecular pattern (PAMP) that binds to Toll-like receptor 2 (TLR2), activating NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and MAPK (mitogen-activated protein kinase) signaling pathways. This activation is particularly pronounced in severe COVID-19 cases, leading to elevated levels of soluble ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), which contribute to endothelial dysfunction and multiorgan damage. Furthermore, the NP is implicated in hyperinflammation and thrombosis-key factors in COVID-19 severity and long COVID. Its potential to bind with MASP-2 (mannan-binding lectin serine protease 2) may also be linked to persistent symptoms in long COVID patients. Understanding these mechanisms, particularly the role of the NP in thrombosis, is essential for developing targeted therapies to manage both acute and chronic effects of COVID-19 effectively. This comprehensive review aims to elucidate the multifaceted roles of the NP, highlighting its contributions to viral pathogenesis, immune evasion, and the exacerbation of thrombotic events, thereby providing insights into potential therapeutic targets for mitigating the severe and long-term impacts of COVID-19.

Cyprinid herpesvirus 2 (CyHV-2) is the main pathogen responsible for the development of herpesviral hematopoietic necrosis disease (HVHND) in crucian carp (Carassius auratus). The CyHV-2 genome encodes approximately 150 genes that are expressed in a well-defined manner during productive infection. However, CyHV-2 open reading frames (ORFs) are primarily derived from sequence and homology analyses, and most lack protein-level evidence to support their properties. In this study, we used high-resolution mass spectrometry followed by proteogenomic mapping to achieve genome re-annotation of CyHV-2. Based on our results, a total of 1,683 MS/MS spectra could be mapped to the CyHV-2 genome through six-frame translation, with 1,665 corresponding to 117 currently annotated protein-coding ORFs. Three of the remaining 18 peptides were mapped to the N-terminal extension region of known ORFs. However, 12 novel CyHV-2 ORFs, designated nORF1-12, were identified and characterized for the first time based on the remaining 15 peptides that could be mapped to previously unannotated regions of the viral genome. And the sequence differences of the novel phosphorylated nORF1, also referred to as ORF25E, in different CyHV-2 strains indicated that the nORF1 is a prospective molecular marker that can monitor the evolution from the Japan (J) to the China (C) genotype of CyHV-2. These findings further validate existing annotations, expand the genomic landscape of CyHV-2, and provide a rich resource for aquatic virology research.IMPORTANCECyHV-2 is a viral pathogen that poses a significant threat to crucian carp farming. CyHV-2 has a large genome with complex sequence features and diverse coding mechanisms, which complicates accurate genome annotation in the absence of protein-level evidence. Here, we employed various protein extraction and separation methods to increase viral protein coverage and performed an integrated proteogenomic analysis to refine the CyHV-2 genome annotation. A total of 129 viral genes were confidently identified, including 117 currently annotated genes and 12 novel genes. For the first time, we present large-scale evidence of peptide presence and levels in the genome of aquatic viruses and confirm the majority of the predicted proteins in CyHV-2. Our findings enhance the understanding of the CyHV-2 genome structure and provide valuable insights for future studies on CyHV-2 biology.

The global SARS-CoV-2 pandemic emphasized the need for accurate pathogen diagnostics. While genomics is the gold standard, integrating mass spectrometry-based proteomics offers additional benefits. However, current proteomic and genomic reference databases are often biased toward specific taxa, such as pathogenic strains or model organisms, and proteomic databases are less comprehensive. These biases and gaps can lead to inaccurate identifications. To address these issues, we introduce MultiStageSearch, a multistep database search method that combines proteome and genome databases for taxonomic analysis. Initially, a generalist proteome database is used to infer potential species. Then, MultiStageSearch generates a specialized proteogenomic database for precise identification. This database is preprocessed to filter duplicates and cluster identical open reading frames to reduce genomic database biases. The workflow operates independently of strain-level NCBI taxonomy, enabling the identification of strains not represented in existing taxonomies. We benchmarked the workflow on viral and bacterial samples, demonstrating its superior performance in strain-level taxonomic inference compared to existing methods. MultiStageSearch offers a flexible and accurate approach for pathogen research and diagnostics, overcoming incomplete search spaces and biases inherent in reference databases.

