SARS-CoV-2 Nucleocapsid Protein Induces Tau Pathological Changes That Can Be Counteracted by SUMO2.

Neurologic manifestations are an immediate consequence of SARS-CoV-2 infection, the etiologic agent of COVID-19, which, however, may also trigger long-term neurological effects. Notably, COVID-19 patients with neurological symptoms show elevated levels of biomarkers associated with brain injury, including Tau proteins linked to Alzheimer's pathology. Studies in brain organoids revealed that SARS-CoV-2 alters the phosphorylation and distribution of Tau in infected neurons, but the mechanisms are currently unknown. We hypothesize that these pathological changes are due to the recruitment of Tau into stress granules (SGs) operated by the nucleocapsid protein (NCAP) of SARS-CoV-2. To test this hypothesis, we investigated whether NCAP interacts with Tau and localizes to SGs in hippocampal neurons in vitro and in vivo. Mechanistically, we tested whether SUMOylation, a posttranslational modification of NCAP and Tau, modulates their distribution in SGs and their pathological interaction. We found that NCAP and Tau colocalize and physically interact. We also found that NCAP induces hyperphosphorylation of Tau and causes cognitive impairment in mice infected with NCAP in their hippocampus. Finally, we found that SUMOylation modulates NCAP SG formation in vitro and cognitive performance in infected mice. Our data demonstrate that NCAP induces Tau pathological changes both in vitro and in vivo. Moreover, we demonstrate that SUMO2 ameliorates NCAP-induced Tau pathology, highlighting the importance of the SUMOylation pathway as a target of intervention against neurotoxic insults, such as Tau oligomers and viral infection.

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with N in PRRSV-Infected PAMs.

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein-protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus-host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines.

Molecular characterization of SARS-CoV-2 nucleocapsid protein.

Corona Virus Disease 2019 (COVID-19) is a highly prevalent and potent infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Until now, the world is still endeavoring to develop new ways to diagnose and treat COVID-19. At present, the clinical prevention and treatment of COVID-19 mainly targets the spike protein on the surface of SRAS-CoV-2. However, with the continuous emergence of SARS-CoV-2 Variants of concern (VOC), targeting the spike protein therapy shows a high degree of limitation. The Nucleocapsid Protein (N protein) of SARS-CoV-2 is highly conserved in virus evolution and is involved in the key process of viral infection and assembly. It is the most expressed viral structural protein after SARS-CoV-2 infection in humans and has high immunogenicity. Therefore, N protein as the key factor of virus infection and replication in basic research and clinical application has great potential research value. This article reviews the research progress on the structure and biological function of SARS-CoV-2 N protein, the diagnosis and drug research of targeting N protein, in order to promote researchers' further understanding of SARS-CoV-2 N protein, and lay a theoretical foundation for the possible outbreak of new and sudden coronavirus infectious diseases in the future.

The cryoEM structure of the Hendra henipavirus nucleoprotein reveals insights into paramyxoviral nucleocapsid architectures.

We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent Ni and Ni+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the Ni+1 N-terminus and Ni-1 C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.

Unveiling potential inhibitors targeting the nucleocapsid protein of SARS-CoV-2: Structural insights into their binding sites.

The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.

Modulation of biophysical properties of nucleocapsid protein in the mutant spectrum of SARS-CoV-2.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Like other types of RNA viruses, the genetic material of SARS-CoV-2 (the agent responsible for COVID-19) is formed of an RNA molecule which is prone to accumulating mutations. This gives SARS-CoV-2 the ability to evolve quickly, and often to remain one step ahead of treatments. Understanding how these mutations shape the behavior of RNA viruses is therefore crucial to keep diseases such as COVID-19 under control. The gene that codes for the protein that 'packages' the genetic information inside SARS-CoV-2 is particularly prone to mutations. This nucleocapsid (N) protein participates in many key processes during the life cycle of the virus, including potentially interfering with the immune response. Exactly how the physical properties of the N-Protein are impacted by the mutations in its genetic sequence remains unclear. To investigate this question, Nguyen et al. predicted the various biophysical properties of different regions of the N-protein based on a computer-based analysis of SARS-CoV-2 genetic databases. This allowed them to determine if specific protein regions were positively or negatively charged in different mutants. The analyses showed that some domains exhibited great variability in their charge between protein variants ­ reflecting the fact that the corresponding genetic sequences showed high levels of plasticity. Other regions remained conserved, however, including across related coronaviruses. Nguyen et al. also conducted biochemical experiments on a range of N-proteins obtained from clinically relevant SARS-CoV-2 variants. Their results highlighted the importance of protein segments with no fixed three-dimensional structure. Mutations in the related sequences created high levels of variation in the physical properties of these 'intrinsically disordered' regions, which had wide-ranging consequences. Some of these genetic changes even gave individual N-proteins the ability to interact with each other in a completely new way. These results shed new light on the relationship between genetic mutations and the variable physical properties of RNA virus proteins. Nguyen et al. hope that this knowledge will eventually help to develop more effective treatments for viral infections.

