In vivo structural characterization of the SARS-CoV-2 RNA genome identifies host proteins vulnerable to repurposed drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding of the RNA virus and its interactions with host proteins could improve therapeutic interventions for COVID-19. By using icSHAPE, we determined the structural landscape of SARS-CoV-2 RNA in infected human cells and from refolded RNAs, as well as the regulatory untranslated regions of SARS-CoV-2 and six other coronaviruses. We validated several structural elements predicted in silico and discovered structural features that affect the translation and abundance of subgenomic viral RNAs in cells. The structural data informed a deep-learning tool to predict 42 host proteins that bind to SARS-CoV-2 RNA. Strikingly, antisense oligonucleotides targeting the structural elements and FDA-approved drugs inhibiting the SARS-CoV-2 RNA binding proteins dramatically reduced SARS-CoV-2 infection in cells derived from human liver and lung tumors. Our findings thus shed light on coronavirus and reveal multiple candidate therapeutics for COVID-19 treatment.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Cerium Oxide Catalyzed Disproportionation of Hydrogen Peroxide: A Closer Look at the Reaction Intermediate.

Cerium oxide nanoparticles (CNPs) have recently gained increasing interest as redox enzyme-mimetics to scavenge the intracellular excess of reactive oxygen species, including hydrogen peroxide (H2 O2 ). Despite the extensive exploration, there remains a notable discrepancy regarding the interpretation of observed redshift of UV-Visible spectroscopy due to H2 O2 addition and the catalase-mimicking mechanism of CNPs. To address this question, we investigated the reaction mechanism by taking a closer look at the reaction intermediate during the catalase mimicking reaction. In this study, we present evidence demonstrating that in aqueous solutions, H2 O2 adsorption at CNP surface triggers the formation of stable intermediates known as cerium-peroxo (Ce-O2 2- ) and/or cerium-hydroperoxo (Ce-OOH- ) complexes as resolved by Raman scattering and UV-Visible spectroscopy. Polymer coating presents steric hinderance for H2 O2 accessibility to the solid-liquid interface limiting further intermediate formation. We demonstrate in depth that the catalytic reactivity of CNPs in the H2 O2 disproportionation reaction increases with the Ce(III)-fraction and decreases in the presence of polymer coatings. The developed approach using UV-Visible spectroscopy for the characterization of the surface peroxide species can potentially serve as a foundation for determining the catalytic reactivity of CNPs in the disproportionation of H2 O2 .

Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV-2.

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs-including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria-could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption.

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

A novel cell culture system modeling the SARS-CoV-2 life cycle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Rapid Construction of Caffeic Acid/p-Phenylenediamine Antifouling Hydrophilic Coating on a PVDF Membrane for Emulsion Separation.

The current methods of constructing modification strategies for hydrophilic membranes are time-consuming, complex in operation, and poor in universality, which limit their application on membranes. In this work, inspired by the adhesion properties and versatility of caffeic acid (CA) and p-phenylenediamine (PPDA), a simple, rapid, and universal method was designed for the separation of oil-in-water emulsion by preparing a stable hydrophilic coating separation membrane. The preparation time of the membrane was shortened to 40 min. The developed PVDF-PCA/PPDA membrane showed superhydrophilic and underwater superoleophobic properties. When applied to petroleum ether-in-water emulsion, isooctane-in-water emulsion, and dodecane-in-water emulsion separation, the oil rejection was more than 99.0%. In the circulating separation of 10 g/L soybean oil-in-water emulsion, the oil rejection was more than 99.3%, and the highest flux was 1036 L·m-2·h-1. The prepared PVDF-PCA/PPDA membrane performed well in the separation test of oily wastewater. The proposed strategy is simple and rapid; it may become a universal method for preparing membranes with super strong antifouling properties against viscous oil and accelerate the research progress of membrane separation of oil-in-water emulsions.

Synthesis of Stable Cerium Oxide Nanoparticles Coated with Phosphonic Acid-Based Functional Polymers.