Upon infection, viruses alter the proteome, creating a hospitable environment for infection. Cells respond to limit viral replication, including through protein regulation by post-translational modifications. We use mass spectrometry to define proteome alterations during West Nile virus (WNV) infection. Our studies identify upregulation of HERPUD1, which restricts WNV replication through a mechanism independent of its role in endoplasmic reticulum (ER)-associated degradation (ERAD). We also identify modifications on viral proteins, including a WNV NS3 phosphorylation site that impacts viral replication. Finally, we reveal activation of two host kinases with antiviral activity. We identify phosphorylation at S108 of AMPKβ1, a non-catalytic subunit that regulates activity of the AMPK complex. We also show activation of PAK2 by phosphorylation at S141, which restricts translation of the viral genome. This work contributes to our understanding of the interplay between host and virus while providing a resource to define the changes to the proteome that regulate viral infection.

The SARS-CoV-2 nucleocapsid protein, or N-protein, is a structural protein that plays an important role in the SARS-CoV-2 life cycle. The N-protein takes part in the regulation of viral RNA replication and drives highly specific packaging of full-length genomic RNA prior to virion formation. One regulatory mechanism that is proposed to drive the switch between these two operating modes is the phosphorylation state of the N-protein. Here, we assess the dynamic behavior of non-phosphorylated and phosphorylated versions of the N-protein homodimer through atomistic molecular dynamics simulations. We show that the introduction of phosphorylation yields a more dynamic protein structure and decreases the binding affinity between the N-protein and RNA. Furthermore, we find that secondary structure is essential for the preferential binding of particular RNA elements from the 5' UTR of the viral genome to the N-terminal domain of the N-protein. Altogether, we provide detailed molecular insights into N-protein dynamics, N-protein:RNA interactions, and phosphorylation. Our results corroborate the hypothesis that phosphorylation of the N-protein serves as a regulatory mechanism that determines N-protein function.

The human metapneumovirus (hMPV), a member of the Pneumoviridae family, is a significant respiratory pathogen that causes severe infections in infants, children, the elderly, adults with chronic illnesses, and individuals with immunocompromised conditions. Globally, hMPV is recognized as the second leading cause of bronchiolitis and pneumonia among children under five. The absence of targeted antiviral treatments or vaccines for hMPV significantly strains the global health-care system. This review summarizes recent advances and scientific findings on hMPV by reviewing the current literature on its life cycle, structure, function, prevention, and treatment options.

Post-translational modifications (PTMs) of proteins play a crucial role in living organisms, altering the properties and functions of proteins. There are over 450 known PTMs involved in various life activities. LSD1 (lysine-specific demethylase 1) is the first identified histone demethylase that can remove monomethylation or dimethylation modifications from histone H3 lysine K4 (H3K4) and histone H3 lysine K9 (H3K9). This ability of LSD1 allows it to inhibit or activate transcription. LSD1 has been found to abnormally express at the protein level in various tumors, making it relevant to multiple diseases. As a PTM enzyme, LSD1 itself undergoes various PTMs, including phosphorylation, acetylation, ubiquitination, methylation, SUMOylation, and S-nitrosylation, influencing its activity and function. Dysregulation of these PTMs has been implicated in a wide range of diseases, including cancer, metabolic disorders, neurological disorders, cardiovascular diseases, and bone diseases. Understanding the species of PTMs and functions regulated by various PTMs of LSD1 provides insights into its involvement in diverse physiological and pathological processes. In this review, we discuss the structural characteristics of LSD1 and amino acid residues that affect its enzyme activity. We also summarize the potential PTMs that occur on LSD1 and their involvement in cellular processes. Furthermore, we describe human diseases associated with abnormal expression of LSD1. This comprehensive analysis sheds light on the intricate interplay between PTMs and the functions of LSD1, highlighting their significance in health and diseases.

