Correction: Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses.

[This corrects the article DOI: 10.1371/journal.ppat.1003231.].

Deciphering Membrane-Protein Interactions and High-Throughput Antigen Identification with Cell Doublets.

Deciphering cellular interactions is essential to both understand the mechanisms underlying a broad range of human diseases, but also to manipulate therapies targeting these diseases. Here, the formation of cell doublets resulting from specific membrane ligand-receptor interactions is discovered. Based on this phenomenon, the study developed DoubletSeeker, a novel high-throughput method for the reliable identification of ligand-receptor interactions. The study shows that DoubletSeeker can accurately identify T cell receptor (TCR)-antigen interactions with high sensitivity and specificity. Notably, DoubletSeeker effectively captured paired TCR-peptide major histocompatibility complex (pMHC) information during a highly complex library-on-library screening and successfully identified three mutant TCRs that specifically recognize the MART-1 epitope. In turn, DoubletSeeker can act as an antigen discovery platform that allows for the development of novel immunotherapy targets, making it valuable for investigating fundamental tumor immunology.

Proteomic and phosphoproteomic analysis of responses to enterovirus A71 infection reveals novel targets for antiviral and viral replication.

Hand, foot, and mouth disease (HFMD) is a common infectious disease in infants and children, especially those under five years of age. EV-A71 is a common pathogen that causes HFMD and the primary pathogen leading to severe or fatal HFMD, which is characterized by neurological complications. However, the underlying mechanisms of EV-A71 pathogenesis remain largely unknown. In this report, we used proteomic and phosphorylated proteomic methods to characterize the proteome and phosphoproteome profiles of EV-A71-infected human neuroblastoma SK-N-SH cells. More than 7744 host proteins and 10069 phosphorylation modification sites were successfully quantified. Among them, 974 proteins and 3648 phosphorylation modification sites were regulated significantly during EV-A71 infection. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that EV-A71 altered cell biological processes, including protein synthesis, RNA splicing and metabolism in SK-N-SH cells. Notably, based on the prediction of upregulated kinases during EV-A71 infection, we identified specific kinase inhibitors approved by the FDA, with ceralasertib, bosutinib, flavin mononucleotide, minocycline, pimasertib and acetylcysteine inhibiting EV-A71 infection. Finally, EV-A71 proteins were found to be phosphorylated during infection, with one site (S184 on 3D polymerase) observed to be crucial for viral replication because a S184A mutation knocked out viral replication. The results improve our understanding of the host response to EV-A71 infection of neuroblastoma cells and provide potential targets for developing anti-EV-A71 strategies.

Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16.

Enterovirus 71 (EV71) and Coxsackie A16 (CVA16) are two major causative agents of hand, foot, and mouth disease (HFMD) in young children. However, the mechanisms regulating the replication and pathogenesis of EV71/CVA16 remain incompletely understood. We performed a genome-wide CRISPR-Cas9 knockout screen and identified Ragulator as a mediator of EV71-induced apoptosis and pyroptosis. The Ragulator-Rag complex is required for EV71 and CVA16 replication. Upon infection, the Ragulator-Rag complex recruits viral 3D protein to the lysosomal surface through the interaction between 3D and RagB. Disruption of the lysosome-tethered Ragulator-Rag-3D complex significantly impairs the replication of EV71/CVA16. We discovered a novel EV71 inhibitor, ZHSI-1, which interacts with 3D and significantly reduces the lysosomal tethering of 3D. ZHSI-1 treatment significantly represses replication of EV71/CVA16 as well as virus-induced pyroptosis associated with viral pathogenesis. Importantly, ZHSI-1 treatment effectively protects against EV71 infection in neonatal and young mice. Thus, our study indicates that targeting lysosome-tethered Ragulator-Rag-3D may be an effective therapeutic strategy for HFMD.

CD74 Interacts with Proteins of Enterovirus D68 To Inhibit Virus Replication.

Enterovirus D68 (EV-D68) is a member of the species Enterovirus D in the genus Enterovirus of the family Picornaviridae. As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Although the intrinsic restriction factors in the cell provide a frontline defense, the molecular nature of virus-host interactions remains elusive. Here, we provide evidence that the major histocompatibility complex class II chaperone, CD74, inhibits EV-D68 replication in infected cells by interacting with the second hydrophobic region of 2B protein, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. 3Cpro cleaves CD74 at Gln-125. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection. IMPORTANCE As an emerging non-polio enterovirus, EV-D68 is widely spread all over the world and causes severe neurological and respiratory illnesses. Here, we report that CD74 inhibits viral replication in infected cells by targeting 2B protein of EV-D68, while EV-D68 attenuates the antiviral role of CD74 through 3Cpro cleavage. The equilibrium between CD74 and EV-D68 3Cpro determines the outcome of viral infection.

Mutagenesis and structural studies reveal the basis for the specific binding of SARS-CoV-2 SL3 RNA element with human TIA1 protein.

Viral RNA-host protein interactions are indispensable during RNA virus transcription and replication, but their detailed structural and dynamical features remain largely elusive. Here, we characterize the binding interface for the SARS-CoV-2 stem-loop 3 (SL3) cis-acting element to human TIA1 protein with a combined theoretical and experimental approaches. The highly structured SARS-CoV-2 SL3 has a high binding affinity to TIA1 protein, in which the aromatic stacking, hydrogen bonds, and hydrophobic interactions collectively direct this specific binding. Further mutagenesis studies validate our proposed 3D binding model and reveal two SL3 variants have enhanced binding affinities to TIA1. And disruptions of the identified RNA-protein interactions with designed antisense oligonucleotides dramatically reduce SARS-CoV-2 infection in cells. Finally, TIA1 protein could interact with conserved SL3 RNA elements within other betacoronavirus lineages. These findings open an avenue to explore the viral RNA-host protein interactions and provide a pioneering structural basis for RNA-targeting antiviral drug design.

A review of PFAS adsorption from aqueous solutions: Current approaches, engineering applications, challenges, and opportunities.

Per- and polyfluoroalkyl substances (PFAS) have drawn great attention due to their wide distribution in water bodies and toxicity to human beings. Adsorption is considered as an efficient treatment technique for meeting the increasingly stringent environmental and health standards for PFAS. This paper systematically reviewed the current approaches of PFAS adsorption using different adsorbents from drinking water as well as synthetic and real wastewater. Adsorbents with large mesopores and high specific surface area adsorb PFAS faster, their adsorption capacities are higher, and the adsorption process are usually more effective under low pH conditions. PFAS adsorption mechanisms mainly include electrostatic attraction, hydrophobic interaction, anion exchange, and ligand exchange. Various adsorbents show promising performances but challenges such as requirements of organic solvents in regeneration, low adsorption selectivity, and complicated adsorbent preparations should be addressed before large scale implementation. Moreover, the aid of decision-making tools including response surface methodology (RSM), techno-economic assessment (TEA), life cycle assessment (LCA), and multi criteria decision analysis (MCDA) were discussed for engineering applications. The use of these tools is highly recommended prior to scale-up to determine if the specific adsorption process is economically feasible and sustainable. This critical review presented insights into the most fundamental aspects of PFAS adsorption that would be helpful to the development of effective adsorbents for the removal of PFAS in future studies and provide opportunities for large-scale engineering applications.

SARS-CoV-2 hijacks cellular kinase CDK2 to promote viral RNA synthesis.

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with nonstructural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp inhibitors.

Removal of perfluorooctanoic acid via polyethyleneimine modified graphene oxide: Effects of water matrices and understanding mechanisms.

