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Background: SARS-CoV-2, a highly contagious coronavirus, is responsible for the global pandemic of COVID-19 in 2019. Currently, it remains uncertain whether SARS-CoV-2 possesses oncogenic or oncolytic potential in influencing tumor progression. Therefore, it is important to evaluate the clinical and functional role of SARS-CoV-2 on tumor progression., Methods: Here, we integrated bioinformatic analysis of COVID-19 RNA-seq data from the GEO database and performed functional studies to explore the regulatory role of SARS-CoV-2 in solid tumor progression, including lung, colon, kidney and liver cancer., Results: Our results demonstrate that infection with SARS-CoV-2 is associated with a decreased expression of genes associated with cancer proliferation and metastasis in lung tissues from patients diagnosed with COVID-19. Several cancer proliferation or metastasis related genes were frequently downregulated in SARS-CoV-2 infected intestinal organoids and human colon carcinoma cells. In vivo and in vitro studies revealed that SARS-CoV-2 nucleocapsid (N) protein inhibits colon and kidney tumor growth and metastasis through the N-terminal (NTD) and the C-terminal domain (CTD). The molecular mechanism indicates that the N protein of SARS-CoV-2 interacts with YBX1, resulting in the recruitment of PKM mRNA into stress granules mediated by G3BP1. This process ultimately destabilizes PKM expression and suppresses glycolysis., Conclusion: Our study reveals a new function of SARS-CoV-2 nucleocapsid protein on tumor progression., (© 2024. The Author(s).)

SARS-CoV-2 uses the receptor binding domain (RBD) of its spike protein to recognize and infect host cells by binding to the cell surface receptor angiotensin converting enzyme 2 (ACE2). The ACE2 receptor is composed of peptidase domain (PD), collectrin-like domain, transmembrane domain, and short cytoplasmic domain, and may exist as a dimer on cell surface. The RBD binding site is located atop of the ACE2 PD, but the involvement of other domains in virus infection is uncertain. We found that the ACE2 PD alone, whether anchored to cell membrane via a glycosylphosphatidylinositol anchor or attached to another surface protein, is fully functional as a receptor for spike-mediated cell fusion and virus infection. However, for ACE2 to function as the viral receptor, the RBD binding site must be positioned in close proximity to the cell membrane. Elevating the surface height of ACE2 using long and rigid protein spacers reduces or eliminates cell fusion and virus infection. Moreover, we found that the RBD-targeting neutralizing antibodies, nanobodies, and de novo designed miniprotein binders, when present on cell surface, also act as viral receptors, facilitating cell fusion and virus infection. Our data demonstrate that RBD binding and close membrane proximity are essential properties for a receptor to effectively mediate SARS-CoV-2 infection. Importantly, we show that soluble RBD-binders can be engineered to make cells either susceptible or resistant to virus infection, which has significant implications for antiviral therapy and various virus-mediated applications., Competing Interests: Competing interests statement:J.Z., H.Z., H.T.T.N., and Z.W. have two provisional patent applications that incorporate discoveries described in this manuscript. One is related to the design of longer version of soluble viral receptors or antibodies as antiviral agents. The other is a method of using soluble viral receptors to introduce cell susceptibility for virus infection and membrane fusion.

The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.

PDZ (PSD-95 [postsynaptic density protein 95]/Dlg [Discs large]/ZO-1 [zonula occludens-1]) domain-containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks and are mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens that express proteins containing PDZ-binding motifs (PDZbms). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of 7 host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of 3 of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and interleukin-16), in human monocyte-derived macrophages and in dendritic cells upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells, and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, supports the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. An inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed., Competing Interests: Conflicts of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Although coronaviruses use diverse receptors, the characterization of coronaviruses with unknown receptors has been impeded by a lack of infection models 1,2 . Here we introduce a strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building artificial receptor scaffolds comprising various modules and generating specific virus-binding domains. We identify key factors for CVRs to functionally mimic native receptors by facilitating spike proteolytic cleavage, membrane fusion, pseudovirus entry and propagation for various coronaviruses. We delineate functional SARS-CoV-2 spike receptor-binding sites for CVR design and reveal the mechanism of cell entry promoted by the N-terminal domain-targeting S2L20-CVR. We generated CVR-expressing cells for 12 representative coronaviruses from 6 subgenera, most of which lack known receptors, and show that a pan-sarbecovirus CVR supports propagation of a propagation-competent HKU3 pseudovirus and of authentic RsHuB2019A 3 . Using an HKU5-specific CVR, we successfully rescued wild-type and ZsGreen-HiBiT-incorporated HKU5-1 (LMH03f) and isolated a HKU5 strain from bat samples. Our study demonstrates the potential of the CVR strategy for establishing native receptor-independent infection models, providing a tool for studying viruses that lack known susceptible target cells., Competing Interests: Competing interests: H.Y. has submitted a patent application to the China National Intellectual Property Administration for the utilization of artificial viral receptors and their applications., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Although various strategies have been used to prevent and treat SARS-CoV-2, the spread and evolution of SARS-CoV-2 is still progressing rapidly. The emerging variants Omicron and its sublineage have a greater ability to spread and escape nearly all current monoclonal antibodies treatments, highlighting an urgent need to develop therapeutics targeting current and emerging Omicron variants or recombinants with breadth and potency. Here, some small molecule drugs are rapidly identified that could covalently binding to receptor binding domain (RBD) protein of Omicron through the combined application of artificial intelligence (AI) and activity-based protein profiling (ABPP) technology. The surface plasmon resonance (SPR) and pseudo-virus neutralization experiments further reveal that an FDA-approved drug gallic acid has robust neutralization potency against Omicron pseudo-virus with the IC 50 values of 23.56 × 10 -6 m. Taken together, a platform combining AI intelligence, biochemical, SPR, molecular docking, and pseudo-virus-based screening for rapid identification and evaluation of potential anti-SARS-CoV-2 small molecule drugs is established and the effectiveness of the platform is validated., (© 2024 Wiley‐VCH GmbH.)