Expression and purification of an NP-hoc fusion protein: Utilizing influenza a nucleoprotein and phage T4 hoc protein.

Influenza poses a substantial health risk, with infants and the elderly being particularly susceptible to its grave impacts. The primary challenge lies in its rapid genetic evolution, leading to the emergence of new Influenza A strains annually. These changes involve punctual mutations predominantly affecting the two main glycoproteins: Hemagglutinin (HA) and Neuraminidase (NA). Our existing vaccines target these proteins, providing short-term protection, but fall short when unexpected pandemics strike. Delving deeper into Influenza's genetic makeup, we spotlight the nucleoprotein (NP) - a key player in the transcription, replication, and packaging of RNA. An intriguing characteristic of the NP is that it is highly conserved across all Influenza A variants, potentially paving the way for a more versatile and broadly protective vaccine. We designed and synthesized a novel NP-Hoc fusion protein combining Influenza A nucleoprotein and T4 phage Hoc, cloned using Gibson assembly in E. coli, and purified via ion affinity chromatography. Simultaneously, we explore the T4 coat protein Hoc, typically regarded as inconsequential in controlled viral replication. Yet, it possesses a unique ability: it can link with another protein, showcasing it on the T4 phage coat. Fusing these concepts, our study designs, expresses, and purifies a novel fusion protein named NP-Hoc. We propose this protein as the basis for a new generation of vaccines, engineered to guard broadly against Influenza A. The excitement lies not just in the immediate application, but the promise this holds for future pandemic resilience, with NP-Hoc marking a significant leap in adaptive, broad-spectrum influenza prevention.

Indiscriminate activities of different henipavirus polymerase complex proteins allow for efficient minigenome replication in hybrid systems.

The henipaviruses, including Nipah virus (NiV) and Hendra virus (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological and respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems that can be safely used in BSL-2 conditions. The nucleocapsid (N), phosphoprotein (P), and large protein (L) of henipaviruses are critical elements of their replication machinery and thus essential support components of the minigenome systems. Here, we tested the effects of diverse combinations of the replication support proteins on the replication capacity of the NiV and HeV minigenomes by exchanging the helper plasmids coding for these proteins among the two viruses. We demonstrate that all combinations including one or more heterologous proteins were capable of replicating both the NiV and HeV minigenomes. Sequence alignment showed identities of 92% for the N protein, 67% for P, and 87% for L. Notably, variations in amino acid residues were not concentrated in the N-P and P-L interacting regions implying that dissimilarities in amino acid composition among NiV and HeV polymerase complex proteins may not impact their interactions. The observed indiscriminate activity of NiV and HeV polymerase complex proteins is different from related viruses, which can support the replication of heterologous genomes only when the whole polymerase complex belongs to the same virus. This newly observed promiscuous property of the henipavirus polymerase complex proteins likely attributed to their conserved interaction regions could potentially be harnessed to develop universal anti-henipavirus antivirals.IMPORTANCEGiven the severity of disease induced by Hendra and Nipah viruses in humans and the continuous emergence of new henipaviruses as well as henipa-like viruses, it is necessary to conduct a more comprehensive investigation of the biology of henipaviruses and their interaction with the host. The replication of henipaviruses and the development of antiviral agents can be studied in systems that allow experiments to be performed under biosafety level 2 conditions. Here, we developed robust minigenome systems for the Nipah virus (NiV) and Hendra virus (HeV) that provide a convenient alternative for studying NiV and HeV replication. Using these systems, we demonstrate that any combination of the three polymerase complex proteins of NiV and HeV could effectively initiate the replication of both viral minigenomes, which suggests that the interaction regions of the polymerase complex proteins could be effective targets for universal and effective anti-henipavirus interventions.

Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure.

Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the sensitivity of influenza to myxovirus resistance protein 1 (MxA), an innate immunity factor that restricts influenza replication. A few critical MxA-resistant mutations have been identified in NP, including the highly conserved proline-283 substitution. This essential proline-283 substitution impairs influenza growth, a fitness defect that becomes particularly prominent at febrile temperature (39°C) when host chaperones are depleted. Here, we biophysically characterize proline-283 NP and serine-283 NP to test whether the fitness defect is caused by the proline-283 substitution introducing folding defects. We show that the proline-283 substitution changes the folding pathway of NP, making NP more aggregation prone during folding, but does not alter the native structure of the protein. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

The RBPome of influenza A virus NP-mRNA reveals a role for TDP-43 in viral replication.

