SARS-unique Domain Research Articles

The SARS-unique domain (SUD) of SARS-CoV-2 nsp3 protein inhibits the antiviral immune responses through the NF-κB pathway.

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.

Nsp3-N interactions are critical for SARS-CoV-2 fitness and virulence

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.

Identification of the SARS-unique domain of SARS-CoV-2 as an antiviral target

SARS-CoV-2 nsp3 is essential for viral replication and host responses. The SARS-unique domain (SUD) of nsp3 exerts its function through binding to viral and host proteins and RNAs. Herein, we show that SARS-CoV-2 SUD is highly flexible in solution. The intramolecular disulfide bond of SARS-CoV SUD is absent in SARS-CoV-2 SUD. Incorporating this bond in SARS-CoV-2 SUD allowed crystal structure determination to 1.35 Å resolution. However, introducing this bond in SARS-CoV-2 genome was lethal for the virus. Using biolayer interferometry, we screened compounds directly binding to SARS-CoV-2 SUD and identified theaflavin 3,3’-digallate (TF3) as a potent binder, Kd 2.8 µM. TF3 disrupted the SUD-guanine quadruplex interactions and exhibited anti-SARS-CoV-2 activity in Vero E6-TMPRSS2 cells with an EC50 of 5.9 µM and CC50 of 98.5 µM. In this work, we provide evidence that SARS-CoV-2 SUD harbors druggable sites for antiviral development.

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction.

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.

Spatial and temporal roles of SARS-CoV PLpro -A snapshot.

SARS‐CoV and SARS‐CoV‐2 encode four structural and accessory proteins (spike, envelope, membrane and nucleocapsid proteins) and two polyproteins (pp1a and pp1ab). The polyproteins are further cleaved by 3C‐like cysteine protease (3CLpro) and papain‐like protease (PLpro) into 16 nonstructural proteins (nsps). PLpro is released from nsp3 through autocleavage, and then it cleaves the sites between nsp1/2, between nsp2/3 and between nsp3/4 with recognition motif of LXGG, and the sites in the C‐terminus of ubiquitin and of protein interferon‐stimulated gene 15 (ISG15) with recognition motif of RLRGG. Alone or together with SARS unique domain (SUD), PLpro can stabilize an E3 ubiquitin ligase, the ring‐finger, and CHY zinc‐finger domain‐containing 1 (RCHY1), through domain interaction, and thus, promote RCHY1 to ubiquitinate its target proteins including p53. However, a dilemma appears in terms of PLpro roles. On the one hand, the ubiquitination of p53 is good for SARS‐CoV because the ubiquitinated p53 cannot inhibit SARS‐CoV replication. On the other hand, the ubiquitination of NF‐κB inhibitor (IκBα), TNF receptor‐associated factors (TRAFs), and stimulator of interferon gene (STING), and the ISGylation of targeted proteins are bad for SARS‐CoV because these ubiquitination and ISGylation initiate the innate immune response and antiviral state. This mini‐review analyzes the dilemma and provides a snapshot on how the viral PLpro smartly manages its roles to avoid its simultaneously contradictory actions, which could shed lights on possible strategies to deal with SARS‐CoV‐2 infections.

Whole Genome Identification of Potential G-Quadruplexes and Analysis of the G-Quadruplex Binding Domain for SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has become a global public health emergency. G-quadruplex, one of the non-canonical secondary structures, has shown potential antiviral values. However, little is known about the G-quadruplexes of the emerging SARS-CoV-2. Herein, we characterized the potential G-quadruplexes in both positive and negative-sense viral strands. The identified potential G-quadruplexes exhibited similar features to the G-quadruplexes detected in the human transcriptome. Within some bat- and pangolin-related betacoronaviruses, the G-tracts rather than the loops were under heightened selective constraints. We also found that the amino acid sequence similar to SUD (SARS-unique domain) was retained in SARS-CoV-2 but depleted in some other coronaviruses that can infect humans. Further analysis revealed that the amino acid residues related to the binding affinity of G-quadruplexes were conserved among 16,466 SARS-CoV-2 samples. Moreover, the dimer of the SUD-homology structure in SARS-CoV-2 displayed similar electrostatic potential patterns to the SUD dimer from SARS. Considering the potential value of G-quadruplexes to serve as targets in antiviral strategy, our fundamental research could provide new insights for the SARS-CoV-2 drug discovery.

Molecular Basis of SARS-CoV-2 Infection and Rational Design of Potential Antiviral Agents: Modeling and Simulation Approaches.

The emergence in late 2019 of the coronavirus SARS-CoV-2 has resulted in the breakthrough of the COVID-19 pandemic that is presently affecting a growing number of countries. The development of the pandemic has also prompted an unprecedented effort of the scientific community to understand the molecular bases of the virus infection and to propose rational drug design strategies able to alleviate the serious COVID-19 morbidity. In this context, a strong synergy between the structural biophysics and molecular modeling and simulation communities has emerged, resolving at the atomistic level the crucial protein apparatus of the virus and revealing the dynamic aspects of key viral processes. In this Review, we focus on how in silico studies have contributed to the understanding of the SARS-CoV-2 infection mechanism and the proposal of novel and original agents to inhibit the viral key functioning. This Review deals with the SARS-CoV-2 spike protein, including the mode of action that this structural protein uses to entry human cells, as well as with nonstructural viral proteins, focusing the attention on the most studied proteases and also proposing alternative mechanisms involving some of its domains, such as the SARS unique domain. We demonstrate that molecular modeling and simulation represent an effective approach to gather information on key biological processes and thus guide rational molecular design strategies.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Structure of the SARS-Unique Domain C From the Bat Coronavirus HKU4.

Coronaviruses (CoVs) that cause infections such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome phylogenetically originate from bat CoVs. The coronaviral nonstructural protein 3 (nsp3) has been implicated in viral replication, polyprotein cleavage, and host immune interference. We report the structure of the C domain from the SARS-Unique Domain of bat CoV HKU4. The protein has a frataxin fold, consisting of 5 antiparallel β strands packed against 2 α helices. Bioinformatics analyses and nuclear magnetic resonance experiments were conducted to investigate the function of HKU4 C. The results showed that HKU4 C engages in protein-protein interactions with the nearby M domain of nsp3. The HKU4 C residues involved in protein-protein interactions are conserved in group 2c CoVs, indicating a conserved function.

P53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication–transcription complex

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the “SARS-unique domain”. The X domain was proposed to be an ADP-ribose-1”-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

SARS Coronavirus Unique Domain: Three-Domain Molecular Architecture in Solution and RNA Binding

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a “SARS-unique domain” (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of 15N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel β-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.

The SARS-unique domain of SARS-CoV contains two macrodomains that bind G-quadruplexes

The SARS-unique domain of SARS-CoV contains two macrodomains that bind G-quadruplexes

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

Nuclear Magnetic Resonance Structure Shows that the Severe Acute Respiratory Syndrome Coronavirus-Unique Domain Contains a Macrodomain Fold

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

The “SARS-unique domain” (SUD) of SARS coronavirus is an oligo(G)-binding protein

Caused by a new coronavirus, severe acute respiratory syndrome (SARS) is a highly contagious disease associated with significant fatality that emerged in 2003. The molecular cause of the unusually high human pathogenicity of the SARS coronavirus (SARS-CoV) is still unknown. In an effort to characterize molecular components of the virus that are absent in other coronaviruses, all of which are considerably less pathogenic for humans, we recombinantly produced the SARS-unique domain (SUD) within non-structural protein 3 (Nsp3) of SARS-CoV and characterized its nucleic-acid binding properties. Zone-interference gel electrophoresis and electrophoretic mobility shift assays revealed a specific affinity of SUD for oligo(G)-strings. A few such segments are present in the SARS-CoV genome, but also in mRNAs of host proteins involved in the regulation of signaling pathways. A putative role of SUD in virus-induced apoptosis or survival of host cells is discussed.