Prenatal SARS-CoV-2 infection is associated with higher rates of pregnancy and birth complications, despite that vertical transmission rates are thought to be low. Here, multi-omics analyses of human placental tissues, cord tissues/plasma, and amniotic fluid from 23 COVID-19 mother-infant pairs revealed robust inflammatory responses in both maternal and fetal compartments. Pronounced expression of complement proteins (C1q, C3, C3b, C4, C5) and inflammatory cytokines (TNF, IL-1α, and IL-17A/E) was detected in the fetal compartment of COVID-19-affected pregnancies. While ~26% of fetal tissues were positive for SARS-CoV-2 RNA, more than 60% of fetal tissues contained SARS-CoV-2 ORF8 proteins, suggesting transplacental transfer of this viral accessory protein. ORF8-positive fetal compartments exhibited increased inflammation and complement activation compared to ORF8-negative COVID-19 pregnancies. In human placental trophoblasts in vitro, exogenous ORF8 exposure resulted in complement activation and inflammatory responses. Co-immunoprecipitation analysis demonstrated that ORF8 binds to C1q specifically by interacting with a 15-peptide region on ORF8 (C37-A51) and the globular domain of C1q subunit A. In conclusion, an ORF8-C1q-dependent complement activation pathway was identified in COVID-19-affected pregnancies, likely contributing to fetal inflammation independently of fetal virus exposure., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. The Author(s).)

SARS-CoV-2 and its variants continue to pose a significant threat to public health. Nanobodies (Nbs) that inhibit the interaction between the receptor-binding domain (RBD) of the spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2) are promising drug candidates. In this study, we report the discovery and structural characterization of a potent Nb that targets the RBD. By screening a phage display alpaca naive Nbs library using the RBD as bait, we identified sixteen candidate Nbs. Of these, nine exhibited nanomolar to micromolar binding affinity and strong neutralizing activity against pseudotyped SARS-CoV-2 viruses, with NbS4 showing the highest neutralization potency. The crystal structure of the SARS-CoV-2 RBD in complex with NbS4 revealed that this Nb binds to a site partially overlapping the ACE2 binding region. Importantly, the key binding residues of NbS4 in the RBD are conserved across most known variants, making it a promising candidate for COVID-19 treatment., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized in vivo into the active form remdesivir triphosphate (RTP), an analogue of adenosine triphosphate (ATP) with a cyano group substitution in the carbon 1' of the ribose (1'-CN). RTP is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked in vitro by RTP, but the low impact in vivo on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (HsPrimPol), which also operates in mitochondria. In this work, we have tested if RTP is a valid substrate for both DNA primase and DNA polymerase activities of HsPrimPol, and its impact in the production of mature DNA primers. RTP resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with ATP. Nevertheless, RTP-initiated primers are abortive, ocassionally reaching a maximal length of 4-5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of ATP, the natural substrate, is much higher than the intracellular concentration of RTP, it is unlikely that HsPrimPol would use RTP for primer synthesis during a remdesivir treatment in real patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Coronavirus 3C-like protease (CoV 3CL pro ) is essential for viral replication, providing an attractive target for monitoring the evolution of CoV and developing anti-CoV drugs. Here, the substrate-binding modes of 3CL pro s from four CoV genera are analyzed and found that the S2 pocket in 3CL pro is highly conserved within each genus but differs between genera. Functionally, the S2 pocket, in conjunction with S4 and S1' pockets, governs the genus-specific substrate selectivity of 3CL pro . Resurrected ancestral 3CL pro s from four CoV genera validate the genus-specific divergence of S2 pocket. Drawing upon the genus-specific S2 pocket as evolutionary marker, eight newly identified 3CL pro s uncover the ancestral state of modern 3CL pro and elucidate the possible evolutionary process for CoV. It is also demonstrated that the S2 pocket is highly correlated with the genus-specific inhibitory potency of PF-07321332 (an FDA-approved drug against COVID-19) on different CoV 3CL pro s. This study on 3CL pro provides novel insights to inform evolutionary mechanisms for CoV and develop genera-specific or broad-spectrum drugs against CoVs., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Furin cleavage site (FCS) of the SARS-CoV-2 S protein, which connects the S1/S2 junction, is essential for facilitating fusion with the host cells. Wild-type (Wt) SARS-CoV-2 S protein, PDB ID: 6yvb, lacks a sequence of amino acid residues, including the FCS that links the S1/S2 junction. For the first time, we demonstrated that a stretch of 14 amino acid residues (677QTNSPRRARSVASQ689) forms an antiparallel β-sheet comprising of PRRAR sequence in the FCS within a short loop. Upon comparing the loop content of the S1/S2 junction with that of Wt SARS-CoV-2 containing PRRAR in the FCS, we observed a decrease in antiparallel β-sheet content and an increase in loop content in the B.1.1.7 variant with HRRAR in the FCS. This short loop within antiparallel β-sheet can serve as a docking site for various proteases, including TMPRSS2 and α1AT. We performed a 300-ns simulation of the SARS-CoV-2 receptor binding domain (RBD) using several antibacterial and antiviral ligands commonly used to treat various infections. Our findings indicate that the receptor binding domain (RBD) comprising the receptor binding motif (RBM) utilizes β6 and a significant portion of the loop to bind with ligands, suggesting its potential for treating SARS-CoV-2 infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome. It consists of multiple domains that perform critical functions during the viral life cycle. CoV-Y is the most C-terminal domain of Nsp3, and it exhibits evolutionary conservation across diverse CoVs; however, the exact biological function of CoV-Y remains unclear. Here, we determined the crystal structure of CoV-Y of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp3 using the single-wavelength anomalous diffraction method. We revealed the interaction between CoV-Y and the host BRCA1-associated protein (BRAP) using immunoprecipitation-mass spectrometry experiments. This interaction was subsequently confirmed in cellular assays, and the precise binding-regions between these two proteins were clarified. We found that this interaction is conserved in SARS-CoV and Middle East respiratory syndrome coronavirus. Next, we demonstrated that CoV-Y enhances IκBα and IκBβ phosphorylation and promotes the nuclear translocation of the downstream NF-κB members p50 and p65 through binding to BRAP. The CoV-Y-BRAP interaction can upregulate the transcript levels of the host inflammatory cytokines. Overall, our findings illustrate the biological function of CoV-Y for the first time and provide novel insights into coronavirus regulation of host inflammatory responses, as well as a possible target for antiviral drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30 kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). In both soluble and condensed forms, N has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide, LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here, assembly has a critical concentration of about 1 μM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS., Competing Interests: Conflict of interest A. Z. is a founder of Assembly Biosciences and a founder and officer of Door Pharmaceuticals. A. Z. has equity in both companies. These are biotech companies that are focused on developing antivirals. All the other authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The COVID-19 pandemic has resulted in a significant toll of deaths worldwide, exceeding seven million individuals, prompting intensive research efforts aimed at elucidating the molecular mechanisms underlying the pathogenesis of SARS-CoV-2 infection. Despite the rapid development of effective vaccines and therapeutic interventions, COVID-19 remains a threat to humans due to the emergence of novel variants and largely unknown long-term consequences. Among the viral proteins, the nucleocapsid protein (N) stands out as the most conserved and abundant, playing the primary role in nucleocapsid assembly and genome packaging. The N protein is promiscuous for the recognition of RNA, yet it can perform specific functions. Here, we discuss the structural basis of specificity, which is directly linked to its regulatory role. Notably, the RNA chaperone activity of N is central to its multiple roles throughout the viral life cycle. This activity encompasses double-stranded RNA (dsRNA) annealing and melting and facilitates template switching, enabling discontinuous transcription. N also promotes the formation of membrane-less compartments through liquid-liquid phase separation, thereby facilitating the congregation of the replication and transcription complex. Considering the information available regarding the catalytic activities and binding signatures of the N protein-RNA interaction, this review focuses on the regulatory role of the SARS-CoV-2 N protein. We emphasize the participation of the N protein in discontinuous transcription, template switching, and RNA chaperone activity, including double-stranded RNA melting and annealing activities., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The connection between SARS-CoV-2 replication-transcription complexes and nucleocapsid (N) protein is critical for regulating genomic RNA replication and virion packaging over the viral life cycle. However, the mechanism that dynamically regulates genomic RNA packaging and replication remains elusive. Here, we demonstrate that the N-terminal domain of SARS-CoV-2 nonstructural protein 3, a core component of viral replication-transcription complexes, binds N protein and displaces RNA in a concentration-dependent manner. This interaction disrupts liquid-liquid phase separation of N protein driven by N protein-RNA interactions which is crucial for virion packaging and viral replication. We also report a high-resolution crystal structure of the nonstructural protein 3 ubiquitin-like domain 1 (Ubl1) at 1.49 Å, which reveals abundant negative charges on the protein surface. Sequence and structural analyses identify several conserved motifs at the Ubl1-N protein interface and a previously unexplored highly negative groove, providing insights into the molecular mechanism of Ubl1-mediated modulation of N protein-RNA binding. Our findings elucidate the mechanism of dynamic regulation of SARS-CoV-2 genomic RNA replication and packaging over the viral life cycle. Targeting the conserved Ubl1-N protein interaction hotspots also promises to aid in the development of broad-spectrum antivirals against pathogenic coronaviruses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV-2. Viral RNA and this protein are bound by electrostatic forces, forming cytoplasmic helical structures known as nucleocapsids. Subsequently, these nucleocapsids interact with the membrane (M) protein, facilitating virus budding into early secretory compartments., Scope of Review: Exploring the role of the N protein in the SARS-CoV-2 life cycle, pathogenesis, post-sequelae consequences, and interaction with host immunity has enhanced our understanding of its function and potential strategies for preventing SARS-CoV-2 infection., Major Conclusion: This review provides an overview of the N protein's involvement in SARS-CoV-2 infectivity, highlighting its crucial role in the virus-host protein interaction and immune system modulation, which in turn influences viral spread., General Significance: Understanding these aspects identifies the N protein as a promising target for developing effective antiviral treatments and vaccines against SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

SARS-CoV-2 mainly infects the respiratory tract but can also target other organs, including the central nervous system. While it was recently shown that cells of the blood-brain-barrier are permissive to SARS-CoV-2 infection in vitro, it remains debated whether neurons can be infected. In this study, we demonstrate that vesicular stomatitis virus particles pseudotyped with the spike protein of SARS-CoV-2 variants WT, Alpha, Delta and Omicron enter the neuronal model cell line SH-SY5Y. Cell biological analyses of the pseudo-virus treated cultures showed marked alterations in microtubules of SH-SY5Y cells. Because the changes in β-tubulin occurred in most cells, but only few were infected, we further asked whether interaction of the cells with spike protein might be sufficient to cause molecular and structural changes. For this, SH-SY5Y cells were incubated with trimeric spike proteins for time intervals of up to 24 h. CellProfiler™-based image analyses revealed changes in the intensities of microtubule staining in spike protein-incubated cells. Furthermore, expression of the spike protein-processing protease cathepsin L was found to be up-regulated by wild type, Alpha and Delta spike protein pseudotypes and cathepsin L was found to be secreted from spike protein-treated cells. We conclude that the mere interaction of the SARS-CoV-2 with neuronal cells can affect cellular architecture and proteolytic capacities. The molecular mechanisms underlying SARS-CoV-2 spike protein induced cytoskeletal changes in neuronal cells remain elusive and require future studies., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The spike protein of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for infecting host cells. It has two segments, S1 and S2. The S1 segment has a receptor-binding domain (RBD) that attaches to the host receptor angiotensin-converting enzyme 2 (ACE2). The S2 segment helps in the fusion of the viral cell membrane by creating a six-helical bundle through the two-heptad repeat domain. To develop effective vaccines and therapeutics against COVID-19, it is critical to express and purify the SARS-CoV-2 Spike protein. Extensive studies have been conducted on expression of a complete recombinant spike protein or its fragments. This review provides an in-depth analysis of the different expression systems employed for spike protein expression, along with their advantages and disadvantages., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The multifaceted interplay between neurodegenerative pathologies, including Alzheimer's disease (AD), and the highly virulent severe acute respiratory syndrome coronavirus (SARS-CoV), is implicated in various conditions. AD and SARS-CoV pathogenesis involve the APOE4 allele, NLRP3 inflammasome, and ACE2-SPIKE complex. APOE4, a genetic polymorphism of the APOE gene, is associated with an increased susceptibility to AD. NLRP3, an inflammatory protein of the innate immune system, plays a pivotal role in immune response cascades. In SARS-CoV, the ACE2 receptor serves as the principal portal for cellular entry, while APOE4 intricately interacts with the ACE2-spike protein complex, enhancing viral internalization process. The interaction of NLRP3 with the ACE2-spike protein complex leads to increased inflammatory signaling. The convergence of APOE4/NLRP3 and ACE2-spike protein complex interactions suggests a possible link between SARS and AD. Therefore, the current research centralizes the association between by utilizing SARS-CoV datasets to explore possible mechanisms that account for the pathogenesis of SARS-CoV and AD. The work is further extended to unveil the molecular interactions of APOE4 and NLRP3 with the ACE2-Spike protein complex at the molecular level by employing molecular dynamics simulation techniques. The therapeutic efficacy of Chyawanprash nutraceuticals is evaluated as their inhibitory potential towards APOE4-ACE2-Spike protein and NLRP3-ACE2-Spike protein complexes. Notably, our simulations unequivocally demonstrate the robust and enduring binding capability of the compound Phyllantidine with the target complexes throughout the simulation period. The findings of the studies further corroborate the primary hypothesis of APOE4 and NLRP3 as driver factors in the pathogenesis of both SARS-CoV and AD. Therefore, this research establishes a paradigm for comprehending the complex interaction between AD and SARS-CoV and lays the groundwork for further study in this domain.Communicated by Ramaswamy H. Sarma.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses., (© 2024 The Author(s). PROTEINS: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

WHO declared severe acute respiratory syndrome coronavirus-2' (SARS-CoV-2) was global health emergency since 2020. In our study eighteen natural compounds were investigated for possible anti-SARS-CoV-2 potential, where the most potent natural compounds were ursolic acid and dioscin with IC 50 value of 4.49 µg/mL and 7.11 µg/mL, respectively. Hesperidin, catechin, diosmin, isorhamnetin-3- O -glucoside and hyperoside showed medium antiviral activity with IC 50 value of 20.87, 22.57, 38.92, 39.62 and 47.10 µg/mL, respectively. Molecular modelling studies including docking study and predictive ADME study were performed on all tested molecules. Their binding energies after docking were calculated and their orientations at the active sites of both SARS-CoV-2 main protease (Mpro) and spike (S) receptors were visualised and compared to the downloaded ligands. Also, the predictive ADME studies showed good pharmacokinetic properties of most of the tested compounds. The obtained in silico results obtained confirmed that many of the tested compounds are promising SARS-CoV-2 inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19). SARS-CoV-2 infection suppresses host innate immunity and impairs cell viability. Among the viral proteins, ORF6 exhibits potent interferon (IFN) antagonistic activity and cellular toxicity. It also interacts with the RNA export factor RAE1, which bridges the nuclear pore complex and nuclear export receptors, suggesting an effect on RNA export. Using the Xenopus oocyte microinjection system, I found that ORF6 blocked the export of not only mRNA but also spliceosomal U snRNA. I further demonstrated that ORF6 affects the interaction between RAE1 and nuclear export receptors and inhibits the RNA binding of RAE1. These effects of ORF6 may cumulatively block the export of several classes of RNA. I also found that ORF6 binds RNA and forms oligomers. These findings provide insights into the suppression of innate immune responses and the reduction in cell viability caused by SARS-CoV-2 infection, contributing to the development of antiviral drugs targeting ORF6., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ichiro Taniguchi. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The neurological symptoms of COVID-19, often referred to as neuro-COVID include neurological pain, memory loss, cognitive and sensory disruption. These neurological symptoms can persist for months and are known as Post-Acute Sequalae of COVID-19 (PASC). The molecular origins of neuro-COVID, and how it contributes to PASC are unknown, however a growing body of research highlights that the self-assembly of protein fragments from SARS-CoV-2 into amyloid nanofibrils may play a causative role. Previously, we identified two fragments from the SARS-CoV-2 proteins, Open Reading Frame (ORF) 6 and ORF10, that self-assemble into neurotoxic amyloid assemblies. Here we further our understanding of the self-assembly mechanisms and nano-architectures formed by these fragments and their biological responses. By solubilising the peptides in a fluorinated solvent, we eliminate insoluble aggregates in the starting materials (seeds) that change the polymorphic landscape of the assemblies. The resultant assemblies are dominated by structures with higher free energies ( e.g. ribbons and amorphous aggregates) that are less toxic to cultured neurons but do affect their mitochondrial respiration. We also show the first direct evidence of cellular uptake of viral amyloids. This work highlights the importance of understanding the polymorphic behaviour of amyloids and the correlation to neurotoxicity, particularly in the context of neuro-COVID and PASC.

Codon bias analysis of SARS-CoV-2 reveals suboptimal adaptation for translation in human cells it infects. The detailed examination of the codons preferentially used by SARS-CoV-2 shows a strong preference for Lys AAA , Gln CAA , Glu GAA , and Arg AGA , which are infrequently used in human genes. In the absence of an adapted tRNA pool, efficient decoding of these codons requires a 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 ) modification at the U 34 wobble position of the corresponding tRNAs (tLys UUU ; tGln UUG ; tGlu UUC ; tArg UCU ). The optimal translation of SARS-CoV-2 open reading frames (ORFs) may therefore require several adjustments to the host's translation machinery, enabling the highly biased viral genome to achieve a more favorable "Ready-to-Translate" state in human cells. Experimental approaches based on LC-MS/MS quantification of tRNA modifications and on alteration of enzymatic tRNA modification pathways provide strong evidence to support the hypothesis that SARS-CoV-2 induces U 34 tRNA modifications and relies on these modifications for its lifecycle. The conclusions emphasize the need for future studies on the evolution of SARS-CoV-2 codon bias and its ability to alter the host tRNA pool through the manipulation of RNA modifications.

The gut microbiota metabolizes flavonoids, amino acids, dietary fiber, and other components of foods to produce a variety of gut microbiota-derived metabolites. Flavonoids are the largest group of polyphenols, and approximately 7000 flavonoids have been identified. A variety of phenolic acids are produced from flavonoids and amino acids through metabolic processes by the gut microbiota. Furthermore, these phenolic acids are easily absorbed. Phenolic acids generally represent phenolic compounds with one carboxylic acid group. Gut microbiota-derived phenolic acids have antiviral effects against several viruses, such as SARS-CoV-2 and influenza. Furthermore, phenolic acids influence the immune system by inhibiting the secretion of proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α. In the nervous systems, phenolic acids may have protective effects against neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Moreover, phenolic acids can improve levels of blood glucose, cholesterols, and triglycerides. Phenolic acids also improve cardiovascular functions, such as blood pressure and atherosclerotic lesions. This review focuses on the current knowledge of the effects of phenolic acids produced from food-derived flavonoids and amino acids by the gut microbiota on health and disease.

The programmed ribosomal frameshift (PRF) region is found in the RNA genome of all coronaviruses and shifts the ribosome reading frame through formation of a three-stem pseudoknot structure, allowing the translation of essential viral proteins. Using NMR spectroscopy, comparative sequence analyses and functional assays we show that, in the absence of the ribosome, a 123-nucleotide sequence encompassing the PRF element of SARS-CoV-2 adopts a well-defined two-stem loop structure that is conserved in all SARS-like coronaviruses. In this conformation, the attenuator hairpin and slippery site nucleotides are exposed in the first stem-loop and two pseudoknot stems are present in the second stem-loop, separated by an 8-nucleotide bulge. Formation of the third pseudoknot stem depends on pairing between bulge nucleotides and base-paired nucleotides of the upstream stem-loop, as shown by a PRF construct where residues of the upstream stem were removed, which formed the pseudoknot structure and had increased frameshifting activity in a dual-luciferase assay. The base-pair switch driving PRF pseudoknot folding was found to be conserved in several human non-SARS coronaviruses. The collective results suggest that the frameshifting pseudoknot structure of these viruses only forms transiently in the presence of the translating ribosome. These findings clarify the frameshifting mechanism in coronaviruses and can have a beneficial impact on antiviral drug discovery., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The development of novel tools to tackle viral processes has become a central focus in global health, during the COVID-19 pandemic. The spike protein is currently one of the main SARS-CoV-2 targets, owing to its key roles in infectivity and virion formation. In this context, exploring innovative strategies to block the activity of essential factors of SARS-CoV-2, such as spike proteins, will strengthen the capacity to respond to current and future threats. In the present work, we developed and tested novel bispecific molecules that encompass: (i) oligonucleotide aptamers S901 and S702, which bind to the spike protein through its S1 domain, and (ii) hydrophobic tags, such as adamantane and tert-butyl-carbamate-based ligands. Hydrophobic tags have the capacity to trigger the degradation of targets recruited in the context of a proteolytic chimera by activating quality control pathways. We observed that S901-adamantyl conjugates promote the degradation of the S1 spike domain, stably expressed in human cells by genomic insertion. These results highlight the suitability of aptamers as target-recognition molecules and the robustness of protein quality control pathways triggered by hydrophobic signals, and place aptamer-Hytacs as promising tools for counteracting coronavirus progression in human cells.

The onset of SARS-CoV-2 infection in 2019 sparked a global COVID-19 pandemic. This infection is marked by a significant rise in both viral and host kinase activity. Our primary objective was to identify a pivotal host kinase essential for COVID-19 infection and the associated phenomenon of the cytokine storm, which may lead to long-term COVID-19 complications irrespective of viral genetic variations. To achieve this, our study tracked kinase phosphorylation dynamics in RAW264.7 macrophages following SPIKE transfection over time. Among the kinases surveyed, p70S6K (RPS6KB1) exhibited a 3.5-fold increase in phosphorylation at S418. This significant change prompted the selection of p70S6K for in silico investigation, utilizing its structure bound to M2698 (PDB: 7N93). M2698, an oral dual Akt/p70S6K inhibitor with an IC 50 of 1.1 nM, exhibited psychosis side effects in phase I clinical trials, potentially linked to its interaction with Akt2. Our secondary objective was to discover a small-molecule analogue of M2698 that exhibits a distinct binding preference for p70S6K over Akt2 through computational modeling and analysis. The in silico part of our project began with validating the prediction accuracy of the docking algorithm, followed by an OCA analysis pinpointing specific atoms on M2698 that could be modified to enhance selectivity. Subsequently, our investigation led to the identification of an analog of M2698, designated as S34, that showed a superior docking score towards p70S6K compared to Akt2. To further assess the stability of S34 in its protein-ligand (PL) complexes with p70S6K and Akt2, MD simulations were conducted. These simulations suggest that S34, on average, forms two hydrogen bond interactions with p70S6K, whereas it only forms one hydrogen bond interaction with Akt2. This difference in hydrogen bond interactions likely contributed to the observed larger root mean square deviation (RMSD) of 0.3 nm in the S34-Akt2 complex, compared to 0.1 nm in the S34-p70S6K complex. Additionally, we calculated free binding energy to predict the strength of the binding interactions of S34 to p70S6K and Akt2, which showed ~2-fold favorable binding affinity of S34 in the p70S6K binding pocket compared to that in the Akt2 binding pocket. These observations may suggest that the S34-p70S6K complex is more stable than the S34-Akt2 complex. Our work focused on identifying a host kinase target and predicting the binding affinity of a novel small molecule to accelerate the development of effective treatments. The wet bench results specifically highlight p70S6K as a compelling anti-COVID-19 target. Meanwhile, our in silico investigations address the known off-target effects associated with M2698 by identifying a close analog called S34. In conclusion, this study presents novel and intriguing findings that could potentially lead to clinical applications with further investigations.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity ( K d ) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

Based on the SARS-CoV-2 fusion peptide (FP) structure determined from the NMR experiment, we created six FP models under different environmental conditions to explore the effects of salt and cholesterol on FP-membrane binding. The all-atom molecular dynamics (MD) simulation results indicated that ionic environments notably impact the FP structure as well as the stability of the helical elements within the peptide. Our findings highlighted the unpredictable influence of ions on the secondary structures and dynamics of the FP, emphasizing the complexity and sensitivity of the peptide's conformations to ionic conditions. When exploring the peptide's interaction with a cholesterol-free phospholipid bilayer membrane, we found that the helical elements of the FP remain stable irrespective of the salt type (Na + or Ca 2+ ). This result emphasizes the crucial role of phospholipid bilayer membranes in supporting the secondary structures of the FP. The MD simulation results showed that Ca 2+ ions facilitated deeper membrane penetration than Na + ions, highlighting the critical role of calcium ions in the FP-membrane binding. Our study indicates the essential role of the aromatic residues (such as Phe833 and Tyr837) in the FP-membrane binding process. Finally, we investigated the FP-membrane binding patterns in the presence of cholesterol. The MD simulation results demonstrated that the coupling of Ca 2+ ions and cholesterol would also benefit the FP-membrane binding. Furthermore, our findings reveal that while the type of ion and cholesterol content exert varied and unpredictable influences on FP-membrane binding patterns, aromatic residues like tyrosine (Tyr) and phenylalanine (Phe) play an essential role in FP-membrane binding. In particular, deep mutational scanning (DMS) experiments have confirmed that mutating phenylalanine in the FP significantly decreases viral mutational fitness, emphasizing the pivotal role of phenylalanine residues in membrane fusion. This knowledge can aid in developing more effective therapeutic strategies targeting the viral fusion peptide and its key amino acids, ultimately contributing to developing treatments and vaccines against the virus.

The cell culture-based isolation of novel coronavirus SARS-CoV-2 from clinical specimens obtained from patients with suspected COVID-19 is important not only for laboratory diagnosis but also for obtaining live virus to characterize emerging variants. Previous studies report that monkey kidney-derived VeroE6/TMPRSS2 cells allow efficient isolation of SARS-CoV-2 from clinical specimens because these cells show stable expression of the receptor molecule monkey ACE2 and the serine-protease TMPRSS2. Here, we demonstrated that VeroE6 cells overexpressing human ACE2 and TMPRSS2 (Vero E6-TMPRSS2-T2A-ACE2 cells) are superior to VeroE6/TMPRSS2 for isolating SARS-CoV-2 from clinical specimens. These cells showed a 1.6-fold increase in efficiency in SARS-CoV-2 isolation, and were particularly effective for clinical specimens with a relatively low viral load (< 10 6 copies/mL). When using vesicular stomatitis virus (VSV) pseudoviruses (VSV/SARS-2pv) bearing the spike proteins of all of the tested SARS-CoV-2 strains, Vero E6-TMPRSS2-T2A-ACE2 cells showed a 2- to fourfold increase in infectivity. Furthermore, the results of virus titration and neutralization antibody assays using Vero E6-TMPRSS2-T2A-ACE2 cells were different from those using VeroE6/TMPRSS2, highlighting the importance of selecting appropriate cell culture systems to determine SARS-CoV-2 infectivity., (© 2024. The Author(s).)

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential., Competing Interests: Competing interests statement:The authors declare no competing interest.

Viral capture and entry to target cells are the first crucial steps that ultimately lead to viral infection. Understanding these events is essential toward the design and development of suitable antiviral drugs and/or vaccines. Viral capture involves dynamic interactions of the virus with specific receptors in the plasma membrane of the target cells. In the last years, single virus tracking has emerged as a powerful approach to assess real time dynamics of viral processes in living cells and their engagement with specific cellular components. However, direct visualization of the early steps of multireceptor viral interactions at the single level has been largely impeded by the technical challenges associated with imaging individual multimolecular systems at relevant spatial (nanometer) and temporal (millisecond) scales. Here, we present a four-color, high-density quantum dot spatiotemporal mapping methodology to capture real-time interactions between individual virus-like-particles (VLPs) and three different viral (co-) receptors on the membrane of primary living immune cells derived from healthy donors. Together with quantitative tools, our approach revealed the existence of a coordinated spatiotemporal diffusion of the three different (co)receptors prior to viral engagement. By varying the temporal-windows of cumulated single-molecule localizations, we discovered that such a concerted diffusion impacts on the residence time of HIV-1 and SARS-CoV-2 VLPs on the host membrane and potential viral infectivity. Overall, our methodology offers the possibility for systematic analysis of the initial steps of viral-host interactions and could be easily implemented for the investigation of other multimolecular systems at the single-molecule level.

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors., Competing Interests: The authors declare no conflict of interest.

Single-molecule force spectroscopy (SMFS) experiments can monitor protein refolding by applying a small force of a few piconewtons (pN) and slowing down the folding process. Bell theory predicts that in the narrow force regime where refolding can occur, the folding time should increase exponentially with increased external force. In this work, using coarse-grained molecular dynamics simulations, we compared the refolding pathways of SARS-CoV-1 RBD and SARS-CoV-2 RBD (RBD refers to the receptor binding domain) starting from unfolded conformations with and without a force applied to the protein termini. For SARS-CoV-2 RBD, the number of trajectories that fold is significantly reduced with the application of a 5 pN force, indicating that, qualitatively consistent with Bell theory, refolding is slowed down when a pulling force is applied to the termini. In contrast, the refolding times of SARS-CoV-1 RBD do not change meaningfully when a force of 5 pN is applied. How this lack of a Bell response could arise at the molecular level is unknown. Analysis of the entanglement changes of the folded conformations revealed that in the case of SARS-CoV-1 RBD, an external force minimizes misfolding into kinetically trapped states, thereby promoting efficient folding and offsetting any potential slowdown due to the external force. These misfolded states contain non-native entanglements that do not exist in the native state of either SARS-CoV-1-RBD or SARS-CoV-2-RBD. These results indicate that non-Bell behavior can arise from this class of misfolding and, hence, may be a means of experimentally detecting these elusive, theoretically predicted states.

Several antiviral therapeutic approaches have been targeted toward the RNA-dependent RNA polymerase (RdRp) complex that is involved in viral genome replication. In SARS-CoV-2, although the RdRp is a multiprotein complex, the focus has been on the ligand binding catalytic core (nonstructural protein nsp12), and not the multiprotein functional dynamics. In this study, we focus on the conformational ensembles of the RdRp complex and their modulation by the presence of RNA, performing comprehensive microsecond-scale atomistic simulations of the apo- and RNA-bound complex. We delineate the differential impact of RNA on the constituent proteins, such as conformational polymorphisms, dominant segment-specific fluctuations, and the switch in dynamical crosstalk within the complex. We distinguish dynamical signatures of nsp7, nsp8, and nsp12 in the apo-state that are reduced in the presence of the RNA and appear to "prime" the complex for activity. Importantly, we identify a unique structural malleability of the nsp8 protein with high conformational heterogeneity in the apo state, especially at three sites (Y71 for nsp8A, and D52 and A66 for nsp8B). Our work highlights the functional implications of the polymorphism of nsp8 structures and reveals possibilities for the development of allosteric inhibitors.

Guanylate-binding protein (GBP) 5 is an interferon-inducible cellular factor with broad anti-viral activity. Recently, GBP5 has been shown to antagonize the glycoproteins of a number of enveloped viruses, in part by disrupting the host enzyme furin. Here we show that GBP5 strongly impairs the infectivity of virus particles bearing not only viral glycoproteins that depend on furin cleavage for infectivity-the envelope (Env) glycoproteins of HIV-1 and murine leukemia virus and the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-but also viral glycoproteins that do not depend on furin cleavage: vesicular stomatitis virus glycoprotein and SARS-CoV S. We observe that GBP5 disrupts proper N -linked protein glycosylation and reduces the incorporation of viral glycoproteins into virus particles. The glycosylation of the cellular protein CD4 is also altered by GBP5 expression. Flow cytometry analysis shows that GBP5 expression reduces the cell-surface levels of HIV-1 Env and the S glycoproteins of SARS-CoV and SARS-CoV-2. Our data demonstrate that, under the experimental conditions used, inhibition of furin-mediated glycoprotein cleavage is not the primary anti-viral mechanism of action of GBP5. Rather, the antagonism appears to be related to impaired trafficking of glycoproteins to the plasma membrane. These results provide novel insights into the broad antagonism of viral glycoprotein function by the cellular host innate immune response., Importance: The surface of enveloped viruses contains viral envelope glycoproteins, an important structural component facilitating virus attachment and entry while also acting as targets for the host adaptive immune system. In this study, we show that expression of GBP5 in virus-producer cells alters the glycosylation, cell-surface expression, and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into the broad impact of the host cell anti-viral factor GBP5 on protein glycosylation and trafficking., Competing Interests: The authors declare no conflict of interest.

The recent COVID-19 pandemics have demonstrated the great therapeutic potential of in vitro transcribed (IVT) mRNAs, but improvements in their biochemical properties, such as cellular stability, reactogenicity and translational activity, are critical for further practical applications in gene replacement therapy and anticancer immunotherapy. One of the strategies to overcome these limitations is the chemical modification of a unique mRNA 5'-end structure, the 5'-cap, which is responsible for regulating translation at multiple levels. This could be achieved by priming the in vitro transcription reaction with synthetic cap analogs. In this study, we combined a highly efficient trinucleotide IVT capping technology with several modifications of the 5' cap triphosphate bridge to synthesize a series of 16 new cap analogs. We also combined these modifications with epigenetic marks (2'-O-methylation and m6Am) characteristic of mRNA 5'-ends in higher eukaryotes, which was not possible with dinucleotide caps. All analogs were compared for their effect on the interactions with eIF4E protein, IVT priming, susceptibility to decapping, and mRNA translation efficiency in model cell lines. The most promising α-phosphorothiolate modification was also evaluated in an in vivo mouse model. Unexpected differences between some of the analogs were analyzed using a protein cell extract pull-down assay., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here we used native mass spectrometry (native MS) to probe a SARS-CoV protease, PLpro, which plays critical roles in coronavirus disease by affecting viral protein production and antagonizing host antiviral responses. Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to localize binding sites of PLpro inhibitors and revealed the stabilizing effects of inhibitors on protein tertiary structure. We compared PLpro from SARS-CoV-1 and SARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern possible differences in protease function. A PLpro mutant lacking a single cysteine was used to localize inhibitor binding, and thermodynamic measurements revealed that inhibitor PR-619 stabilized the folded PLpro structure. These results will inform further development of PLpro as a therapeutic target against SARS-CoV-2 and other emerging coronaviruses.

The precise cellular mechanisms underlying heightened proinflammatory cytokine production during coronavirus infection remain incompletely understood. Here we identify the envelope (E) protein in severe coronaviruses (SARS-CoV-2, SARS, or MERS) as a potent inducer of interleukin-1 release, intensifying lung inflammation through the activation of TMED10-mediated unconventional protein secretion (UcPS). In contrast, the E protein of mild coronaviruses (229E, HKU1, or OC43) demonstrates a less pronounced effect. The E protein of severe coronaviruses contains an SS/DS motif, which is not present in milder strains and facilitates interaction with TMED10. This interaction enhances TMED10-oligomerization, facilitating UcPS cargo translocation into the ER-Golgi intermediate compartment (ERGIC)-a pivotal step in interleukin-1 UcPS. Progesterone analogues were identified as compounds inhibiting E-enhanced release of proinflammatory factors and lung inflammation in a Mouse Hepatitis Virus (MHV) infection model. These findings elucidate a molecular mechanism driving coronavirus-induced hyperinflammation, proposing the E-TMED10 interaction as a potential therapeutic target to counteract the adverse effects of coronavirus-induced inflammation., (© 2024. The Author(s).)

Since 2019, SARS-CoV-2 has undergone mutations, resulting in pandemic and epidemic waves. The SARS-CoV-2 spike protein, crucial for cellular entry, binds to the ACE2 receptor exclusively when its receptor-binding domain (RBD) adopts the up-conformation. However, whether ACE2 also interacts with the RBD in the down-conformation to facilitate the conformational shift to RBD-up remains unclear. Herein, we present the structures of the BA.2.86 and the JN.1 spike proteins bound to ACE2. Notably, we successfully observed the ACE2-bound down-RBD, indicating an intermediate structure before the RBD-up conformation. The wider and mobile angle of RBDs in the up-state provides space for ACE2 to interact with the down-RBD, facilitating the transition to the RBD-up state. The K356T, but not N354-linked glycan, contributes to both of infectivity and neutralizing-antibody evasion in BA.2.86. These structural insights the spike-protein dynamics would help understand the mechanisms underlying SARS-CoV-2 infection and its neutralization., (© 2024. The Author(s).)

