Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.
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ABSTRACT:
SUBMITTER: Stevaert A
PROVIDER: S-EPMC8084268 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.
| S-EPMC7946191 | biostudies-literature
Project description:Coronaviruses encode an endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ∼1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.
| S-EPMC3318636 | biostudies-literature
Project description:Endoribonuclease non-structural protein 15 (nsp15) (EndoU) is conserved among coronaviruses (CoVs) and is crucial for viral replication, evasion of the innate immune system, and virulence. EndoU-deficient CoVs can activate the interferon (IFN) response and attenuate their virulence, and nsp15 is considered the target of attenuated vaccine development. Among alpha-CoVs, transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) cause lethal diseases in piglets and cats, but the role of EndoU in viral propagation and virulence remains unclear. Here, we verified the TGEV and FIPV EndoU active sites His226 and His241 and found that the antagonization of SeV-induced IFN-β production by nsp15 depends on its EndoU activity. Furthermore, we constructed infectious clones of wild-type (WT) and EndoU-deficient (EnUmt) TGEV and FIPV. Unexpectedly, we found that both the WT and EnUmt viruses propagated efficiently in multiple types of immunocompetent (PK-15, IPI-2I, ST, CRFK, F81, and Fcwf-4) cells. Moreover, the results of infection experiments showed that compared with piglets and cats infected with the WT, the EnUmt virus-infected piglets and cats did not exhibit significantly reduced mortality, tissue injury, or viral shedding. Specially, the death of cats infected with EnUmt-FIPV occurred earlier than that of cats infected with WT. Hence, our results suggest that the function of EndoU is conserved, but nsp15-mediated regulation of the propagation and pathogenesis of CoVs are diverse. Our findings provide a reference for an in-depth understanding of EndoU-mediated immune escape and pathogenicity in CoVs.IMPORTANCEUnderstanding the role of the endoribonuclease non-structural protein 15 (nsp15) (EndoU) in coronavirus (CoV) infection and pathogenesis is essential for vaccine target discovery. Whether the EndoU activity of CoV nsp15, as a virulence-related protein, has a diverse effect on viral virulence needs to be further explored. Here, we found that the transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) nsp15 proteins antagonize SeV-induced interferon-β (IFN-β) production in human embryonic kidney 293 cells. Interestingly, compared with wild-type infection, infection with EnUmt-TGEV or EnUmt-FIPV did not change the IFN-β response or reduce viral propagation in immunocompetent cells. The results of animal experiments showed that EnUmt viruses did not reduce the clinical presentation and mortality caused by TGEV and FIPV. Our findings enrich the understanding of nsp15-mediated regulation of alpha-CoV propagation and virulence and reveal that the conserved functions of nonstructural proteins have diverse effects on the pathogenicity of CoVs.
| S-EPMC10715224 | biostudies-literature
Project description:The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.
| S-EPMC8740563 | biostudies-literature
Project description:Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 in vitro Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins.IMPORTANCE The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.
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Project description:Facing the lack in specific antiviral treatment, it is necessary to develop new means of prevention. In the case of the Coronaviridae this family is now recognized as including potent human pathogens causing upper and lower respiratory tract infections as well as nosocomial ones. Within the purpose of developing new antiseptics molecules, the antiseptic virucidal activity of two calix[4]arene derivatives, the tetra-para-sulfonato-calix[4]arene (C[4]S) and the 1,3-bis(bithiazolyl)-tetra-para-sulfonato-calix[4]arene (C[4]S-BTZ) were evaluated toward the human coronavirus 229E (HCoV 229E). Comparing these results with some obtained previously with chlorhexidine and hexamidine, (i) these two calixarenes did not show any cytotoxicity contrary to chlorhexidine and hexamidine, (ii) C[4]S showed as did hexamidine, a very weak activity against HCoV 229E, and (iii) the C[4]S-BTZ showed a stronger activity than chlorhexidine, i.e. 2.7 and 1.4log?? reduction in viral titer after 5min of contact with 10?³mol L?¹ solutions of C[4]S-BTZ and chlorhexidine, respectively. Thus, the C[4]S-BTZ appeared as a promising virucidal (antiseptic) molecule.
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Project description:The ongoing spread of the COVID-19 coronavirus infection requires us to find new tools and methods for detecting and studying the infection and preventing morbidity (new analytical procedures and tests). With the aim of developing probes for the detection of SARS-CoV-2 coronavirus by modeling in silico (molecular docking), the noncovalent binding of cyanine and squarylium dyes with different molecular charges and different types of heterocyclic residues and substituents (42 compounds in total) with different variants of the NSP15 endoribonuclease of the SARS-CoV-2 coronavirus (COVID-19) of the original (wild) type and mutant types is studied. The interaction energies and spatial configurations of the dye molecules in complexes with NSP15 are determined. Some dyes with negative values of the total energy of the complex Etot are promising for further practical study as probes for coronavirus.
| S-EPMC9022896 | biostudies-literature
Project description:Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, optimal HIV-1 gene silencing by siRNA requires precise complementarity with most of the target sequence. The emergence of mutations in the targeted gene could lead to rapid viral escape from the siRNA. In the present study, Escherichia coli endoribonuclease III (RNase III) or mammalian Dicer was used to cleave double-stranded RNA into endoribonuclease-prepared siRNA (esiRNA). esiRNAs generate a variety of siRNAs which can efficiently and specifically target multiple sites in the cognate RNA. esiRNAs targeting the region encoding the HIV-1 reverse transcriptase (RT) reduced viral replication by 90%. The inhibition was dose dependent and sequence specific because several irrelevant esiRNAs did not inhibit HIV-1 replication. Importantly, esiRNAs obtained from the prototypic RT sequence of the HXB2 strain and from highly mutated RT sequences showed similar degrees of viral inhibition, suggesting that the heterogeneous population of esiRNAs could overcome individual mismatches in the RT sequence. Finally, esiRNAs generated by Dicer cleavage were five times more potent than those generated by bacterial RNase III digestion. These results show that esiRNAs are potent HIV-1 inhibitors. Moreover, sequence targets do not need to be highly conserved to reach a high level of viral replication inhibition.
| S-EPMC2045487 | biostudies-literature
Project description:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.
| S-EPMC525082 | biostudies-literature