Functional polymers, such as poly(ethylene glycol) (PEG), terminated with a single phosphonic acid, hereafter PEGik-Ph are often applied to coat metal oxide surfaces during post-synthesis steps but are not sufficient to stabilize sub-10 nm particles in protein-rich biofluids. The instability is attributed to the weak binding affinity of post-grafted phosphonic acid groups, resulting in a gradual detachment of the polymers from the surface. Here, we assess these polymers as coating agents using an alternative route, namely, the one-step wet-chemical synthesis, where PEGik-Ph is introduced with cerium precursors during the synthesis. Characterization of the coated cerium oxide nanoparticles (CNPs) indicates a core-shell structure, where the cores are 3 nm cerium oxide and the shell consists of functionalized PEG polymers in a brush configuration. Results show that CNPs coated with PEG1k-Ph and PEG2k-Ph are of potential interest for applications as nanomedicines due to their high Ce(III) content and increased colloidal stability in cell culture media. We further demonstrate that the CNPs in the presence of hydrogen peroxide show an additional absorbance band in the UV-vis spectrum, which is attributed to Ce-O22- peroxo-complexes and could be used in the evaluation of their catalytic activity for scavenging reactive oxygen species.

Hepatitis E Virus Life Cycle.

Hepatitis E virus (HEV) infects over 20 million people worldwide per year, leading to 30,000-40,000 deaths. In most cases HEV infection in a self-limited, acute illness. However, chronic infections could occur in immunocompromised individuals. Due to scarcity of robust cell culture models in vitro and genetic tractable animal models in vivo, the details of HEV life cycle, as well as its interaction with host cells still remain elusive, which dampens antivirals discovery. In this chapter, we present an update in the HEV infectious cycle steps: entry, genome replication/subgenomic RNA transcription, assembly, and release. Moreover, we discussed the future prospective on HEV research and illustrates important questions urgently to be addressed.

Encapsulating Metal Nanoparticles into a Layered Zeolite Precursor with Surface Silanol Nests Enhances Sintering Resistance.

Supported metal nanoparticles are used as heterogeneous catalysts but often deactivated due to sintering at high temperatures. Confining metal species into a porous matrix reduces sintering, yet supports rarely provide additional stabilization. Here, we used the silanol-rich layered zeolite IPC-1P to stabilize ultra-small Rh nanoparticles. By adjusting the IPC-1P interlayer space through swelling, we prepared various architectures, including microporous and disordered mesoporous. In situ scanning transmission electron microscopy confirmed that Rh nanoparticles are resistant to sintering at high temperature (750 °C, 6 hrs). Rh clusters strongly bind to surface silanol quadruplets at IPC-1P layers by hydrogen transfer to clusters, while high silanol density hinders their migration based on density functional theory calculations. Ultimately, combining swelling with long-chain surfactant and utilizing metal-silanol interactions resulted in a novel, catalytically active material-Rh@IPC_C22.

Identification of functional cis-acting RNA elements in the hepatitis E virus genome required for viral replication.

There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5' and 3' untranslated regions and three open reading frames (ORF). HEV genome has 5' cap and 3' poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains cis-acting elements that can be recognized by the virally encoded polymerase as "self" for replication. To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific "signal" that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector.

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Heterogeneity and Specificity of Nanoscale Adhesion Forces Measured between Self-Assembled Monolayers and Lignocellulosic Substrates: A Chemical Force Microscopy Study.

Lack of fundamental understanding of cellulase interactions with different plant cell wall components during cellulose saccharification hinders progress toward achieving an economic production of biofuels from renewable plant biomass. Here, chemical force microscopy (CFM) was utilized to quantify the interactions between two surfaces that model either hydrophilic or hydrophobic functional groups of cellulases and a set of lignocellulosic substrates prepared through Kraft, sulfite, or organosolv pulping with defined chemical composition. The measured forces were then decoupled into specific and nonspecific components using the Poisson statistical approach. Heterogeneities in the distributions of forces as a function of the pretreatment method were mapped. Our results showed that hydrophobic domains and chemical moieties involved in hydrogen bonding and polar interactions were homogeneously distributed on all substrates but with distribution densities that varied with the type of the pretreatment method used to prepare substrates. In addition, we showed that increasing surface lignin coverage increased the heterogeneity of the substrates. When forces were decoupled, our results indicated that xylan reduced the strength of hydrogen bonding between the hydrophilic model surface and substrates. Permanent dipole-dipole interactions dominated the adhesion of the hydrophilic model surface to lignosulfonates, whereas hydrophobic interactions facilitated the adhesion of the hydrophobic model surface to Kraft lignin. We further showed that the structure of lignin determines the type of forces that dominate lignocellulosic interactions with other surfaces. Our findings suggest that nonproductive binding of cellulases to lignocellulosic biomass can be reduced by altering the hydrophobicity and/or chemical moieties involved in the polar interactions and by utilizing organosolv as a pretreatment method.

Investigating commercial cellulase performances toward specific biomass recalcitrance factors using reference substrates.

Three commercial cellulase preparations, Novozymes Cellic(®) Ctec2, Dupont Accellerase(®) 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic(®) Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.

Single Atom Engineering for Nanorobotics.

The fields of single atom engineering represent cutting-edge areas in nanotechnology and materials science, pushing the boundaries of how small we can go in engineering functional devices and materials. Nanorobots, or nanobots, are robotic systems scaled down to the nanometer level and designed to perform tasks at similarly small scales. Single atom engineering, on the other hand, involves manipulating individual atoms to create precise materials and devices with controlled properties and functionalities. By integrating single atom engineering into nanorobotics, we unlock the potential to enable the precise incorporation of multiple functionalities onto these minuscule machines with nanometer-level precision. In this perspective, we describe the nascent field of single atom engineering in nanorobotics.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger.

Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl α-1,3-mannosyltransferase (also known as "asparagine-linked glycosylation 3", or ALG3) is involved in early N-linked glycan synthesis and thus is essential for formation of N-linked protein glycosylation. In this study, we examined the effects of alg3 gene deletion (alg3Δ) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3Δ delayed spore germination in liquid cultures of complete medium (CM), potato dextrose (PD), minimal medium (MM) and CM with addition of cAMP (CM+cAMP), and resulted in significant reduction of hyphal growth on CM, potato dextrose agar (PDA), and CM+cAMP and spore production on CM. The alg3Δ also led to a significant accumulation of red pigment on both liquid and solid CM cultures. The relative abundances of 54 of the total 215 proteins identified in the secretome were significantly altered as a result of alg3Δ, 63% of which were secreted at higher levels in alg3Δ strain than the parent. The rCel7A expressed in the alg3Δ mutant was smaller in size than that expressed in both wild-type and parental strains, but still larger than T. reesei Cel7A. The circular dichroism (CD)-melt scans indicated that change in glycosylation of rCel7A does not appear to impact the secondary structure or folding. Enzyme assays of Cel7A and rCel7A on nanocrystalline cellulose and bleached kraft pulp demonstrated that the rCel7As have improved activities on hydrolyzing the nanocrystalline cellulose. Overall, the results suggest that alg3 is critical for growth, sporulation, pigment production, and protein secretion in A. niger, and demonstrate the feasibility of this alternative approach to evaluate the roles of N-linked glycosylation in glycoprotein secretion and function.

Design and characterization of novel SARS-CoV-2 fusion inhibitors with N-terminally extended HR2 peptides.

Development of potent and broad-spectrum antivirals against SARS-CoV-2 remains one of top priorities, especially in the case of that current vaccines cannot effectively prevent viral transmission. We previously generated a group of fusion-inhibitory lipopeptides, with one formulation being evaluated under clinical trials. In this study, we dedicated to characterize the extended N-terminal motif (residues 1161-1168) of the so-called spike (S) heptad repeat 2 (HR2) region. Alanine scanning analysis of this motif verified its critical roles in S protein-mediated cell-cell fusion. Using a panel of HR2 peptides with the N-terminal extensions, we identified a peptide termed P40, which contained four extended N-terminal residues (VDLG) and exhibited improved binding and antiviral activities, whereas the peptides with further extensions had no such effects. Then, we developed a new lipopeptide P40-LP by modifying P40 with cholesterol, which exhibited dramatically increased activities in inhibiting SARS-CoV-2 variants including divergent Omicron sublineages. Moreover, P40-LP displayed a synergistic effect with IPB24 lipopeptide that was designed containing the C-terminally extended residues, and it could effectively inhibit other human coronaviruses, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Taken together, our results have provided valuable insights for understanding the structure-function relationship of SARS-CoV-2 fusion protein and offered novel antiviral strategies to fight against the COVID-19 pandemic.

SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the global pandemic of COVID-19. SARS-CoV-2 genome encodes a main protease (nsp5, also called Mpro) and a papain-like protease (nsp3, also called PLpro), which are responsible for processing viral polyproteins to assemble a functional replicase complex. In this study, we found that Mpro of SARS-CoV-2 can cleave human MAGED2 and other mammalian orthologs at Gln-263. Moreover, SARS-CoV and MERS-CoV Mpro can also cleave human MAGED2, suggesting MAGED2 cleavage by Mpro is an evolutionarily conserved mechanism of coronavirus infection in mammals. Intriguingly, Mpro from Beta variant cleaves MAGED2 more efficiently than wild type, but Omicron Mpro is opposite. Further studies show that MAGED2 inhibits SARS-CoV-2 infection at viral replication step. Mechanistically, MAGED2 is associated with SARS-CoV-2 nucleocapsid protein through its N-terminal region in an RNA-dependent manner, and this disrupts the interaction between SARS-CoV-2 nucleocapsid protein and viral genome, thus inhibiting viral replication. When MAGED2 is cleaved by Mpro, the N-terminal of MAGED2 will translocate into the nucleus, and the truncated MAGED2 is unable to suppress SARS-CoV-2 replication. This work not only discovers the antiviral function of MAGED2 but also provides new insights into how SARS-CoV-2 Mpro antagonizes host antiviral response. IMPORTANCE Host factors that restrict severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain elusive. Here, we found that MAGED2 can be cleaved by SARS-CoV-2 main protease (Mpro) at Gln-263. SARS-CoV and MERS-CoV Mpro can also cleave MAGED2, and MAGED2 from multiple species can be cleaved by SARS-CoV-2 Mpro. Mpro from Beta variant cleaves MAGED2 more efficiently efficiently than wild type, but Omicron is the opposite. MAGED2 depletion enhances SARS-CoV-2 infection, suggesting its inhibitory role in SARS-CoV-2 infection. Mechanistically, MAGED2 restricts SARS-CoV-2 replication by disrupting the interaction between nucleocapsid and viral genomes. When MAGED2 is cleaved, its N-terminal will translocate into the nucleus. In this way, Mpro relieves MAGED2' inhibition on viral replication. This study improves our understanding of complex viral-host interaction and provides novel targets to treat SARS-CoV-2 infection.

A tissue specific-infection mouse model of SARS-CoV-2.

Animal models play crucial roles in the rapid development of vaccines/drugs for the prevention and therapy of COVID-19, but current models have some deficits when studying the pathogenesis of SARS-CoV-2 on some special tissues or organs. Here, we generated a human ACE2 and SARS-CoV-2 NF/F knockin mouse line that constitutively expresses human ACE2 and specifically expresses SARS-CoV-2 N gene induced by Cre-recombinase. By crossing with Cre transgenic lines allowing for lung-specific and constitutive expression, we generated lung-specific (Sftpc-hACE2-NF/F) and constitutive SARS-CoV-2 N (EIIa-hACE2-NF/F) expressing mice. Upon intranasal infection with a SARS-CoV-2 GFP/ΔN strain which can only replicate in SARS-CoV-2 N expressed cells, we demonstrated that both the Sftpc-hACE2-NF/F and EIIa-hACE2-NF/F mice support viral replication. Consistent with our design, viral replication was limited to the lung tissues in Sftpc-hACE2-NF/F mice, while the EIIa-hACE2-NF/F mice developed infections in multiple tissues. Furthermore, our model supports different SARS-CoV-2 variants infection, and it can be successfully used to evaluate the effects of therapeutic monoclonal antibodies (Ab1F11) and antiviral drugs (Molnupiravir). Finally, to test the effect of SARS-CoV-2 infection on male reproduction, we generated Sertoli cell-specific SARS-CoV-2 N expressed mice by crossing with AMH-Cre transgenic line. We found that SARS-CoV-2 GFP/ΔN strain could infect Sertoli cells, led to spermatogenic defects due to the destruction of blood-testis barrier. Overall, combining with different tissue-specific Cre transgenic lines, the human ACE2 and SARS-CoV-2 NF/F line enables us to evaluate antivirals in vivo and study the pathogenesis of SARS-CoV-2 on some special tissues or organs.