This research aimed to evaluate the adsorption behaviors and mechanisms of perfluorooctanoic acid (PFOA) onto polyethyleneimine modified graphene oxide (GO-PEI) from aqueous solutions. The adsorption capacity was significantly improved by doping polyethyleneimine (PEI) onto graphene oxide (GO). The Brunauer-Emmett-Teller (BET) isotherm model was considered as the best isotherm model in describing the PFOA adsorption onto GO-PEI3 (wPEI/wGO = 3). GO-PEI3 exhibited high adsorption capacity (qe = 368.2 mg/g, calculated from BET isotherm model) and excellent stability. The maximum monolayer amount of PFOA adsorption onto GO-PEI3 (qm = 231.2 mg/g) was successfully evaluated. The calculated saturated concentration (Cs = 169.9 mg/L) of PFOA on GO-PEI3 closely agrees with its critical micelle concentration (CMC = 157.0 mg/L), suggesting the formation of multilayer hemi-micelles or micelles PFOA structures on the surface of GO-PEI3. PFOA adsorption onto GO-PEI3 was inhibited by several factors including: the presence of humic acid (HA) by competing with the adsorption sites, background salts through the double-layer compression effect, and the competition from soluble ions for the amine or amide functional groups on GO-PEI3. Finally, both the FT-IR and XPS results confirmed that the adsorption of PFOA onto GO-PEI3 was through electrostatic attraction and hydrophobic interaction (physical adsorption), but not chemical adsorption. This work provides fundamental knowledge both in understanding the adsorption behavior through the BET isotherm model and in developing a stable adsorbent for PFOA adsorption. In addition, the findings highlight the potential of PFOA remediation from wastewater systems using GO-PEI in engineering applications.

Surface-bound hydroxyl radical-dominated degradation of sulfamethoxazole in the amorphous FeOOH/ peroxymonosulfate system: The key role of amorphous structure enhancing electron transfer.

In this study, activation of peroxymonosulfate (PMS) by amorphous FeOOH to degrade sulfamethoxazole (SMX) was investigated. The amorphous FeOOH showed a better performance in the decomposition of PMS and the degradation of SMX than the crystallized α-FeOOH and ß-FeOOH. The quenching experiments and EPR measurements suggested that the mechanism of PMS activation by amorphous FeOOH was mainly the surface-bound radicals (●OH and SO4●-). Basically, the surface-bound ●OH radicals were the dominate reactive oxide species in this system, which were mainly generated via the decomposition of amorphous FeOOH-PMS complexes. The degradation of SMX was significantly inhibited with the presence of H2PO4-, and this adverse impact was negligibly affected by the increase of H2PO4- concentration, implying that the inhibition of SMX degradation was caused by competitive adsorption. Consequently, the Fe-OH bonds on the amorphous FeOOH were proposed as the reactive sites for forming amorphous FeOOH-PMS complexes. Besides, the amorphous FeOOH showed a better performance in the degradation of SMX in the acid conditions than that in the base conditions due to the surface charge of amorphous FeOOH. More importantly, the reduction efficiency of Fe(III) was significantly enhanced due to the excellent conductivity of amorphous FeOOH.

SARS-CoV-2 fusion-inhibitory lipopeptides maintain high potency against divergent variants of concern including Omicron.

The emergence of SARS-CoV-2 Omicron and other variants of concern (VOCs) has brought huge challenges to control the COVID-19 pandemic, calling for urgent development of effective vaccines and therapeutic drugs. In this study, we focused on characterizing the impacts of divergent VOCs on the antiviral activity of lipopeptide-based fusion inhibitors that we previously developed. First, we found that pseudoviruses bearing the S proteins of five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) and one variant of interest (Lambda) exhibited greatly decreased infectivity relative to the wild-type (WT) strain or single D614G mutant, especially the Omicron pseudovirus. Differently, the most of variants exhibited an S protein with significantly enhanced cell fusion activity, whereas the S protein of Omicron still mediated decreased cell-cell fusion. Next, we verified that two lipopeptide-based fusion inhibitors, IPB02V3 and IPB24, maintained the highly potent activities in inhibiting various S proteins-driven cell fusion and pseudovirus infection. Surprisingly, both IPB02V3 and IPB24 lipopeptides displayed greatly increased potencies against the infection of authentic Omicron strain relative to the WT virus. The results suggest that Omicron variant evolves with a reduced cell fusion capacity and is more sensitive to the inhibition of fusion-inhibitory lipopeptides; thus, IPB02V3 and IPB24 can be further developed as potent, broad-spectrum antivirals for combating Omicron and the potential future outbreak of other emerging variants.

Influenza A virus NS1 protein hijacks YAP/TAZ to suppress TLR3-mediated innate immune response.

