RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Sistemas CRISPR-Cas , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Células A549 , Profilaxis Antibiótica/métodos , Secuencia de Bases , Betacoronavirus/genética , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Simulación por Computador , Secuencia Conservada , Coronavirus/efectos de los fármacos , Coronavirus/genética , Coronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/tratamiento farmacológico , Proteínas de la Nucleocápside de Coronavirus , ARN Polimerasa Dependiente de ARN de Coronavirus , Células Epiteliales/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Pulmón/patología , Pulmón/virología , Proteínas de la Nucleocápside/genética , Pandemias , Fosfoproteínas , Filogenia , Neumonía Viral/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/genética , SARS-CoV-2 , Proteínas no Estructurales Virales/genéticaRESUMEN
Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell.
Asunto(s)
Orthohantavirus , Proteoma , Humanos , Codón Iniciador , Proteoma/metabolismo , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Células Endoteliales/metabolismo , Regiones no Traducidas 5' , Orthohantavirus/genética , ARN Mensajero/genética , Péptidos/metabolismo , Biosíntesis de ProteínasRESUMEN
The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.
Asunto(s)
Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Euterios/virología , Evolución Molecular , Genoma Viral/genética , Homología de Secuencia de Ácido Nucleico , Animales , Betacoronavirus/clasificación , COVID-19 , China , Quirópteros/virología , Chlorocebus aethiops , Proteínas de la Envoltura de Coronavirus , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Proteínas M de Coronavirus , Proteínas de la Nucleocápside de Coronavirus , Reservorios de Enfermedades/virología , Genómica , Especificidad del Huésped , Humanos , Pulmón/patología , Pulmón/virología , Malasia , Proteínas de la Nucleocápside/genética , Pandemias , Fosfoproteínas , Filogenia , Neumonía Viral/epidemiología , Neumonía Viral/transmisión , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa , Recombinación Genética , SARS-CoV-2 , Alineación de Secuencia , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genética , Zoonosis/transmisión , Zoonosis/virologíaRESUMEN
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.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , Multimerización de Proteína , ARN Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , Unión Proteica , Sitios de Unión , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ensamble de Virus/genética , Humanos , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , COVID-19/virologíaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN , Virus de la Influenza A , ARN Mensajero , ARN Viral , Proteínas de Unión al ARN , Replicación Viral , Humanos , Replicación Viral/genética , ARN Viral/metabolismo , ARN Viral/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Virus de la Influenza A/metabolismo , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Células HEK293 , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Unión Proteica , AnimalesRESUMEN
Nucleoprotein (N) is an immunodominant antigen in many enveloped virus infections. While the diagnostic value of anti-N antibodies is clear, their role in immunity is not. This is because while they are non-neutralising, they somehow clear infection by coronavirus, influenza and LCMV in vivo. Here, we show that anti-N immune protection is mediated by the cytosolic Fc receptor and E3 ubiquitin ligase TRIM21. Exploiting LCMV as a model system, we demonstrate that TRIM21 uses anti-N antibodies to target N for cytosolic degradation and generate cytotoxic T cells (CTLs) against N peptide. These CTLs rapidly eliminate N-peptide-displaying cells and drive efficient viral clearance. These results reveal a new mechanism of immune synergy between antibodies and T cells and highlights N as an important vaccine target.
Asunto(s)
Anticuerpos Antivirales/inmunología , Inmunidad Celular , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas de la Nucleocápside/inmunología , Ribonucleoproteínas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/genética , Ratones , Ratones Noqueados , Proteínas de la Nucleocápside/genética , Ribonucleoproteínas/genética , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
The swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused significant disruptions in porcine breeding and raised concerns about potential human infection. The nucleocapsid (N) protein of SADS-CoV plays a vital role in viral assembly and replication, but its structure and functions remain poorly understood. This study utilized biochemistry, X-ray crystallography, and immunization techniques to investigate the N protein's structure and function in SADS-CoV. Our findings revealed distinct domains within the N protein, including an RNA-binding domain, two disordered domains, and a dimerization domain. Through biochemical assays, we confirmed that the N-terminal domain functions as an RNA-binding domain, and the C-terminal domain is involved in dimerization, with the crystal structure analysis providing visual evidence of dimer formation. Immunization experiments demonstrated that the disordered domain 2 elicited a significant antibody response. These identified domains and their interactions are crucial for viral assembly. This comprehensive understanding of the N protein in SADS-CoV enhances our knowledge of its assembly and replication mechanisms, enabling the development of targeted interventions and therapeutic strategies. IMPORTANCE: SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.