Among biomimetic strategies shaping engineering designs, molecularly imprinted polymer (MIP) technology stands out, involving chemically synthesised receptors emulating natural antigen-antibody interactions. These versatile 'designer polymers' with remarkable stability and low cost, are pivotal for in vitro diagnostics. Amid the recent global health crisis, we probed MIPs' potential to capture SARS-CoV-2 virions. Large biotemplates complicate MIP design, influencing generated binding site specificity. To precisely structure recognition sites within polymers, we innovated an epitope imprinting method supplemented by in silico polymerization component screening. A viral surface Spike protein informed epitope selection was targeted for MIP development. A novel multi-monomer docking approach (MMSD) was employed to simulate classical receptor-ligand interactions, mimicking binding reinforcement across multiple amino acids. Around 40 monomer combinations were docked to the epitope sequence and top performers experimentally validated via rapid fluorescence binding assays. Notably, high imprinting factor polymers correlated with MMSD predictions, promising rational MIP design applicable to diverse viral pathologies., (© 2024. The Author(s).)

The majority of viruses classified as pandemic threats are enveloped viruses which enter the cell through receptor-mediated endocytosis and take advantage of endosomal acidification to activate their fusion machinery. Here we report that the endosomal fusion of low pH-requiring viruses is highly dependent on TRPM7, a widely expressed TRP channel that is located on the plasma membrane and in intracellular vesicles. Using several viral infection systems expressing the envelope glycoproteins of various viruses, we find that loss of TRPM7 protects cells from infection by Lassa, LCMV, Ebola, Influenza, MERS, SARS-CoV-1, and SARS-CoV-2. TRPM7 ion channel activity is intrinsically necessary to acidify virus-laden endosomes but is expendable for several other endosomal acidification pathways. We propose a model wherein TRPM7 ion channel activity provides a countercurrent of cations from endosomal lumen to cytosol necessary to sustain the pumping of protons into these virus-laden endosomes. This study demonstrates the possibility of developing a broad-spectrum, TRPM7-targeting antiviral drug to subvert the endosomal fusion of low pH-dependent enveloped viruses., (© 2024. The Author(s).)

Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions., Competing Interests: Competing interests statement:P.K. is a nonexecutive director, shareholder of and consultant to Refeyn Ltd., J.L.P.B. and W.B.S. are shareholders of and consultants to Refeyn Ltd. W.B.S. and P.K. received the University of Oxford’s COVID-19 Research Response Fund. P.K. has filed a patent for the contrast enhancement methodology and its application to mass measurement of single biomolecules. G.R.S. is on the GSK Vaccines Scientific Advisory Board, a founder shareholder of RQ biotechnology and Jenner investigator. Oxford University holds intellectual property related to the Oxford-AstraZeneca vaccine.

The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3'-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications., (© 2024. The Author(s).)

Spike (S) glycoprotein is the largest structural protein of SARS-CoV-2 virus and the main one involved in anchoring of the host receptor ACE2 through the receptor binding domain (RBD). S protein secondary structure is of great interest for shedding light on various aspects, from functionality to pathogenesis, finally to spectral fingerprint for the design of optical biosensors. In this paper, the secondary structure of SARS-CoV-2 S protein and its constituting components, namely RBD, S1 and S2 regions, are investigated at serological pH by measuring their amide I infrared absorption bands through Attenuated Total Reflection Infrared (ATR-IR) spectroscopy. Experimental data in combination with MultiFOLD predictions, Define Secondary Structure of Proteins (DSSP) web server and Gravy value calculations, provide a comprehensive understanding of RBD, S1, S2, and S proteins in terms of their secondary structure content, conformational order, and interaction with the solvent., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralising antibodies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microtubule affinity-regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS-CoV-2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co-expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b-mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The dynamic evolution of SARS-CoV-2 variants necessitates ongoing advancements in therapeutic strategies. Despite the promise of monoclonal antibody (mAb) therapies like bebtelovimab, concerns persist regarding resistance mutations, particularly single-to-multipoint mutations in the receptor-binding domain (RBD). Our study addresses this by employing interface-guided computational protein design to predict potential bebtelovimab-resistance mutations. Through extensive physicochemical analysis, mutational preferences, precision-recall metrics, protein-protein docking, and energetic analyses, combined with all-atom, and coarse-grained molecular dynamics (MD) simulations, we elucidated the structural-dynamics-binding features of the bebtelovimab-RBD complexes. Identification of susceptible RBD residues under positive selection pressure, coupled with validation against bebtelovimab-escape mutations, clinically reported resistance mutations, and viral genomic sequences enhances the translational significance of our findings and contributes to a better understanding of the resistance mechanisms of SARS-CoV-2., (© 2024 Federation of European Biochemical Societies.)

The SARS-CoV-2 pandemic, responsible for approximately 7 million deaths worldwide, highlights the urgent need to understand the molecular mechanisms of the virus in order to prevent future outbreaks. The Spike glycoprotein of SARS-CoV-2, which is critical for viral entry through its interaction with ACE2 and other host cell receptors, has been a focus of this study. The present research goes beyond receptor recognition to explore Spike's influence on cellular metabolism. AP-MS interactome analysis revealed an interaction between the Spike S1 domain and lactate dehydrogenase B (LDHB), which was further confirmed by co-immunoprecipitation and immunofluorescence, indicating colocalisation in cells expressing the S1 domain. The study showed that Spike inhibits the catalytic activity of LDHB, leading to increased lactate levels in HEK-293T cells overexpressing the S1 subunit. In the hypothesised mechanism, Spike deprives LDHB of NAD + , facilitating a metabolic switch from aerobic to anaerobic energy production during infection. The Spike-NAD + interacting region was characterised and mainly involves the W436 within the RDB domain. This novel hypothesis suggests that the Spike protein may play a broader role in altering host cell metabolism, thereby contributing to the pathophysiology of viral infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)