The Hippo signaling pathway, which is historically considered as a dominator of organ development and homeostasis has recently been implicated as an immune regulator. However, its role in host defense against influenza A virus (IAV) has not been widely investigated. Here, we found that IAV could activate the Hippo effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) through physical binding of the IAV non-structural protein 1 (NS1) with C-terminal domain of YAP/TAZ, facilitating their nuclear location. Meanwhile, YAP/TAZ downregulated the expression of pro-inflammatory and anti-viral cytokines against IAV infection, therefore benefiting virus replication and host cell apoptosis. A mouse model of IAV infection further demonstrated Yap deficiency protected mice against IAV infection, relieving lung injury. Mechanistically, YAP/TAZ blocked anti-viral innate immune signaling via downregulation of Toll-like receptor 3 (TLR3) expression. YAP directly bound to the putative TEADs binding site on the promoter region of TLR3. The elimination of acetylated histone H3 occupancy in the TLR3 promoter resulted in its transcriptional silence. Moreover, treatment of Trichostatin A, a histone deacetylases (HDACs) inhibitor or disruption of HDAC4/6 reversed the inhibition of TLR3 expression by YAP/TAZ, suggesting HDAC4/6 mediated the suppression function of YAP/TAZ. Taken together, we uncovered a novel immunomodulatory mechanism employed by IAV, where YAP/TAZ antagonize TLR3-mediated innate immunity.

Screening for inhibitors against SARS-CoV-2 and its variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, generating new variants that pose a threat to global health; therefore, it is imperative to obtain safe and broad-spectrum antivirals against SARS-CoV-2 and its variants. To this end, we screened compounds for their ability to inhibit viral entry, which is a critical step in virus infection. Twenty compounds that have been previously reported to inhibit SARS-CoV-2 replication were tested by using pseudoviruses containing the spike protein from the original strain (SARS-CoV-2-WH01). The cytotoxicity of these compounds was determined. Furthermore, we identified six compounds with strong antagonistic activity against the WH01 pseudovirus, and low cytotoxicity was identified. These compounds were then evaluated for their efficacy against pseudoviruses expressing the spike protein from B.1.617.2 (Delta) and B.1.1.529 (Omicron), the two most prevalent circulating strains. These assays demonstrated that two phenothiazine compounds, trifluoperazine 2HCl and thioridazine HCl, inhibit the infection of Delta and Omicron pseudoviruses. Finally, we discovered that these two compounds were highly effective against authentic SARS-CoV-2 viruses, including the WH01, Delta, and Omicron strains. Our study identified potential broad-spectrum SARS-CoV-2 inhibitors and provided insights into the development of novel therapeutics.

Gasdermin E is required for induction of pyroptosis and severe disease during enterovirus 71 infection.

Pyroptosis is an inflammatory form of programmed cell death that is executed by the gasdermin (GSDM)-N domain of GSDM family proteins, which form pores in the plasma membrane. Although pyroptosis acts as a host defense against invasive pathogen infection, its role in the pathogenesis of enterovirus 71 (EV71) infection is unclear. In the current study, we found that EV71 infection induces cleavage of GSDM E (GSDME) by using western blotting analysis, an essential step in the switch from caspase-3-mediated apoptosis to pyroptosis. We show that this cleavage is independent of the 3C and 2A proteases of EV71. However, caspase-3 activation is essential for this cleavage, as GSDME could not be cleaved in caspase-3-KO cells upon EV71 infection. Further analyses showed that EV71 infection induced pyroptosis in WT cells but not in caspase-3/GSDME double-KO cells. Importantly, GSDME is required to induce severe disease during EV71 infection, as GSDME deficiency in mice was shown to alleviate pathological symptoms. In conclusion, our results reveal that GSDME is important for the pathogenesis of EV71 via mediating initiation of pyroptosis.

ID2 inhibits innate antiviral immunity by blocking TBK1- and IKKε-induced activation of IRF3.