Asunto(s)
Proteínas de la Nucleocápside , Multimerización de Proteína , Animales , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Cristalografía por Rayos X , Porcinos , Epítopos/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Unión Proteica , Anticuerpos Antivirales/inmunología , Humanos , Dominios Proteicos , Modelos MolecularesRESUMEN
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.
Asunto(s)
Genoma Viral , Virus Nipah , Replicación Viral , Virus Nipah/genética , Virus Nipah/fisiología , Humanos , Proteínas Virales/metabolismo , Proteínas Virales/genética , Virus Hendra/genética , Virus Hendra/metabolismo , Virus Hendra/fisiología , Animales , Henipavirus/genética , Henipavirus/metabolismo , Infecciones por Henipavirus/virología , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Línea CelularRESUMEN
Nipah virus (NiV) is a highly pathogenic paramyxovirus causing frequently lethal encephalitis in humans. The NiV genome is encapsidated by the nucleocapsid (N) protein. RNA synthesis is mediated by the viral RNA-dependent RNA polymerase (RdRP), consisting of the polymerase (L) protein complexed with the homo-tetrameric phosphoprotein (P). The advance of the polymerase along its template requires iterative dissolution and reformation of transient interactions between P and N protomers in a highly regulated process that remains poorly understood. This study applied functional and biochemical NiV polymerase assays to the problem. We mapped three distinct protein interfaces on the C-terminal P-X domain (P-XD), which form a triangular prism and engage L, the C-terminal N tail, and the globular N core, respectively. Transcomplementation assays using NiV L and N-tail binding-deficient mutants revealed that only one XD of a P tetramer binds to L, whereas three must be available for N-binding for efficient polymerase activity. The dissolution of the N-tail complex with P-XD was coordinated by a transient interaction between N-core and the α-1/2 face of this XD but not unoccupied XDs of the P tetramer, creating a timer for coordinated polymerase advance. IMPORTANCE: Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors.
Asunto(s)
Genoma Viral , Virus Nipah , Fosfoproteínas , ARN Polimerasa Dependiente del ARN , Virus Nipah/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/química , Humanos , Unión Proteica , ARN Viral/metabolismo , ARN Viral/genética , Dominios Proteicos , Replicación Viral , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genéticaRESUMEN
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.
Asunto(s)
Virus de la Influenza A , Proteínas de la Nucleocápside , Empaquetamiento del Genoma Viral , Animales , Perros , Humanos , Sustitución de Aminoácidos , Línea Celular , Genoma Viral , Virus de la Influenza A/química , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Lisina/genética , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , ARN Viral/metabolismo , Empaquetamiento del Genoma Viral/genética , Virión/química , Virión/genética , Virión/metabolismo , Mutación , Electricidad EstáticaRESUMEN
There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Humanos , Chlorocebus aethiops , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Células Vero , Replicación Viral , ARN ViralRESUMEN
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N∗ or Δ(1-209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we use mass photometry and negative stain electron microscopy to show that purified Δ(1-209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of Δ(1-209) vRNPs requires the leucine-rich helix of the central disordered region and that this helix promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to Δ(1-209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by phosphorylation.
Asunto(s)
Proteínas de la Nucleocápside , SARS-CoV-2 , Humanos , COVID-19/virología , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/ultraestructura , ARN Viral/metabolismo , ARN Viral/ultraestructura , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestructura , Fosforilación , Ensamble de Virus/genéticaRESUMEN
Porcine deltacoronavirus (PDCoV), an enteropathogenic coronavirus, causes severe watery diarrhoea, dehydration and high mortality in piglets, which has the potential for cross-species transmission in recent years. Growth factor receptor-bound protein 2 (Grb2) is a bridging protein that can couple cell surface receptors with intracellular signal transduction events. Here, we investigated the reciprocal regulation between Grb2 and PDCoV. It is found that Grb2 regulates PDCoV infection and promotes IFN-ß production through activating Raf/MEK/ERK/STAT3 pathway signalling in PDCoV-infected swine testis cells to suppress viral replication. PDCoV N is capable of interacting with Grb2. The proline-rich motifs in the N- or C-terminal region of PDCoV N were critical for the interaction between PDCoV-N and Grb2. Except for Deltacoronavirus PDCoV N, the Alphacoronavirus PEDV N protein could interact with Grb2 and affect the regulation of PEDV replication, while the N protein of Betacoronavirus PHEV and Gammacoronavirus AIBV could not interact with Grb2. PDCoV N promotes Grb2 degradation by K48- and K63-linked ubiquitin-proteasome pathways. Overexpression of PDCoV N impaired the Grb2-mediated activated effect on the Raf/MEK/ERK/STAT3 signal pathway. Thus, our study reveals a novel mechanism of how host protein Grb2 protein regulates viral replication and how PDCoV N escaped natural immunity by interacting with Grb2.