Genome-wide approaches have significantly advanced our knowledge of the repertoire of RNA-binding proteins (RBPs) that associate with cellular polyadenylated mRNAs within eukaryotic cells. Recent studies focusing on the RBP interactomes of viral mRNAs, notably SARS-Cov-2, have revealed both similarities and differences between the RBP profiles of viral and cellular mRNAs. However, the RBPome of influenza virus mRNAs remains unexplored. Herein, we identify RBPs that associate with the viral mRNA encoding the nucleoprotein (NP) of an influenza A virus. Focusing on TDP-43, we show that it binds several influenza mRNAs beyond the NP-mRNA, and that its depletion results in lower levels of viral mRNAs and proteins within infected cells, and a decreased yield of infectious viral particles. We provide evidence that the viral polymerase recruits TDP-43 onto viral mRNAs through a direct interaction with the disordered C-terminal domain of TDP-43. Notably, other RBPs found to be associated with influenza virus mRNAs also interact with the viral polymerase, which points to a role of the polymerase in orchestrating the assembly of viral messenger ribonucleoproteins.

Impact of mutations on the stability of SARS-CoV-2 nucleocapsid protein structure.

The nucleocapsid (N) protein of SARS-CoV-2 is known to participate in various host cellular processes, including interferon inhibition, RNA interference, apoptosis, and regulation of virus life cycles. Additionally, it has potential as a diagnostic antigen and/or immunogen. Our research focuses on examining structural changes caused by mutations in the N protein. We have modeled the complete tertiary structure of native and mutated forms of the N protein using Alphafold2. Notably, the N protein contains 3 disordered regions. The focus was on investigating the impact of mutations on the stability of the protein's dimeric structure based on binding free energy calculations (MM-PB/GB-SA) and RMSD fluctuations after MD simulations. The results demonstrated that 28 mutations out of 37 selected mutations analyzed, compared with wild-type N protein, resulted in a stable dimeric structure, while 9 mutations led to destabilization. Our results are important to understand the tertiary structure of the N protein dimer of SARS-CoV-2 and the effect of mutations on it, their behavior in the host cell, as well as for the research of other viruses belonging to the same genus additionally, to anticipate potential strategies for addressing this viral illness․.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging. IMPORTANCE: Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.

Two Phylogenetic Cohorts of the Nucleocapsid Protein NP and Their Correlation with the Host Range of Influenza A Viruses.

Influenza A virus has a wide natural areal among birds, mammals, and humans. One of the main regulatory adaptors of the virus host range is the major NP protein of the viral nucleocapsid. Phylogenetic analysis of the NP protein of different viruses has revealed the existence of two phylogenetic cohorts in human influenza virus population. Cohort I includes classical human viruses that caused epidemics in 1957, 1968, 1977. Cohort II includes the H1N1/2009pdm virus, which had a mixed avian-swine origin but caused global human pandemic. Also, the highly virulent H5N1 avian influenza virus emerged in 2021 and caused outbreaks of lethal infections in mammals including humans, appeared to have the NP gene of the second phylogenetic cohort and, therefore, by the type of adaptation to human is similar to the H1N1/2009pdm virus and seems to possess a high epidemic potential for humans. The data obtained shed light on pathways and dynamics of adaptation of avian influenza viruses to humans and propose phylogenetic algorithm for systemic monitoring of dangerous virus strains to predict epidemic harbingers and take immediate preventive measures.

The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

Vimentin inhibits peste des petits ruminants virus replication by interaction with nucleocapsid protein.

The Peste des petits ruminant virus (PPRV) is a member of the Paramyxoviridae family and is classified into the genus Measles virus. PPRV predominantly infects small ruminants, leading to mortality rates of nearly 100%, which have caused significant economic losses in developing countries. Host proteins are important in virus replication, but the PPRV nucleocapsid (N) protein-host interacting partners for regulating PPRV replication remain unclear. The present study confirmed the interaction between PPRV-N and the host protein vimentin by co-immunoprecipitation and co-localization experiments. Overexpression of vimentin suppressed PPRV replication, whereas vimentin knockdown had the opposite effect. Mechanistically, N was subjected to degradation via the ubiquitin/proteasome pathway, where vimentin recruits the E3 ubiquitin ligase NEDD4L to fulfill N-ubiquitination, resulting in the degradation of the N protein. These findings suggest that the host protein vimentin and E3 ubiquitin ligase NEDD4L have an anti-PPRV effect.

RBM14 inhibits the replication of porcine epidemic diarrhea virus by recruiting p62 to degrade nucleocapsid protein through the activation of autophagy and interferon pathway.

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV.