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Nuclear factor κB (NF‐κB) plays a crucial role in various cellular processes, including inflammatory and immune responses. Its activation is tightly regulated by the IKK (IκB kinase) complex. Upon severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, the virus is initially recognized by the innate immune system and typically activates the NF‐κB pathway, leading to a severe inflammatory response. However, the influence of viral proteins upon pro‐inflammatory pathway is complicated. Here, we demonstrated that the viral protein nsp3 of SARS‐CoV‐2 exhibits an unusual function, which attenuated the NF‐κB‐mediated inflammatory response against SARS‐CoV‐2 infection in a unique manner. nsp3 interacted with the essential NF‐κB modulator NEMO/IKKγ and promoted its polyubiquitylation via the E3 ubiquitin ligase CBL (Cbl Proto‐Oncogene). Consequently, polyubiquitylated NEMO undergoes proteasome‐dependent degradation, which disrupts NF‐κB activation. Moreover, we found that the SARS unique domain (SUD) in nsp3 of SARS‐CoV‐2 is essential for inducing NEMO degradation, whereas this function is absent in SUD of SARS‐CoV. The reduced activation of pro‐inflammatory response at an early stage could mask the host immune response and faciliate excessive viral replication. Conversely, this finding may partially explain why SARS‐CoV‐2 causes a less inflammatory reaction than SARS‐CoV, resulting in more mild or moderate COVID‐19 cases and greater transmissibility. Given that NEMO is important for NF‐κB activation, we propose that inhibiting polyubiquitylation and degradation of NEMO upon SARS‐CoV‐2 infection is a novel strategy to modulate the host inflammatory response. [ABSTRACT FROM AUTHOR]

Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear. Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins. Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in doublemembrane vesicle formation--a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization. Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections. [ABSTRACT FROM AUTHOR]

Abstract Over the course of the COVID-19 pandemic, several SARS-CoV-2 variants have emerged that may exhibit different etiological effects such as enhanced transmissibility and infectivity. However, genetic variations that reduce virulence and deteriorate viral fitness have not yet been thoroughly investigated. The present study sought to evaluate the effects of viral genetic makeup on COVID-19 epidemiology in Pakistan, where the infectivity and mortality rate was comparatively lower than other countries during the first pandemic wave. For this purpose, we focused on the comparative analyses of 7096 amino-acid long polyprotein pp1ab. Comparative sequence analysis of 203 SARS-CoV-2 genomes, sampled from Pakistan during the first wave of the pandemic revealed 179 amino acid substitutions in pp1ab. Within this set, 38 substitutions were identified within the Nsp3 region of the pp1ab polyprotein. Structural and biophysical analysis of proteins revealed that amino acid variations within Nsp3’s macrodomains induced conformational changes and modified protein-ligand interactions, consequently diminishing the virulence and fitness of SARS-CoV-2. Additionally, the epistatic effects resulting from evolutionary substitutions in SARS-CoV-2 proteins may have unnoticed implications for reducing disease burden. In light of these findings, further characterization of such deleterious SARS-CoV-2 mutations will not only aid in identifying potential therapeutic targets but will also provide a roadmap for maintaining vigilance against the genetic variability of diverse SARS-CoV-2 strains circulating globally. Furthermore, these insights empower us to more effectively manage and respond to potential viral-based pandemic outbreaks of a similar nature in the future.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted puri fied nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negativestain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFoldpredicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically devastating pathogen that has evolved various strategies to evade innate immunity. Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5 (MDA5), a receptor that senses viral RNA. In this study, the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed, and the detailed mechanisms were explored. We found that the interaction between P62 and MDA5 is enhanced due to two factors: the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2α and the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells. As a result of these modifications, the classic P62-mediated autophagy is triggered. Additionally, porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2 (CCT2), which is enhanced by PRRSV nsp3. This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination. In summary, enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways: the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2, leading to intense innate immune suppression. The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies. [ABSTRACT FROM AUTHOR]

SARS-CoV, an RNA virus, is contagious and displays a remarkable degree of adaptability, resulting in intricate disease presentations marked by frequent genetic mutations that can ultimately give rise to drug resistance. Targeting its viral replication cycle could be a potential therapeutic option to counter its viral growth in the human body leading to the severe infectious stage. The Mpro of SARS-CoV-2 is a promising target for therapeutic development as it is crucial for viral transcription and replication. The derivatives of β-diketone and coumarin have already been reported for their antiviral potential and, thus, are considered as a potential scaffold in the current study for the computational design of potential analogs for targeting the viral replication of SARS-CoV-2. In our study, we used novel diketone-hinged coumarin derivatives against the SARS-CoV-2 MPro to develop a broad-spectrum antiviral agent targeting SARS-CoV-2. Through an analysis of pharmacokinetics and docking studies, we identified a list of the top 10 compounds that demonstrated effectiveness in inhibiting the SARS-CoV-2 MPro virus. On the basis of the pharmacokinetics and docking analyses, the top 5 novel coumarin analogs were synthesized and characterized. The thermodynamic stability of compounds KS82 and KS94 was confirmed by their molecular dynamics, and the stability of the simulated system indicated their inhibitory nature. Molecules KS82 and KS94 were further evaluated for their anti-viral potential using Vero E6 cells followed by RT-PCR assay against SARS-CoV-2. The test compound KS82 was the most active with the potential to inhibit SARS-CoV-2 replication in Vero E6 cells. These data indicate that KS82 prevents the attack of the virus and emerges as the primary candidate with promising antiviral properties. [ABSTRACT FROM AUTHOR]

Over the course of the COVID-19 pandemic, several SARS-CoV-2 variants have emerged that may exhibit different etiological effects such as enhanced transmissibility and infectivity. However, genetic variations that reduce virulence and deteriorate viral fitness have not yet been thoroughly investigated. The present study sought to evaluate the effects of viral genetic makeup on COVID-19 epidemiology in Pakistan, where the infectivity and mortality rate was comparatively lower than other countries during the first pandemic wave. For this purpose, we focused on the comparative analyses of 7096 amino-acid long polyprotein pp1ab. Comparative sequence analysis of 203 SARS-CoV-2 genomes, sampled from Pakistan during the first wave of the pandemic revealed 179 amino acid substitutions in pp1ab. Within this set, 38 substitutions were identified within the Nsp3 region of the pp1ab polyprotein. Structural and biophysical analysis of proteins revealed that amino acid variations within Nsp3's macrodomains induced conformational changes and modified protein-ligand interactions, consequently diminishing the virulence and fitness of SARS-CoV-2. Additionally, the epistatic effects resulting from evolutionary substitutions in SARS-CoV-2 proteins may have unnoticed implications for reducing disease burden. In light of these findings, further characterization of such deleterious SARS-CoV-2 mutations will not only aid in identifying potential therapeutic targets but will also provide a roadmap for maintaining vigilance against the genetic variability of diverse SARS-CoV-2 strains circulating globally. Furthermore, these insights empower us to more effectively manage and respond to potential viral-based pandemic outbreaks of a similar nature in the future. [ABSTRACT FROM AUTHOR]

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome. Synopsis: The SARS-CoV-2 NSP3 protein binds directly to fragile X mental retardation proteins (FMRPs) to support viral replication. NSP3 binding disrupts FMRP interaction with the stress granule protein UBAP2L, thereby preventing FMRP localization to these structures. The SARS-CoV-2 NSP3 protein binds to the KH domains of FMRPs through a short peptide motif. Engineered SARS-CoV-2 viruses unable to bind FMRPs have reduced replication. NSP3 binding to FMRPs disrupts their localization to stress granules through competition with UBAP2L. The SARS-CoV-2 NSP3 protein binds directly to fragile X mental retardation proteins (FMRPs) to support viral replication. NSP3 binding disrupts FMRP interaction with the stress granule protein UBAP2L, thereby preventing FMRP localization to these structures. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19. [ABSTRACT FROM AUTHOR]

Since the emergence of SARS-CoV-2, many genetic variations within its genome have been identified, but only a few mutations have been found in nonstructural proteins (NSPs). Among this class of viral proteins, NSP3 is a multidomain protein with 16 different domains, and its largest domain is known as the macrodomain or Mac1 domain. In this study, we present a virtual screening campaign in which we computationally evaluated the NCI anticancer library against the NSP3 Mac1 domain, using Molegro Virtual Docker. The top hits with the best MolDock and Re-Rank scores were selected. The physicochemical analysis and drug-like potential of the top hits were analyzed using the SwissADME data server. The binding stability and affinity of the top NSC compounds against the NSP3 Mac1 domain were analyzed using molecular dynamics (MD) simulation, using Desmond software, and their interaction energies were analyzed using the MM/GBSA method. In particular, by applying subsequent computational filters, we identified 10 compounds as possible NSP3 Mac1 domain inhibitors. Among them, after the assessment of binding energies (ΔGbind) on the whole MD trajectories, we identified the four most interesting compounds that acted as strong binders of the NSP3 Mac1 domain (NSC-358078, NSC-287067, NSC-123472, and NSC-142843), and, remarkably, it could be further characterized for developing innovative antivirals against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Alphavirus infections cause multiple alterations in the intracellular environment that can have both positive and negative effects on viral replication. The Old World alphaviruses, such as Sindbis (SINV), chikungunya (CHIKV), and Semliki Forest viruses, hinder the ability of vertebrate cells to form stress granules (SGs). Previously, this inhibitory function was attributed to the hypervariable domain (HVD) of nsP3, which sequesters the key SG components, G3BP1 and G3BP2, and to the nsP3 macro domain. The macro domain possesses ADP-ribosylhydrolase activity, which can diminish the ADP-ribosylation of G3BP1 during viral replication. However, our recent findings do not support the prevailing notions. We demonstrate that the interactions between SINV- or CHIKV-specific nsP3s and G3BPs, and the ADP-ribosylhydrolase activity are not major contributors to the inhibitory process, at least when nsP3 is expressed at biologically relevant levels. Instead, the primary factors responsible for suppressing SG formation are virus-induced transcriptional and translational shutoffs that rapidly develop within the first few hours post infection. Poorly replicating SINV variants carrying mutated nsP3 HVD still inhibit SG development even in the presence of sodium arsenite (NaAs). Conversely, SINV mutants lacking transcription and/or translation inhibitory functions lose their ability to inhibit SG formation, despite expressing high levels of wt nsP3. Moreover, we found that stable cell lines expressing GFP-nsP3 fusions retain the capacity to form SGs when exposed to NaAs. However, our results do not rule out the possibility that additional alphavirus-induced changes in cell biology may contribute to the suppression of SG formation. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like other coronaviruses, replicates their genome in virus-induced cytosolic membrane-bound replication organelles (ROs). SARS-CoV-2 promotes the biogenesis of ROs by inducing the rearrangement of endoplasmic reticulum (ER) membranes. NSP3, NSP4, and NSP6 are transmembrane viral non-structural proteins (NSPs) and essential players in the formation of ROs. To understand how these three NSPs work synergistically with host-binding proteins, we performed affinity purifications followed by mass spectrometry analyses to study the host-viral protein-protein interactome of NSP3, NSP4, and NSP6 expressed individually and in combination. Through this analysis, we identified two host transmembrane proteins, REEP5 and TRAM1, as critical interacting partners of NSP3 that localize at the membrane of the RO. REEP5 interacts with TRAM1 endogenously and binds NSP3 during SARS-CoV-2 infection. REEP5 knockout reduces ER membrane rearrangements and inhibits SARS-CoV-2 replication. Collectively, our study shows that the host REEP5/TRAM1 complex binds NSP3, promoting RO biogenesis and viral replication. IMPORTANCE Generation of virus-host protein–protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Among the different drug targets of SARS-CoV-2, a multi-domain protein known as NSP3 is a critical element of the translational and replication machinery. The macrodomain-I, in particular, has been reported to have an essential role in the viral attack on the innate immune response. In this study, we explore natural medicinal compounds and identify potential inhibitors to target the SARS-CoV-2–NSP3 macrodomain-I. Computational modeling and simulation tools were utilized to investigate the structural-dynamic properties using triplicates of 100 ns MD simulations. In addition, the MM/GBSA method was used to calculate the total binding free energy of each inhibitor bound to macrodomain-I. Two significant hits were identified: 3,5,7,4′-tetrahydroxyflavanone 3′-(4-hydroxybenzoic acid) and 2-hydroxy-3-O-beta-glucopyranosyl-benzoic acid. The structural-dynamic investigation of both compounds with macrodomain-I revealed stable dynamics and compact behavior. In addition, the total binding free energy for each complex demonstrated a robust binding affinity, of ΔG −61.98 ± 0.9 kcal/mol for Compound A, while for Compound B, the ΔG was −45.125 ± 2.8 kcal/mol, indicating the inhibitory potential of these compounds. In silico bioactivity and dissociation constant (KD) determination for both complexes further validated the inhibitory potency of each compound. In conclusion, the aforementioned natural products have the potential to inhibit NSP3, to directly rescue the host immune response. The current study provides the basis for novel drug development against SARS-CoV-2 and its variants. [ABSTRACT FROM AUTHOR]

SARS-CoV, an RNA virus, is contagious and displays a remarkable degree of adaptability, resulting in intricate disease presentations marked by frequent genetic mutations that can ultimately give rise to drug resistance. Targeting its viral replication cycle could be a potential therapeutic option to counter its viral growth in the human body leading to the severe infectious stage. The Mpro of SARS-CoV-2 is a promising target for therapeutic development as it is crucial for viral transcription and replication. The derivatives of β-diketone and coumarin have already been reported for their antiviral potential and, thus, are considered as a potential scaffold in the current study for the computational design of potential analogs for targeting the viral replication of SARS-CoV-2. In our study, we used novel diketone-hinged coumarin derivatives against the SARS-CoV-2 MPro to develop a broad-spectrum antiviral agent targeting SARS-CoV-2. Through an analysis of pharmacokinetics and docking studies, we identified a list of the top 10 compounds that demonstrated effectiveness in inhibiting the SARS-CoV-2 MPro virus. On the basis of the pharmacokinetics and docking analyses, the top 5 novel coumarin analogs were synthesized and characterized. The thermodynamic stability of compounds KS82 and KS94 was confirmed by their molecular dynamics, and the stability of the simulated system indicated their inhibitory nature. Molecules KS82 and KS94 were further evaluated for their anti-viral potential using Vero E6 cells followed by RT-PCR assay against SARS-CoV-2. The test compound KS82 was the most active with the potential to inhibit SARS-CoV-2 replication in Vero E6 cells. These data indicate that KS82 prevents the attack of the virus and emerges as the primary candidate with promising antiviral properties.

SARS-CoV-2, the causative agent of COVID-19 encodes at least 16 nonstructural proteins of variably understood function. Nsp3, the largest nonstructural protein contains several domains, including a SARS-unique domain (SUD), which occurs only in Sarbecovirus. The SUD has a role in preferentially enhancing viral translation. During isolation of mouse-adapted SARS-CoV-2, we isolated an attenuated virus that contained a single mutation in a linker region of nsp3 (nsp3-S676T). The S676T mutation decreased virus replication in cultured cells and primary human cells and in mice. Nsp3-S676T alleviated the SUD translational enhancing ability by decreasing the interaction between two translation factors, Paip1 and PABP1. We also identified a compensatory mutation in the nucleocapsid (N) protein (N-S194L) that restored the virulent phenotype, without directly binding to SUD. Together, these results reveal an aspect of nsp3-N interactions, which impact both SARS-CoV-2 replication and, consequently, pathogenesis. [ABSTRACT FROM AUTHOR]

Remdesivir is the first FDA-approved drug for treating severe SARS-CoV-2 infection and targets RNA-dependent RNA polymerase (RdRp) that is required for viral replication. To monitor for the development of mutations that may result in remdesivir resistance during prolonged treatment, we sequenced SARS-CoV-2 specimens collected at different treatment time points in two transplant patients with severe COVID-19. In the first patient, an allogeneic hematopoietic stem cell transplant recipient, a transient RdRp catalytic subunit mutation (nsp12:A449V) was observed that has not previously been associated with remdesivir resistance. As no in vitro study had been conducted to elucidate the phenotypic effect of nsp12:A449V, its clinical significance is unclear. In the second patient, two other transient RdRp mutations were detected: one in the catalytic subunit (nsp12:V166A) and the other in an accessory subunit important for processivity (nsp7:D67N). This is the first case report for a potential link between the nsp12:V166A mutation and remdesivir resistance in vivo, which had only been previously described by in vitro studies. The nsp7:D67N mutation has not previously been associated with remdesivir resistance, and whether it has a phenotypic effect is unknown. Our study revealed SARS-CoV-2 genetic dynamics during remdesivir treatment in transplant recipients that involved mutations in the RdRp complex (nsp7 and nsp12), which may be the result of selective pressure. These results suggest that close monitoring for potential resistance during the course of remdesivir treatment in highly vulnerable patient populations may be beneficial. Development and utilization of diagnostic RdRp genotyping tests may be a future direction for improving the management of chronic COVID-19. [ABSTRACT FROM AUTHOR]

The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid‐inducible gene I, and melanoma differentiation‐associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) hijacks GSK‐3β to facilitate its replication. GSK‐3β‐induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK‐3β‐N‐nsp3 cascade promotes viral replication. Although SARS‐CoV‐2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK‐3β, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK‐3β and the interaction between PACT and GSK‐3β is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK‐3β independent of its well‐studied double‐stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS‐CoV‐2 replication through the blockage of GSK‐3β‐N‐nsp3 cascade. [ABSTRACT FROM AUTHOR]

The coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is affecting human life in an unprecedented manner and has become a global public health emergency. Identification of novel inhibitors of viral infection/replication is the utmost priority to curtail COVID-19 progression. A pre-requisite for such inhibitors is good bioavailability, non-toxicity and serum stability. Computational studies have shown that curcumin can be a candidate inhibitor of certain SARS-CoV-2 proteins; however, poor bio-availability of curcumin limits its possible therapeutic application. To circumvent this limitation, we have used mitocurcumin (MC), a triphenyl phosphonium conjugated curcumin derivative, to study the ability to inhibit SARS-CoV-2 infection using molecular docking and molecular dynamics (MD) simulation. MC is serum stable and several fold more potent as compared to curcumin. Molecular docking studies revealed that MC can bind at active site of SARS-CoV-2 ADP Ribose Phosphatase (NSP3) and SARS-CoV-2 methyltransferase (NSP10-NSP16 complex) with a high binding energy of − 10.3 kcal/mol and − 10.4 kcal/mol, respectively. MD simulation (100 ns) studies revealed that binding of MC to NSP3 and NSP16 resulted in a stable complex. MC interacted with critical residues of NSP3 macro-domain and NSP10-NSP16 complex and occupied their active sites. NSP3 is known to suppress host immune responses whereas NSP10-NSP16 complex is known to prevent immune recognition of viral mRNA. Our study suggests that MC can potentially inhibit the activity of NSP3 and NSP10-NSP16 complex, resulting in compromised viral immune evasion mechanism, and thereby accentuate the innate immune mediated clearance of viral load. [ABSTRACT FROM AUTHOR]

RNA interference in vertebrates acts as an antiviral mechanism only in undifferentiated embryonic stem cells and is mediated by microRNAs. In somatic cells, host microRNAs also bind to the genomes of RNA viruses, regulating their translation and replication. It has been shown that viral (+)RNA can evolve under the influence of host cell miRNAs. In more than two years of the pandemic, the SARS-CoV-2 virus has mutated significantly. It is quite possible that some mutations could be retained in the virus genome under the influence of miRNAs produced by alveolar cells. We demonstrated that microRNAs in human lung tissue exert evolutionary pressure on the SARS-CoV-2 genome. Moreover, a significant number of sites of host microRNA binding with the virus genome are located in the NSP3-NSP5 region responsible for autoproteolysis of viral polypeptides. [ABSTRACT FROM AUTHOR]

During coronavirus infection, three non-structural proteins, nsp3, nsp4, and nsp6, are of great importance as they induce the formation of double-membrane vesicles where the replication and transcription of viral gRNA takes place, and the interaction of nsp3 and nsp4 lumenal regions triggers membrane pairing. However, their structural states are not well-understood. We investigated the interactions between nsp3 and nsp4 by predicting the structures of their lumenal regions individually and in complex using AlphaFold2 as implemented in ColabFold. The ColabFold prediction accuracy of the nsp3–nsp4 complex was increased compared to nsp3 alone and nsp4 alone. All cysteine residues in both lumenal regions were modelled to be involved in intramolecular disulphide bonds. A linker region in the nsp4 lumenal region emerged as crucial for the interaction, transitioning to a structured state when predicted in complex. The key interactions modelled between nsp3 and nsp4 appeared stable when the transmembrane regions of nsp3 and nsp4 were added to the modelling either alone or together. While molecular dynamics simulations (MD) demonstrated that the proposed model of the nsp3 lumenal region on its own is not stable, key interactions between nsp and nsp4 in the proposed complex model appeared stable after MD. Together, these observations suggest that the interaction is robust to different modelling conditions. Understanding the functional importance of the nsp4 linker region may have implications for the targeting of double membrane vesicle formation in controlling coronavirus infection. [ABSTRACT FROM AUTHOR]

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks. [ABSTRACT FROM AUTHOR]

Green tea catechin compounds (Camellia sinensis L. Kuntze) have an antiviral activity such as influenza, hepatitis B, hepatitis C, herpes simplex virus, HIV, and proven in vitro antiviral influenza against NSP5 in SARS CoV. These considerations are used in this study using Non-structural Protein (NSP), namely NSP3 and NSP5 in SARS CoV-2, which have a role in viral replication and transcription. This study aims to predict the physicochemical properties according to the five rules of Lipinski's using swissADME. Prediction of toxicity with LD50 classification using the Protox II online tool. Catechin compound activity based on ligand interaction with NSP3 (PDB ID: 6W6Y) and NSP5 (PDB ID: 6M2N) receptors using Molegro Virtual Docker (MVD) 6.0. The results showed the predictions of physicochemical properties of the (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epicatechin (EC) compounds fulfilled the five rules of Lipinski's. Catechin compounds have toxicity at levels 4 and 6. The activity of catechin compounds on NSP3 (PDB ID: 6W6Y) and NSP5 (PDB ID: 6M2N) receptors indicated that all catechin compounds had inhibitory activity. The best potential activity compound is (-)-epigallocatechin-3-gallate (EGCG) with a rerank score of -102.8200 and -134.1800 Kcal/mol so that EGCG can be recommended as a candidate for the SARS CoV-2 antiviral compound.

Stress granules (SGs) are cytoplasmic aggregates of proteins and mRNA that form in response to diverse environmental stressors, including viral infections. Several viruses possess the ability to block the formation of stress granules by targeting the SGs marker protein G3BP. However, the molecular functions and mechanisms underlying the regulation of SGs formation by Getah virus (GETV) remain unclear. In this study, we found that GETV infection triggered the formation of Nsp3-G3BP aggregates, which differed in composition from SGs. Further studies revealed that the presence of these aggregates was dependent on the activation of the PKR/eIF2α signaling pathway. Interestingly, we found that Nsp3 HVD domain blocked the formation of SGs by binding to G3BP NTF2 domain. Moreover, knockout of G3BP in NCI-H1299 cells had no effect on GETV replication, while overexpression of G3BP to form the genuine SGs significantly inhibited GETV replication. Overall, our study elucidates a novel role GETV Nsp3 to change the composition of SG as well as cellular stress response. [Display omitted] • GETV replication induces G3BP aggregates that resemble SGs. • The PKR/eIF2a signaling pathway is essential for GETV induced G3BP aggregates. • GETV Nsp3 HVD domain interacts with the G3BP1 NTF2 domain. • Overexpression of G3BP significantly inhibited GETV replication. [ABSTRACT FROM AUTHOR]

Since the emergence of SARS-CoV-2, many genetic variations within its genome have been identified, but only a few mutations have been found in nonstructural proteins (NSPs). Among this class of viral proteins, NSP3 is a multidomain protein with 16 different domains, and its largest domain is known as the macrodomain or Mac1 domain. In this study, we present a virtual screening campaign in which we computationally evaluated the NCI anticancer library against the NSP3 Mac1 domain, using Molegro Virtual Docker. The top hits with the best MolDock and Re-Rank scores were selected. The physicochemical analysis and drug-like potential of the top hits were analyzed using the SwissADME data server. The binding stability and affinity of the top NSC compounds against the NSP3 Mac1 domain were analyzed using molecular dynamics (MD) simulation, using Desmond software, and their interaction energies were analyzed using the MM/GBSA method. In particular, by applying subsequent computational filters, we identified 10 compounds as possible NSP3 Mac1 domain inhibitors. Among them, after the assessment of binding energies (ΔGbind) on the whole MD trajectories, we identified the four most interesting compounds that acted as strong binders of the NSP3 Mac1 domain (NSC-358078, NSC-287067, NSC-123472, and NSC-142843), and, remarkably, it could be further characterized for developing innovative antivirals against SARS-CoV-2.

Among the different drug targets of SARS-CoV-2, a multi-domain protein known as NSP3 is a critical element of the translational and replication machinery. The macrodomain-I, in particular, has been reported to have an essential role in the viral attack on the innate immune response. In this study, we explore natural medicinal compounds and identify potential inhibitors to target the SARS-CoV-2–NSP3 macrodomain-I. Computational modeling and simulation tools were utilized to investigate the structural-dynamic properties using triplicates of 100 ns MD simulations. In addition, the MM/GBSA method was used to calculate the total binding free energy of each inhibitor bound to macrodomain-I. Two significant hits were identified: 3,5,7,4′-tetrahydroxyflavanone 3′-(4-hydroxybenzoic acid) and 2-hydroxy-3-O-beta-glucopyranosyl-benzoic acid. The structural-dynamic investigation of both compounds with macrodomain-I revealed stable dynamics and compact behavior. In addition, the total binding free energy for each complex demonstrated a robust binding affinity, of ΔG −61.98 ± 0.9 kcal/mol for Compound A, while for Compound B, the ΔG was −45.125 ± 2.8 kcal/mol, indicating the inhibitory potential of these compounds. In silico bioactivity and dissociation constant (KD) determination for both complexes further validated the inhibitory potency of each compound. In conclusion, the aforementioned natural products have the potential to inhibit NSP3, to directly rescue the host immune response. The current study provides the basis for novel drug development against SARS-CoV-2 and its variants.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. In infected cells, its positive-sense RNA genome is translated into polyproteins that are subsequently processed into four nonstructural proteins (nsP1 to 4), the virus-encoded subunits of the RNA replicase. However, for RNA replication, interactions between nsPs and host proteins are also needed. These interactions are mostly mediated through the intrinsically disordered C-terminal hypervariable domain (HVD) in nsP3. Duplicate FGDF motifs in the HVD are required for interaction with mammalian RasGAP SH3-binding proteins (G3BPs) and their mosquito homolog Rin; these interactions are crucial for CHIKV RNA replication. In this study, we inactivated G3BP/Rin-binding motifs in the HVD and inserted peptides containing either native or inactivated G3BP/Rin-binding motifs into flexible regions of nsP1, nsP2, or nsP4. Insertion of native motifs into nsP1 or nsP2 but not into the C terminus of nsP4 activated CHIKV RNA replication in human cells in a G3BP-dependent manner. In mosquito cells, activation also resulted from the insertion of inactive motifs after residue 8 or 466 in nsP2; however, the effect was significantly larger when the inserted sequence contained native motifs. Nonetheless, CHIKV mutants harboring mutations in the HVD and containing insertions of native motifs in nsP2 were not viable in mosquito cells. In contrast, mutant genomes containing native motifs after residue 466 or 618 in nsP2 replicated in BHK-21 cells, with the latter mutant forming infectious progeny. Thus, the binding of G3BPs to nsP2 can support CHIKV RNA replication and restore the infectivity of viruses lacking G3BP-binding motifs in the HVD of nsP3. [ABSTRACT FROM AUTHOR]

Coronavirus nonstructural protein 3 (nsp3) is a multi-functional protein, playing a critical role in viral replication and in regulating host antiviral innate immunity. In this study, we demonstrate that nsp3 from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian coronavirus infectious bronchitis virus (IBV) directly interacts with melanoma differentiation-associated gene 5 (MDA5), rendering an inhibitory effect on the MDA5-mediated type I interferon (IFN) response. By the co-expression of MDA5 with wild-type and truncated nsp3 constructs, at least three interacting regions mapped to the papain-like protease (PLpro) domain and two other domains located at the N- and C-terminal regions were identified in SARS-CoV-2 nsp3. Furthermore, by introducing point mutations to the catalytic triad, the deubiquitylation activity of the PLpro domain from both SARS-CoV-2 and IBV nsp3 was shown to be responsible for the suppression of the MDA5-mediated type I IFN response. It was also demonstrated that both MDA5 and nsp3 were able to interact with ubiquitin and ubiquitinated proteins, contributing to the interaction between the two proteins. This study confirms the antagonistic role of nsp3 in the MDA5-mediated type I IFN signaling, highlighting the complex interaction between a multi-functional viral protein and the innate immune response. [ABSTRACT FROM AUTHOR]

The world is now facing intolerable damage in all sectors of life because of the deadly COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2. The discovery and development of anti-SARS-CoV-2 drugs have become pragmatic in the time needed to fight against this pandemic. The non-structural protein 3 is essential for the replication of transcriptase complex (RTC) and may be regarded as a possible target against SARS-CoV-2. Here, we have used a comprehensive in silico technique to find potent drug molecules against the NSP3 receptor of SARS-CoV-2. Virtual screening of 150 Isatin derivatives taken from PubChem was performed based on their binding affinity estimated by docking simulations, resulting in the selection of 46 ligands having binding energy greater than −7.1 kcal/mol. Moreover, the molecular interactions of the nine best-docked ligands having a binding energy of ≥ −8.5 kcal/mol were analyzed. The molecular interactions showed that the three ligands (S5, S16, and S42) were stabilized by forming hydrogen bonds and other significant interactions. Molecular dynamic simulations were performed to mimic an in vitro protein-like aqueous environment and to check the stability of the best three ligands and NSP3 complexes in an aqueous environment. The binding energy of the S5, S16, and S42 systems obtained from the molecular mechanics Poisson–Boltzmann surface area also favor the system's stability. The MD and MM/PBSA results explore that S5, S16, and S42 are more stable and can be considered more potent drug candidates against COVID-19 disease. [ABSTRACT FROM AUTHOR]

Remdesivir is the first FDA-approved drug for treating severe SARS-CoV-2 infection and targets RNA-dependent RNA polymerase (RdRp) that is required for viral replication. To monitor for the development of mutations that may result in remdesivir resistance during prolonged treatment, we sequenced SARS-CoV-2 specimens collected at different treatment time points in two transplant patients with severe COVID-19. In the first patient, an allogeneic hematopoietic stem cell transplant recipient, a transient RdRp catalytic subunit mutation (nsp12:A449V) was observed that has not previously been associated with remdesivir resistance. As no in vitro study had been conducted to elucidate the phenotypic effect of nsp12:A449V, its clinical significance is unclear. In the second patient, two other transient RdRp mutations were detected: one in the catalytic subunit (nsp12:V166A) and the other in an accessory subunit important for processivity (nsp7:D67N). This is the first case report for a potential link between the nsp12:V166A mutation and remdesivir resistance in vivo, which had only been previously described by in vitro studies. The nsp7:D67N mutation has not previously been associated with remdesivir resistance, and whether it has a phenotypic effect is unknown. Our study revealed SARS-CoV-2 genetic dynamics during remdesivir treatment in transplant recipients that involved mutations in the RdRp complex (nsp7 and nsp12), which may be the result of selective pressure. These results suggest that close monitoring for potential resistance during the course of remdesivir treatment in highly vulnerable patient populations may be beneficial. Development and utilization of diagnostic RdRp genotyping tests may be a future direction for improving the management of chronic COVID-19.

Ras-GTPase-activating SH3 domain-binding-proteins 1 (G3BP1) and 2 (G3BP2) are multifunctional RNA-binding proteins involved in stress granule nucleation, previously identified as essential cofactors of Old World alphaviruses. They are recruited to viral replication complexes formed by the Chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) via an interaction with a duplicated FGxF motif conserved in the hypervariable domain (HVD) of virus-encoded nsP3. According to mutagenesis studies, this FGxF duplication is strictly required for G3BP binding and optimal viral growth. Contrasting with this scenario, nsP3 encoded by Mayaro virus (MAYV), an arthritogenic virus grouped with Old World alphaviruses, contains a single canonical FGxF sequence. In light of this unusual feature, we questioned MAYV nsP3/G3BPs relationships. We report that G3BP1 and G3BP2 are both required for MAYV growth in human cells and bind nsP3 protein. In infected cells, they are recruited to nsP3-containing cytosolic foci and active replication complexes. Unexpectedly, deletion of the single FGxF sequence in MAYV nsP3 did not abolish these phenotypes. Using mutagenesis and in silico modeling, we identify an upstream FGAP amino acid sequence as an additional MAYV nsP3/G3BP interaction motif required for optimal viral infectivity. These results, therefore, highlight a non-conventional G3BP binding sequence in MAYV nsP3. [ABSTRACT FROM AUTHOR]

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (,34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously. [ABSTRACT FROM AUTHOR]

Macrodomain-I of the NSP3 (non-structural protein 3) is responsible for immune response hijacking in the SARS-CoV-2 infection known as COVID-19. In the omicron variant (B.1.1.529), this domain harbors a new mutation, V1069I, which may increase the binding of ADPr and consequently the infection severity. This macrodomain-I, due to its significant role in infection, is deemed to be an important drug target. Hence, using structural bioinformatics and molecular simulation approaches, we performed a virtual screening of the traditional Chinese medicines (TCM) database for potential anti-viral drugs. The screening of 57,000 compounds yielded the 10 best compounds with docking scores better than the control ADPr. Among the top ten, the best three hits—TCM42798, with a docking score of −13.70 kcal/mol, TCM47007 of −13.25 kcal/mol, and TCM30675 of −12.49 kcal/mol—were chosen as the best hits. Structural dynamic features were explored including stability, compactness, flexibility, and hydrogen bonding, further demonstrating the anti-viral potential of these hits. Using the MM/GBSA approach, the total binding free energy for each complex was reported to be −69.78 kcal/mol, −50.11 kcal/mol, and −47.64 kcal/mol, respectively, which consequently reflect the stronger binding and inhibitory potential of these compounds. These agents might suppress NSP3 directly, allowing the host immune system to recuperate. The current study lays the groundwork for the development of new drugs to combat SARS-CoV-2 and its variants. [ABSTRACT FROM AUTHOR]

Abstract Background Analysis of viral protein–protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein–protein interactions. Results Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. Conclusions Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

Ras-GTPase-activating SH3 domain-binding-proteins 1 (G3BP1) and 2 (G3BP2) are multifunctional RNA-binding proteins involved in stress granule nucleation, previously identified as essential cofactors of Old World alphaviruses. They are recruited to viral replication complexes formed by the Chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) via an interaction with a duplicated FGxF motif conserved in the hypervariable domain (HVD) of virus-encoded nsP3. According to mutagenesis studies, this FGxF duplication is strictly required for G3BP binding and optimal viral growth. Contrasting with this scenario, nsP3 encoded by Mayaro virus (MAYV), an arthritogenic virus grouped with Old World alphaviruses, contains a single canonical FGxF sequence. In light of this unusual feature, we questioned MAYV nsP3/G3BPs relationships. We report that G3BP1 and G3BP2 are both required for MAYV growth in human cells and bind nsP3 protein. In infected cells, they are recruited to nsP3-containing cytosolic foci and active replication complexes. Unexpectedly, deletion of the single FGxF sequence in MAYV nsP3 did not abolish these phenotypes. Using mutagenesis and in silico modeling, we identify an upstream FGAP amino acid sequence as an additional MAYV nsP3/G3BP interaction motif required for optimal viral infectivity. These results, therefore, highlight a non-conventional G3BP binding sequence in MAYV nsP3.

The worldwide COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonstructural protein 3 (nsp3) has 1945 residues and is the largest protein encoded by SARS-CoV-2. It comprises more than a dozen independent domains with various functions. Many of these domains were studied in the closely-related virus SARS-CoV following an earlier outbreak. Nonetheless structural and functional information on the C-terminal region of nsp3 containing two transmembrane and three extra-membrane domains remains incomplete. This part of the protein appears to be involved in initiation of double membrane vesicle (DMV) formation, membranous organelles the virus builds to hide its replication-transcription complex from host immune defenses. Here we present the near-complete backbone and Ile, Leu, and Val methyl group chemical shift assignments of the most C-terminal domain of nsp3, CoV-Y. As the exact function and binding partners of CoV-Y remain unknown, our data provide a basis for future NMR studies of protein–protein interactions to elucidate the molecular mechanism of DMV formation. [ABSTRACT FROM AUTHOR]

The emergence of SARS-CoV-2 causing the COVID-19 pandemic, has highlighted how a combination of urgency, collaboration and building on existing research can enable rapid vaccine development to fight disease outbreaks. However, even countries with high vaccination rates still see surges in case numbers and high numbers of hospitalized patients. The development of antiviral treatments hence remains a top priority in preventing hospitalization and death of COVID-19 patients, and eventually bringing an end to the SARS-CoV-2 pandemic. The SARS-CoV-2 proteome contains several essential enzymatic activities embedded within its non-structural proteins (nsps). We here focus on nsp3, that harbours an essential papain-like protease (PLpro) domain responsible for cleaving the viral polyprotein as part of viral processing. Moreover, nsp3/PLpro also cleaves ubiquitin and ISG15 modifications within the host cell, derailing innate immune responses. Small molecule inhibition of the PLpro protease domain significantly reduces viral loads in SARS-CoV-2 infection models, suggesting that PLpro is an excellent drug target for next generation antivirals. In this review we discuss the conserved structure and function of PLpro and the ongoing efforts to design small molecule PLpro inhibitors that exploit this knowledge. We first discuss the many drug repurposing attempts, concluding that it is unlikely that PLpro-targeting drugs already exist. We next discuss the wealth of structural information on SARS-CoV-2 PLpro inhibition, for which there are now ∼30 distinct crystal structures with small molecule inhibitors bound in a surprising number of distinct crystallographic settings. We focus on optimisation of an existing compound class, based on SARS-CoV PLpro inhibitor GRL-0617, and recapitulate how new GRL-0617 derivatives exploit different features of PLpro, to overcome some compound liabilities.

The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.

With up to 17 domains, non-structural protein 3 (nsp3) is the largest protein of SARS-CoV-2. In part due to its large size, many of its functions still remain a mystery. It is known that nsp3 fulfils several essential functions in the cycle of infection, however most of its domains have not been structurally determined. One of its essential functions is to cleave the polyprotein, which is translated first upon infection, into other functional non-structural proteins. Nsp3 is also involved in the evasion of the host immune system and forms large pore complexes important for viral replication. Furthermore, it interacts with more than 30 other host and viral proteins, resulting in a multitude of potential ways to affect the host cell and viral replication. Themany roles of this coronaviral Swiss army knife make it a promising drug target. In this review, we aim to clarify naming conventions and give an overview on the structures and functions of its domains as a starting point for further research. [ABSTRACT FROM AUTHOR]

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07–2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear., Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins., Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation-a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization., Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ma-Lauer, Li, Niemeyer, Richter, Pusl, von Brunn, Ru, Xiang, Schwinghammer, Liu, Baral, Berthold, Qiu, Roy, Kremmer, Flaswinkel, Drosten, Jin and von Brunn.)

During coronavirus infection, three non-structural proteins, nsp3, nsp4, and nsp6, are of great importance as they induce the formation of double-membrane vesicles where the replication and transcription of viral gRNA takes place, and the interaction of nsp3 and nsp4 lumenal regions triggers membrane pairing. However, their structural states are not well-understood. We investigated the interactions between nsp3 and nsp4 by predicting the structures of their lumenal regions individually and in complex using AlphaFold2 as implemented in ColabFold. The ColabFold prediction accuracy of the nsp3–nsp4 complex was increased compared to nsp3 alone and nsp4 alone. All cysteine residues in both lumenal regions were modelled to be involved in intramolecular disulphide bonds. A linker region in the nsp4 lumenal region emerged as crucial for the interaction, transitioning to a structured state when predicted in complex. The key interactions modelled between nsp3 and nsp4 appeared stable when the transmembrane regions of nsp3 and nsp4 were added to the modelling either alone or together. While molecular dynamics simulations (MD) demonstrated that the proposed model of the nsp3 lumenal region on its own is not stable, key interactions between nsp and nsp4 in the proposed complex model appeared stable after MD. Together, these observations suggest that the interaction is robust to different modelling conditions. Understanding the functional importance of the nsp4 linker region may have implications for the targeting of double membrane vesicle formation in controlling coronavirus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the disease coronavirus-19 disease (COVID-19) has wreaked havoc on the health and economy of humanity. In addition, the disease is observed in domestic and wild animals. The disease has impacted directly and indirectly every corner of the planet. Currently, there are no effective therapies for the treatment of COVID-19. Vaccination to protect against COVID-19 started in December 2020. SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of 29.8 kb. More than two-thirds of the genome comprise Orf1ab encoding 16 nonstructural proteins (nsps) followed by mRNAs encoding structural proteins, spike (S), envelop (E), membrane (M), and nucleocapsid (N). These genes are interspaced with several accessory genes (open reading frames [Orfs] 3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and 10). The functions of these proteins are of particular interest for understanding the pathogenesis of SARS-CoV-2. Several of the nsps (nsp3, nsp4, and nsp6) and Orf3a are transmembrane proteins involved in regulating the host immunity, modifying host cell organelles for viral replication and escape and hence considered drug targets. In this paper, we report mapping the transmembrane structure of the nsps of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Coronavirus nonstructural protein 3 (nsp3) is a multi-functional protein, playing a critical role in viral replication and in regulating host antiviral innate immunity. In this study, we demonstrate that nsp3 from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian coronavirus infectious bronchitis virus (IBV) directly interacts with melanoma differentiation-associated gene 5 (MDA5), rendering an inhibitory effect on the MDA5-mediated type I interferon (IFN) response. By the co-expression of MDA5 with wild-type and truncated nsp3 constructs, at least three interacting regions mapped to the papain-like protease (PLpro) domain and two other domains located at the N- and C-terminal regions were identified in SARS-CoV-2 nsp3. Furthermore, by introducing point mutations to the catalytic triad, the deubiquitylation activity of the PLpro domain from both SARS-CoV-2 and IBV nsp3 was shown to be responsible for the suppression of the MDA5-mediated type I IFN response. It was also demonstrated that both MDA5 and nsp3 were able to interact with ubiquitin and ubiquitinated proteins, contributing to the interaction between the two proteins. This study confirms the antagonistic role of nsp3 in the MDA5-mediated type I IFN signaling, highlighting the complex interaction between a multi-functional viral protein and the innate immune response.

Romania officially declared its first Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) case on February 26, 2020. The first and largest coronavirus disease 2019 (COVID-19) outbreak in Romania was recorded in Suceava, North-East region of the country, and originated at the Suceava regional county hospital. Following sheltering-in-place measures, infection rates decreased, only to rise again after relaxation of measures. This study describes the spread of SARS-CoV-2 in Suceava and other parts of Romania and analyses the mutations and their association with clinical manifestation of the disease during the period of COVID-19 outbreak. Sixty-two samples were sequenced via high-throughput platform and screened for variants. For selected mutations, putative biological significance was assessed, and their effects on disease severity. Phylogenetic analysis was conducted on Romanian genomes (n = 112) and on sequences originating from Europe, United Kingdom, Africa, Asia, South, and North America (n = 876). The results indicated multiple introduction events for SARS-CoV-2 in Suceava, mainly from Italy, Spain, United Kingdom, and Russia although some sequences were also related to those from the Czechia, Belgium, and France. Most Suceava genomes contained mutations common to European lineages, such as A20268G, however, approximately 10% of samples were missing such mutations, indicating a possible different arrival route. While overall genome regions ORF1ab, S, and ORF7 were subject to most mutations, several recurring mutations such as A105V were identified, and these were mainly present in severe forms of the disease. Non-synonymous mutations, such as T987N (Thr987Asn in NSP3a domain), associated with changes in a protein responsible for decreasing viral tethering in human host were also present. Patients with diabetes and hypertension exhibited higher risk ratios (RR) of acquiring severe forms of the disease and these were mainly related to A105V mutation. This study identified the arrival routes of SARS-CoV-2 in Romania and revealed potential associations between the SARS-CoV-2 genomic organization circulating in the country and the clinical manifestation of COVID-19 disease.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. [ABSTRACT FROM AUTHOR]

Background: Analysis of viral protein–protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein–protein interactions. Results: Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. Conclusions: Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development. [ABSTRACT FROM AUTHOR]

Romania officially declared its first Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) case on February 26, 2020. The first and largest coronavirus disease 2019 (COVID-19) outbreak in Romania was recorded in Suceava, North-East region of the country, and originated at the Suceava regional county hospital. Following sheltering-in-place measures, infection rates decreased, only to rise again after relaxation of measures. This study describes the spread of SARS-CoV-2 in Suceava and other parts of Romania and analyses the mutations and their association with clinical manifestation of the disease during the period of COVID-19 outbreak. Sixty-two samples were sequenced via high-throughput platform and screened for variants. For selected mutations, putative biological significance was assessed, and their effects on disease severity. Phylogenetic analysis was conducted on Romanian genomes (n = 112) and on sequences originating from Europe, United Kingdom, Africa, Asia, South, and North America (n = 876). The results indicated multiple introduction events for SARS-CoV-2 in Suceava, mainly from Italy, Spain, United Kingdom, and Russia although some sequences were also related to those from the Czechia, Belgium, and France. Most Suceava genomes contained mutations common to European lineages, such as A20268G, however, approximately 10% of samples were missing such mutations, indicating a possible different arrival route. While overall genome regions ORF1ab, S, and ORF7 were subject to most mutations, several recurring mutations such as A105V were identified, and these were mainly present in severe forms of the disease. Non-synonymous mutations, such as T987N (Thr987Asn in NSP3a domain), associated with changes in a protein responsible for decreasing viral tethering in human host were also present. Patients with diabetes and hypertension exhibited higher risk ratios (RR) of acquiring severe forms of the disease and these were mainly related to A105V mutation. This study identified the arrival routes of SARS-CoV-2 in Romania and revealed potential associations between the SARS-CoV-2 genomic organization circulating in the country and the clinical manifestation of COVID-19 disease. [ABSTRACT FROM AUTHOR]

Novel COVID-19 is a highly infectious disease that is caused by the recently discovered SARS-CoV-2. It is a fast-spreading disease that urgently requires therapeutics. The current study employed computational regression methods to target the ADP-ribose phosphatase (ADRP) domain of Nsp3 using FDA-approved drugs. Identified leads were further investigated using molecular dynamics simulation (MDS). The screening and MDS results suggest that trifluperidol could be a novel inhibitor of the ADRP domain of Nsp3. Trifluperidol could, therefore, be used to help control the spread of COVID-19, either alone or in combination with antiviral agents. [ABSTRACT FROM AUTHOR]

Abstract Background Transmissible gastroenteritis virus (TGEV), a member of the family Coronaviridae, causes lethal watery diarrhea in piglets. Previous studies have revealed that the coronaviruses develop various strategies to evade the host innate immunity through the inhibition of nuclear factor kappa B (NF-κB) signaling pathway. However, the ability of TGEV to inhibit the host innate immune response by modulating the NF-κB signaling pathway is not clear. Methods In this study, a dual luciferase reporter assay was used to confirm the inhibition of NF-κB by TGEV infection and to identify the major viral proteins involved in the inhibition of NF-κB signaling. Real-time quantitative PCR was used to quantify the mRNA expression of inflammatory factors. The deubiquitination of Nsp3 domains and its effect on IκBα and p65 were analyzed by western blotting. The ubiquitination level of IκBα was analyzed by immunoprecipitation. Results In ST and IPEC-J2 cells, TGEV exhibited a dose-dependent inhibition of NF-κB activity. Individual TGEV protein screening revealed the high potential of non-structural protein 3 (Nsp3) to inhibit NF-κB signaling, and leading to the downregulation of the NF-κB-induced cytokine production. We demonstrated that the inhibitory effect of Nsp3 was mainly mediated through the suppression of IκBα degradation as well as the inhibition of p65 phosphorylation and nuclear translocation. Furthermore, the amino acid residues at positions 590–1,215 in Nsp3 were demonstrated to inhibit the degradation of IκBα by inhibiting the IκBα ubiquitination. Conclusion TGEV infection can inhibit the activation of the NF-κB signaling pathway, which is mainly mediated by Nsp3 through the canonical pathway. The amino acid residues at positions 590–1,215 in Nsp3 compose the critical domain that mediates NF-κB inhibition. We speculate that this inhibitory effect is likely to be related to the structure of PLP2 with deubiquitinating enzyme activity of the amino acid residues at positions 590–1,215 in Nsp3. Our study provides a better understanding of the TGEV-mediated innate immune modulation and lays the basis for studies on the pathogenesis of coronavirus.

Macrodomain-I of the NSP3 (non-structural protein 3) is responsible for immune response hijacking in the SARS-CoV-2 infection known as COVID-19. In the omicron variant (B.1.1.529), this domain harbors a new mutation, V1069I, which may increase the binding of ADPr and consequently the infection severity. This macrodomain-I, due to its significant role in infection, is deemed to be an important drug target. Hence, using structural bioinformatics and molecular simulation approaches, we performed a virtual screening of the traditional Chinese medicines (TCM) database for potential anti-viral drugs. The screening of 57,000 compounds yielded the 10 best compounds with docking scores better than the control ADPr. Among the top ten, the best three hits—TCM42798, with a docking score of −13.70 kcal/mol, TCM47007 of −13.25 kcal/mol, and TCM30675 of −12.49 kcal/mol—were chosen as the best hits. Structural dynamic features were explored including stability, compactness, flexibility, and hydrogen bonding, further demonstrating the anti-viral potential of these hits. Using the MM/GBSA approach, the total binding free energy for each complex was reported to be −69.78 kcal/mol, −50.11 kcal/mol, and −47.64 kcal/mol, respectively, which consequently reflect the stronger binding and inhibitory potential of these compounds. These agents might suppress NSP3 directly, allowing the host immune system to recuperate. The current study lays the groundwork for the development of new drugs to combat SARS-CoV-2 and its variants.