The transcription regulator ID2 plays an essential role in the development and differentiation of immune cells. Here, we report that ID2 also negatively regulates antiviral innate immune responses. During viral infection of human epithelial cells, ID2 bound to TANK-binding kinase 1 (TBK1) and to inhibitor of nuclear factor κB kinase ε (IKKε). These interactions inhibited the recruitment and activation of interferon (IFN) regulatory factor 3 (IRF3) by TBK1 or IKKε, leading to a reduction in the expression of IFN-ß1 (IFNB1). IFN-ß induced the nuclear export of ID2 to form a negative feedback loop. Knocking out ID2 in human cells enhanced innate immune responses and suppressed infection by different viruses, including SARS-CoV-2. Mice with a myeloid-specific deficiency of ID2 produced more IFN-α in response to viral infection and were more resistant to viral infection than wild-type mice. Our findings not only establish ID2 as a modulator of IRF3 activation induced by TBK1 and/or IKKε but also introduce a mechanism for cross-talk between innate immunity and cell development and differentiation.

Insight into the activation mechanisms of biochar by boric acid and its application for the removal of sulfamethoxazole.

Sulfamethoxazole (SMX) is frequently detected in the environment and causes a huge threaten to human health. Biochar (BC) is a metal-free adsorbent and generally exhibits a good adsorption capacity for SMX. However, the current activated methods usually result in the high energy consumption and low yield of the biochar. In this study, biochar was activated by boric acid under limited oxygen condition. The yield of biochar was increased by 103% after the activated by boric acid. The specific surface area of BC was significantly increased from 766.6 m2·g-1 to 1190.6 m2·g-1. The intensity of the (111) diamond peak of B-BC was higher than that of BC, suggesting that boric acid affected the surface pyrolysis temperature of biochar. The proposed roles of boric acid in the activation process were to: 1) enhance the generation of micropores during the pyrolysis process; 2) improve the yield of biochar via the transformation pathways of C-corresponding bonds and physical blocking. The boric acid activated biochar (B-BC) had a higher adsorption capacity for SMX than BC under the various aqueous conditions. Hence, boric acid activated biochar is a promising porous adsorbent to enhance the removal of SMX and achieve practical application.

An antibody-based proximity labeling map reveals mechanisms of SARS-CoV-2 inhibition of antiviral immunity.

The global epidemic caused by the coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in the infection of over 200 million people. To extend the knowledge of interactions between SARS-CoV-2 and humans, we systematically investigate the interactome of 29 viral proteins in human cells by using an antibody-based TurboID assay. In total, 1,388 high-confidence human proximal proteins with biotinylated sites are identified. Notably, we find that SARS-CoV-2 manipulates the antiviral and immune responses. We validate that the membrane protein ITGB1 associates angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 entry. Moreover, we reveal that SARS-CoV-2 proteins inhibit activation of the interferon pathway through the mitochondrial protein mitochondrial antiviral-signaling protein (MAVS) and the methyltransferase SET domain containing 2, histone lysine methyltransferase (SETD2). We propose 111 potential drugs for the clinical treatment of coronavirus disease 2019 (COVID-19) and identify three compounds that significantly inhibit the replication of SARS-CoV-2. The proximity labeling map of SARS-CoV-2 and humans provides a resource for elucidating the mechanisms of viral infection and developing drugs for COVID-19 treatment.

Enhanced adsorption of perfluorooctanoate (PFOA) onto low oxygen content ordered mesoporous carbon (OMC): Adsorption behaviors and mechanisms.

The pollution of perfluorooctanoic acid (PFOA) in water bodies has been a serious threat to environment and human health. Ordered mesoporous carbons (OMCs) with different oxygen contents were prepared and first used for adsorbing PFOA from aqueous solutions. The OMC-900 with a lower oxygen content has a higher PFOA adsorption capacity than the oxygen-rich OMC-700. OMCs require a much shorter time to reach the adsorption equilibrium comparing with other adsorbents reported in literature. The mesopores play an important role in this rapid adsorption kinetics. The pseudo-second-order model better fitted the kinetic data. The multilayers adsorption was proposed for the adsorption of PFOA onto OMCs since the Freundlich isotherm model fits the experimental data well. The micelle or hemi-micelle structures may be formed during the adsorption. Various background salts showed a positive effect on PFOA adsorption due to the salting-out and divalent bridge effects. The humic acid can lead to a discernible reduction in PFOA adsorption by competing for adsorption sites on OMCs. The hydrophobic interaction and electrostatic interaction adsorption mechanisms were proposed and verified by the adsorption data. The high adsorption capacity and fast adsorption kinetics of the OMC make it a potential adsorbent for PFOA removal in engineering applications.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection.

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.