Asunto(s)
Proteína Adaptadora GRB2 , Proteínas de la Nucleocápside , Replicación Viral , Animales , Porcinos , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de la Nucleocápside/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/metabolismo , Deltacoronavirus/metabolismo , Deltacoronavirus/genética , Sistema de Señalización de MAP Quinasas , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/metabolismo , Humanos , Transducción de Señal , Línea Celular , Quinasas raf/metabolismo , Quinasas raf/genética , Células HEK293RESUMEN
Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.
Asunto(s)
Genética Inversa , Tospovirus , Replicación Viral , Animales , Genética Inversa/métodos , ARN Polimerasa Dependiente del ARN , Tospovirus/genética , Estados Unidos , Replicación Viral/genética , ARN Viral/genética , Proteínas de la Nucleocápside/genéticaRESUMEN
The Nucleocapsid Protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not only the core structural protein required for viral packaging, but also participates in the regulation of viral replication, and its post-translational modifications such as phosphorylation have been shown to be an important strategy for regulating virus proliferation. Our previous work identified NP could be ubiquitinated, as confirmed by two independent studies. But the function of NP ubiquitination is currently unknown. In this study, we first pinpointed TRIM6 as the E3 ubiquitin ligase responsible for NP ubiquitination, binding to NP's CTD via its RING and B-box-CCD domains. TRIM6 promotes the K29-typed polyubiquitination of NP at K102, K347, and K361 residues, increasing its binding to viral genomic RNA. Consistently, functional experiments such as the use of the reverse genetic tool trVLP model and gene knockout of TRIM6 further confirmed that blocking the ubiquitination of NP by TRIM6 significantly inhibited the proliferation of SARS-CoV-2. Notably, the NP of coronavirus is relatively conserved, and the NP of SARS-CoV can also be ubiquitinated by TRIM6, indicating that NP could be a broad-spectrum anti-coronavirus target. These findings shed light on the intricate interaction between SARS-CoV-2 and the host, potentially opening new opportunities for COVID-19 therapeutic development.
Asunto(s)
COVID-19 , Genoma Viral , SARS-CoV-2 , Ubiquitina-Proteína Ligasas , Humanos , Proliferación Celular , COVID-19/genética , COVID-19/virología , Proteínas de la Nucleocápside/genética , ARN Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/metabolismoRESUMEN
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.
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COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Empaquetamiento del Genoma Viral/fisiología , Animales , COVID-19/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Genoma Viral , Genómica , Células HEK293 , Humanos , Proteínas de la Nucleocápside/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , ARN Viral/genética , SARS-CoV-2/genética , Células VeroRESUMEN
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.
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Bacteriófago T4 , Escherichia coli , Proteínas Recombinantes de Fusión , Bacteriófago T4/genética , Bacteriófago T4/química , Bacteriófago T4/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/biosíntesis , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/química , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: ⢠A new anti-PEDV N protein monoclonal antibody strain was prepared ⢠Establishment of a more sensitive double antibody sandwich quantitative ELISA ⢠DAS-qELISA was found to be useful for controlling the PEDV spread.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Infecciones por Coronavirus , Ensayo de Inmunoadsorción Enzimática , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Virus de la Diarrea Epidémica Porcina/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/genéticaRESUMEN
The coronavirus' (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit (211DxE213) and trans-Golgi network (TGN) retention (199KxGxYR204). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the 199KxGxYR204 motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M-E interaction. Hampering the ER export of M by mutating its 211DxE213 motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein-protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process.
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Proteínas de la Membrana , Coronavirus del Síndrome Respiratorio de Oriente Medio , Proteínas de la Membrana/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Ensamble de Virus/genéticaRESUMEN
This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.