The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation.

Inflammation is a pathogenic driver of many diseases, including atherosclerosis and rheumatoid arthritis. Hyperinflammation can be seen as any inflammatory response that is deleterious to the host, regardless of cause. In medicine, hyperinflammation is defined as severe, deleterious, and fluctuating systemic or local inflammation with presence of a cytokine storm. It has been associated with rare autoinflammatory disorders. However, advances in omics technologies, including genomics, proteomics, and metabolomics, have revealed it to be more common, occurring in sepsis and severe coronavirus disease 2019. With a focus on proteomics, this review highlights the key role of omics in this shift. Through an exploration of research, we present how omics technologies have contributed to improved diagnostics, prognostics, and targeted therapeutics in the field of hyperinflammation. We also discuss the integration of advanced technologies, multiomics approaches, and artificial intelligence in analyzing complex datasets to develop targeted therapies, and we address their potential for revolutionizing the clinical aspects of hyperinflammation. We emphasize personalized medicine approaches for effective treatments and outline challenges, including the need for standardized methodologies, robust bioinformatics tools, and ethical considerations regarding data privacy. This review aims to provide a comprehensive overview of the molecular mechanisms underpinning hyperinflammation and underscores the potential of omics technologies in enabling successful clinical management.

SARS-CoV-2 has demonstrated a remarkable capacity for immune evasion. While initial studies focused on the Wuhan variant and adaptive immunity, later emerging strains such as Omicron exhibit mutations that may alter their immune-modulatory properties. We performed a comprehensive review of immune evasion mechanisms associated with SARS-CoV-2 viral proteins to focus on the evolutionary dynamics of immune modulation. We systematically analyzed and compared the impact of all currently known Wuhan and Omicron SARS-CoV-2 proteins on type I interferon (IFN) responses using a dual-luciferase reporter assay carrying an interferon-inducible promoter. Results revealed that Nsp1, Nsp5, Nsp14, and ORF6 are potent type I IFN inhibitors conserved across Wuhan and Omicron strains. Notably, we identified strain-specific differences, with Nsp6 and Spike proteins exhibiting enhanced IFN suppression in Omicron, whereas the Envelope protein largely retained this function. To extend these findings, we investigated selected proteins in primary human endothelial cells and also observed strain-specific differences in immune response with higher type I IFN response in cells expressing the Wuhan strain variant, suggesting that Omicron's adaptational mutations may contribute to a damped type I IFN response in the course of the pandemic's trajectory.

Rift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus that can cause substantial morbidity and mortality in livestock and humans, for which there are no currently available licensed human therapeutics or vaccines. Therefore, the development of safe and effective antivirals is both necessary and urgent. The Gc protein is the primary target of the neutralizing antibody response related to Rift Valley fever virus. Here, we report one Gc-specific neutralizing antibody (NA137) isolated from an alpaca and one bispecific antibody (E2-NA137), the protective efficacies of which we evaluated in A129 mice. In this prophylactic study, the survival rates of the NA137 and E2-NA137 groups were both 80%, and in the treatment study, the survival rates were 20% and 60%, respectively. Altogether, our results emphasize that NA137 and E2-NA137 provide a potential approach for treating RVFV either prophylactically or therapeutically.

IFNγ plays both positive and negative roles in the regulation of innate and adaptive immune responses against tumors and virally infected tissues by upregulating CXCL10 and PD-L1 expression.

To identify novel pathways and drugs that regulate the IFNγ-dependent PD-L1, we expressed GFP under the control of mouse PD-L1 promoter in mouse cancer cells that up regulate PD-L1 and CXCL10 in response to IFNγ stimulation. Using these cells, we screened an FDA approved library of 1496 small molecules known for their ability to inhibit IFNγ-dependent increase in PD-L1.

We identified 46 drugs that up regulated and 4 that down regulated IFNγ-dependent PD-L1 expression. We discovered that in addition to the known JAK inhibitors Ruxolitinib and Baricitinib, Dinaciclib, a CDK1/2/5/9 inhibitor, and Ganetespib, a Hsp90 inhibitor, significantly inhibit both PD-L1 and CXCL10 expression in the model cells. Furthermore, both drugs suppressed IFNγ-dependent CXCL10 and PD-L1 expression in-vitro in primary human lung cells and human cancer cells. These drugs also significantly inhibited delayed-type hypersensitivity (DTH) in-vivo in an inflammation mouse model.

Our novel screening platform can therefore be used in the future to identify novel immunomodulators and pathways in cancer and inflammation, expanding therapeutic horizons.

The capacity of host cells to sustain or restrict virus infection is influenced by their proteome. Understanding the compendium of proteins defining cellular permissiveness is key to many questions in fundamental virology. Here, we apply a multi-omic approach to determine the proteins that are associated with highly permissive, intermediate, and hostile cellular states. We observed two groups of differentially regulated genes: (i) with robust changes in mRNA and protein levels and (ii) with protein/RNA discordances. While many of the latter are classified as interferon-stimulated genes (ISGs), most exhibit no antiviral effects in overexpression screens. This suggests that IFN-dependent protein changes can be better indicators of antiviral function than mRNA levels. Phosphoproteomics revealed an additional regulatory layer involving non-signaling proteins with altered phosphorylation. Indeed, we confirmed that several permissiveness-associated proteins with changes in abundance or phosphorylation regulate infection fitness. Altogether, our study provides a comprehensive and systematic map of the cellular alterations driving virus susceptibility.

After the coronavirus disease 2019 (COVID-19) pandemic, the postacute effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have gradually attracted attention. To precisely evaluate the health status of convalescent patients with COVID-19, we analyzed symptom and proteome data of 442 plasma samples from healthy controls, hospitalized patients, and convalescent patients 6 or 12 months after SARS-CoV-2 infection. Symptoms analysis revealed distinct relationships in convalescent patients. Results of plasma protein expression levels showed that C1QA, C1QB, C2, CFH, CFHR1, and F10, which regulate the complement system and coagulation, remained highly expressed even at the 12-month follow-up compared with their levels in healthy individuals. By combining symptom and proteome data, 442 plasma samples were categorized into three subtypes: S1 (metabolism-healthy), S2 (COVID-19 retention), and S3 (long COVID). We speculated that convalescent patients reporting hair loss could have a better health status than those experiencing headaches and dyspnea. Compared to other convalescent patients, those reporting sleep disorders, appetite decrease, and muscle weakness may need more attention because they were classified into the S2 subtype, which had the most samples from hospitalized patients with COVID-19. Subtyping convalescent patients with COVID-19 may enable personalized treatments tailored to individual needs. This study provides valuable plasma proteomic datasets for further studies associated with long COVID.

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health and the economy, underscoring the urgent need for extensive research into the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry and the development of effective therapeutic interventions.

We established a cell line expressing human angiotensin-converting enzyme 2 (ACE2). We used it as a model of pseudotyped viral entry using murine leukemia virus (MLV) expressing SARS-CoV-2 spike (S) protein on its surface and firefly luciferase as a reporter. We screened an U.S. Food and Drug Administration (FDA)-approved compound library for inhibiting ACE2-dependent SARS-CoV-2 pseudotyped viral entry and identified several drug-repurposing candidates.

We identified 18 drugs and drug candidates, including 14 previously reported inhibitors of viral entry and four novel candidates. Pyridoxal 5'-phosphate, Dovitinib, Adefovir dipivoxil, and Biapenem potently inhibit ACE2-dependent viral entry with inhibitory concentration 50% (IC50) values of 57nM, 74 nM, 130 nM, and 183 nM, respectively.

We identified four novel FDA-approved candidate drugs for anti-SARS-CoV-2 combination therapy. Our findings contribute to the growing body of evidence supporting drug repurposing as a viable strategy for rapidly developing COVID-19 treatments.

COVID-19, caused by the SARS-CoV-2 virus, is not only characterized by respiratory symptoms but is also associated with a wide range of systemic complications, including significant hematologic abnormalities. This is a comprehensive review of the current literature, using PubMed and Google Scholar, on the pathophysiology and incidence of thromboembolic events in COVID-19 patients and thromboprophylaxis. COVID-19 infection induces a prothrombotic state in patients through the dysregulation of the renin-angiotensin-aldosterone system (RAAS), endothelial dysfunction, elevated von Willebrand factor (vWF), and a dysregulated immune response involving the complement system and neutrophil extracellular traps (NETs). As a result, thromboembolic complications have emerged in COVID-19 cases, occurring more frequently in severe cases and hospitalized patients. These thrombotic events affect both venous and arterial circulation, with increased incidences of deep venous thrombosis (DVT), pulmonary embolism (PE), systemic arterial thrombosis, and myocardial infarction (MI). While DVT and PE are more common, the literature highlights the potential lethal consequences of arterial thromboembolism (ATE). This review also briefly examines the ongoing discussions regarding the use of anticoagulants for the prevention of thrombotic events in COVID-19 patients. While theoretically promising, current studies have yielded varied outcomes: Some suggest potential benefits, whereas others report an increased risk of bleeding events among hospitalized patients. Therefore, further large-scale studies are needed to assess the efficacy and safety of anticoagulants for thromboprophylaxis in COVID-19 patients.

ApoE4 is the primary risk factor for Alzheimer Disease (AD). Early AD pathological events first affect the neuronal endolysosomal system, which in turn causes neuronal protein aggregation and cell death. Despite the crucial influence of lysosomes upon AD pathophysiology, and that apoE4 localizes to lysosomes, the influence of apoE4 on lysosomal function remains unexplored. We find that expression of apoE4 in neuronal cell lines results in lysosomal alkalinization and impaired lysosomal function. To identify driving factors for these defects, we performed quantitative lysosomal proteome profiling. This revealed that apoE4 expression results in differential regulation of numerous lysosomal proteins, correlating with apoE allele status and disease severity in AD brains. In particular, apoE4 expression results in the depletion of lysosomal Lgals3bp and the accumulation of lysosomal Tmed5. We additionally validated that these lysosomal protein changes can be targeted to modulate lysosomal function. Taken together, this work thereby reveals that apoE4 causes widespread lysosomal defects through remodeling the lysosomal proteome, with the lysosomal Tmed5 accumulation and Lgals3bp depletion manifesting as lysosomal alkalinization in apoE4 neurons.

Avian infectious bronchitis virus (IBV) belongs to family Coronaviridae, genus Gammacoronavirus and is one of the most predominant causes of respiratory disease in poultry. Its high mutation rate constantly leads to the emergence of novel variants that complicate disease control. In 2016, a GA13-like IBV outbreak occurred in Costa Rica, prompting the introduction of the 4/91 live-attenuated vaccine. The objective of this research was to perform a molecular characterization of IBV variants circulating in the country six years after the introduction of the 4/91 vaccine. A total of 177 samples from symptomatic birds were analyzed, with 43 testing positive for IBV. Seven complete S1 sequences were obtained and clustered within the GI-13 lineage by phylogenetic analysis. Sequence analysis showed high genetic similarity to the 4/91 vaccine strain, with nucleotide and amino acid sequence identities over 99.13% and 97.96%, respectively, despite these samples being taken from unvaccinated birds. Post-translational modification analysis of the S1 protein revealed conserved N-glycosylation and palmitoylation sites, while two serine phosphorylation changes were predicted between the obtained sequences and the vaccine strain. Selective pressure analysis identified 10 sites under positive selection, mainly located within the receptor-binding domain and hypervariable regions of the S1 subunit. The presence of 4/91-like variants in unvaccinated birds needs attention, and its relation to observed pathology requires further research. Continuous surveillance is essential to monitor for potential vaccine escape mutants and mitigate their impact.

The online version contains supplementary material available at 10.1007/s13337-025-00910-4.

The global response to the COVID-19 pandemic, caused by the novel SARS-CoV-2 virus, has seen an unprecedented increase in the development of antiviral therapies. Traditional antiviral strategies have primarily focused on direct-acting antivirals (DAAs), which specifically target viral components. In recent years, increasing attention was given to an alternative approach aiming to exploit host cellular pathways or immune responses to inhibit viral replication, which has led to development of so-called host-targeted antivirals (HTAs). The emergence of SARS-CoV-2 and COVID-19 has promoted a boost in this field. Numerous HTAs have been tested and demonstrated their potential against SARS-CoV-2 through in vitro and in vivo studies. However, in striking contrast, only a limited number have successfully progressed to advanced clinical trial phases (2-4), and even less have entered clinical practice. This review aims to explore the current landscape of HTAs targeting SARS-CoV-2 that have reached phase 2-4 clinical trials. Additionally, it will explore the challenges faced in the development of HTAs and in gaining regulatory approval and market availability.

Open Reading Frame 9b (Orf9b), an accessory protein of SARS-CoV and -2, is involved in innate immune suppression through its binding to the mitochondrial receptor Translocase of Outer Membrane 70 (Tom70). Previous structural studies of Orf9b in isolation revealed a β-sheet-rich homodimer, however, structures of Orf9b in complex with Tom70 revealed a monomeric helical fold. Here, we developed a biophysical model that quantifies how Orf9b switches between these conformations and binds to Tom70, a requirement for suppressing the type 1 interferon response. We used this model to characterize the effect of lipid binding and mutations in variants of concern to the Orf9b:Tom70 equilibrium. We found that the binding of a lipid to the Orf9b homodimer biases the Orf9b monomer:dimer equilibrium towards the dimer by reducing the dimer dissociation rate ∼100-fold. We also found that mutations in variants of concern can alter different microscopic rate constants without significantly affecting binding to Tom70. Together our results highlight how perturbations to different steps in these coupled equilibria can affect the apparent affinity of Orf9b to Tom70, with potential downstream implications for interferon signaling in coronavirus infection.

It is a common strategy for viruses to block the host cell cycle to favour their DNA replication. Baculovirus, being a double-stranded DNA virus, can arrest the cell cycle in the G2/M phase to facilitate its replication. However, the key viral genes and mechanisms crucial for inducing cell cycle arrest remain poorly understood. Here, we initially examined the impacts of several Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication-associated genes: ie1, lef-1, lef-2, lef-3, lef-4, odv-ec27 and dbp. We assessed their effects on both the host cells' DNA replication and cell cycle. Our findings reveal that when the lef-2 gene was overexpressed, it led to a significant increase in the number of cells in the G2/M phase and a reduction in the number of cells in the S phase. Furthermore, we discovered that the LEF-2 protein is located in the virogenic stroma and confirmed its involvement in viral DNA replication. Additionally, by employing interference and overexpression experiments, we found that LEF-2 influences host cell DNA replication and blocks the cell cycle in the G2/M phase by regulating the expression of CyclinB and CDK1. Finally, we found that BmNPV lef-2 triggered a DNA damage response in the host cell, and inhibiting this response removed the cell cycle block caused by BmNPV LEF-2. Thus, our findings indicate that the BmNPV lef-2 gene plays a crucial role in viral DNA replication and can regulate host cell cycle processes. This study furthers our understanding of baculovirus-host cell interactions and provides new insight into the molecular mechanisms of antiviral research.

Infectious Bursal Disease Virus (IBDV), a double-stranded RNA virus of the Avibirnavirus genus, causes significant vaccine failures in immunocompromised young poultry. The VP1 protein of IBDV undergoes post-translational modifications that are critical for viral RNA transcription, genome replication, and overall viral proliferation. Phosphorylation enhances the ability of the IBDV polymerase VP1 and facilitates viral replication, while the specific mechanisms underlying VP1 phosphorylation and its role in the IBDV life cycle remain largely unexplored. This study shows that SRPK1 phosphorylates VP1 at the serine 48 (S48) residue in the N-terminal 46SPSR49 motif, enhancing polymerase activity and promoting replication. During IBDV infection, VP1 recruits SRPK1 and co-localizes with it. Inhibiting or deleting SRPK1 greatly reduced VP1 polymerase activity, a leading to a decrease in viral replication. Mutant strains S48A and S48E displayed impaired replication, highlighting the crucial role of SRPK1-mediated phosphorylation in VP1 function. These findings emphasize the key role of SRPK1-mediated VP1 phosphorylation in IBDV replication, providing new insights into viral-host interactions and potential therapeutic targets.

Human enteric α-defensin 5 (HD5) is an immune system peptide that acts as an important antimicrobial factor but is also known to promote pathogen infections by enhancing adhesion of the pathogens. The mechanistic basis of these conflicting functions is unknown. Here we show that HD5 induces abundant filopodial extensions in epithelial cells that capture Shigella, a major human enteroinvasive pathogen that is able to exploit these filopodia for invasion, revealing a mechanism for HD5-augmented bacterial invasion. Using multi-omics screening and in vitro, organoid, dynamic gut-on-chip and in vivo models, we identify the HD5 receptor as P2Y11, a purinergic receptor distributed apically on the luminal surface of the human colonic epithelium. Inhibitor screening identified cAMP-PKA signalling as the main pathway mediating the cytoskeleton-regulating activity of HD5. In illuminating this mechanism of Shigella invasion, our findings raise the possibility of alternative intervention strategies against HD5-augmented infections.

Viral accessory proteins play critical roles in viral escape from host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, and ORF10), strongly activates inflammasome-dependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.

The co-chaperone BAG3 plays critical roles in maintaining cellular proteostasis. It associates with 14-3-3 proteins during the trafficking of aggregation-prone proteins and facilitates their degradation through chaperone-assisted selective autophagy in cooperation with small heat shock proteins. Although reversible phosphorylation regulates BAG3 function, the involved phosphatases remain unknown. Here, we used affinity purification mass spectrometry to identify phosphatases that target BAG3. Of the hits, we evaluated the involvement of protein phosphatase-1 (PP1) using chemical inhibitors and activators in in vitro and cellular approaches. Our results demonstrate that PP1 can dephosphorylate BAG3-pS136 in cells and counteract 14-3-3γ association with BAG3 at this motif. Furthermore, protein phosphatase-5 (PP5) co-enriched with proteostasis-related proteins, and it has the capacity to dephosphorylate a BAG3 phosphorylation-site cluster regulating the interaction of BAG3 with small heat shock proteins and BAG3-mediated protein degradation. Our findings provide new insights into the regulation of BAG3 by phosphatases. This paves the way for future research focused on the precise control of BAG3 function through its regulatory proteins, potentially holding therapeutic promise for diseases characterized by disrupted proteostasis.

Filopodia, widely distributed on cell surfaces, are distinguished by their dynamic extensions, playing pivotal roles in a myriad of biological processes. Their functions span from mechanosensing and guidance to cell-cell communication during cellular organization in the early embryo. Filopodia have significant roles in pathogenic processes, such as cancer invasion and viral dissemination. Molecular mapping of the filopodome has revealed generic components essential for filopodia functions. In parallel, recent insights into biophysical mechanisms governing filopodia dynamics have provided the foundation for broader investigations of filopodia's biological functions. We highlight recent discoveries of engagement of filopodia in various stages of development and pathogenesis and present an overview of intricate molecular and physical features of these cellular structures across a spectrum of cellular activities.

The first vaccines approved against SARS-CoV-2, mRNA-1273 and BNT162b2, utilized mRNA platforms. However, little is known about the proteomic markers and pathways associated with host immune responses to mRNA vaccination. In this proof-of-concept study, sera from male and female vaccine recipients were evaluated for proteomic and immunologic responses 1-month and 6-months following homologous third vaccination.

An aptamer-based (7,289 marker) proteomic assay coupled with traditional serology was leveraged to generate a comprehensive evaluation of systemic responsiveness in 64 and 68 healthy recipients of mRNA-1273 and BNT162b2 vaccines, respectively.

Sera from female recipients of mRNA-1273 showed upregulated indicators of inflammatory and immunological responses at 1-month post-third vaccination, and sera from female recipients of BNT162b2 demonstrated upregulated negative regulators of RNA sensors at 1-month. Sera from male recipients of mRNA-1273 showed no significant upregulation of pathways at 1-month post-third vaccination, though there were multiple significantly upregulated proteomic markers. Sera from male recipients of BNT162b2 demonstrated upregulated markers of immune response to doublestranded RNA and cell-cycle G(2)/M transition at 1-month. Random Forest analysis of proteomic data from pre-third-dose sera identified 85 markers used to develop a model predictive of robust or weaker IgG responses and antibody levels to SARS-CoV-2 spike protein at 6-months following boost; no specific markers were individually predictive of 6-month IgG response. Thirty markers that contributed most to the model were associated with complement cascade and activation; IL-17, TNFR pro-apoptotic, and PI3K signaling; and cell cycle progression.

These results demonstrate the utility of proteomics to evaluate correlates or predictors of serological responses to SARS-CoV-2 vaccination.

Characterization of shared patterns of RNA expression between genes across conditions has led to the discovery of regulatory networks and novel biological functions. However, it is unclear if such coordination extends to translation, a critical step in gene expression. Here, we uniformly analyzed 3,819 ribosome profiling datasets from 117 human and 94 mouse tissues and cell lines. We introduce the concept of Translation Efficiency Covariation (TEC), identifying coordinated translation patterns across cell types. We nominate potential mechanisms driving shared patterns of translation regulation. TEC is conserved across human and mouse cells and helps uncover gene functions. Moreover, our observations indicate that proteins that physically interact are highly enriched for positive covariation at both translational and transcriptional levels. Our findings establish translational covariation as a conserved organizing principle of mammalian transcriptomes.

Future pandemic threats may be caused by novel coronaviruses and influenza A viruses. Here we show that when directly added to a cell culture, 12mer guanine RNA (G12) and its phosphorothioate-linked derivatives (G12(S)), rapidly entered cytoplasm and suppressed the propagation of human coronaviruses and influenza A viruses to between 1/100 and nearly 1/1000 of normal virus infectivity without cellular toxicity and induction of innate immunity. Moreover, G12(S) alleviated the weight loss caused by coronavirus infection in mice. G12(S) might exhibit a stable G-tetrad with left-handed parallel-stranded G-quadruplex, and inhibit the replication process by impeding interaction between viral nucleoproteins and viral RNA in the cytoplasm. Unlike previous antiviral strategies that target the G-quadruplexes of the viral genome, we now show that excess exogenous G-quadruplex-forming small RNA displaces genomic RNA from ribonucleoprotein, effectively inhibiting viral replication. The approach has the potential to facilitate the creation of versatile middle-molecule antivirals featuring lipid nanoparticle-free delivery.

Protein phosphorylation involves the reversible modification of a protein (substrate) residue by another protein (kinase). Liquid chromatography-mass spectrometry studies are rapidly generating massive protein phosphorylation datasets across multiple conditions. Researchers then must infer kinases responsible for changes in phosphosites of each substrate. However, tools that infer kinase-substrate interactions (KSIs) are not optimized to interactively explore the resulting large and complex networks, significant phosphosites, and states. There is thus an unmet need for a tool that facilitates user-friendly analysis, interactive exploration, visualization, and communication of phosphoproteomics datasets. We present PhosNetVis, a web-based tool for researchers of all computational skill levels to easily infer, generate, and interactively explore KSI networks in 2D or 3D by streamlining phosphoproteomics data analysis steps within a single tool. PhostNetVis lowers barriers for researchers by rapidly generating high-quality visualizations to gain biological insights from their phosphoproteomics datasets. It is available at https://gumuslab.github.io/PhosNetVis/.