Variable oligomerization modes in coronavirus non-structural protein 9

Non-structural protein 9 (Nsp9) of coronaviruses is believed to bind single-stranded RNA in the viral replication complex. The crystal structure of Nsp9 of human coronavirus (HCoV) 229E reveals a novel disulfide-linked homodimer, which is very different from the previously reported Nsp9 dimer of SARS coronavirus. In contrast, the structure of the Cys69Ala mutant of HCoV-229E Nsp9 shows the same dimer organization as the SARS-CoV protein. In the crystal, the wild-type HCoV-229E protein forms a trimer of dimers, whereas the mutant and SARS-CoV Nsp9 are organized in rod-like polymers. Chemical cross-linking suggests similar modes of aggregation in solution. In zone-interference gel electrophoresis assays and surface plasmon resonance experiments, the HCoV-229E wild-type protein is found to bind oligonucleotides with relatively high affinity, whereas binding by the Cys69Ala and Cys69Ser mutants is observed only for the longest oligonucleotides. The corresponding mutations in SARS-CoV Nsp9 do not hamper nucleic acid binding. From the crystal structures, a model for single-stranded RNA binding by Nsp9 is deduced. We propose that both forms of the Nsp9 dimer are biologically relevant; the occurrence of the disulfide-bonded form may be correlated with oxidative stress induced in the host cell by the viral infection.     

Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.     

RNA recognition and cleavage by the SARS coronavirus endoribonuclease

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn(2+). The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 A structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.     

Membrane binding mechanism of an RNA virus-capping enzyme

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.     

Crystal structures of two coronavirus ADP-ribose-1'-monophosphatases and their complexes with ADP-Ribose: a systematic structural analysis of the viral ADRP domain

The coronaviruses are a large family of plus-strand RNA viruses that cause a wide variety of diseases both in humans and in other organisms. The coronaviruses are composed of three main lineages and have a complex organization of nonstructural proteins (nsp's). In the coronavirus, nsp3 resides a domain with the macroH2A-like fold and ADP-ribose-1"-monophosphatase (ADRP) activity, which is proposed to play a regulatory role in the replication process. However, the significance of this domain for the coronaviruses is still poorly understood due to the lack of structural information from different lineages. We have determined the crystal structures of two viral ADRP domains, from the group I human coronavirus 229E and the group III avian infectious bronchitis virus, as well as their respective complexes with ADP-ribose. The structures were individually solved to elucidate the structural similarities and differences of the ADRP domains among various coronavirus species. The active-site residues responsible for mediating ADRP activity were found to be highly conserved in terms of both sequence alignment and structural superposition, whereas the substrate binding pocket exhibited variations in structure but not in sequence. Together with data from a previous analysis of the ADRP domain from the group II severe acute respiratory syndrome coronavirus and from other related functional studies of ADRP domains, a systematic structural analysis of the coronavirus ADRP domains was realized for the first time to provide a structural basis for the function of this domain in the coronavirus replication process.     

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.     

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.     

Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent RNA polymerase activity

Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His₆-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/β domain and a long-α-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His₆ tag. Remarkably, the same protein harboring the sequence GPLG instead of the His₆ tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.     

Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5′ UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.     

Identification of phytochemicals as potential therapeutic agents that binds to Nsp15 protein target of coronavirus (SARS-CoV-2) that are capable of inhibiting virus replication

BackgroundCoronavirus disease 2019 (COVID-19) playing havoc across the globe caused 585,727 deaths and 13,616,593 confirmed cases so far as per World Health Organization data released till 17th July 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is responsible for causing this pandemic across different continents. It is not only impacting the world economy but also quarantined millions of people in their homes or hospitals.PurposeAt present, there is no Food and Drug Administration-approved drug or vaccine available to treat this disease. Still, people are trying various pre-existing medicines that are known to have anti-viral or anti-parasitic effects. In view of this, the present study aimed to study the binding potential of various phytochemicals present in multiple natural plant extract as a secondary metabolite to non-structural protein 15 (Nsp15) protein, a drug target known to play a crucial role in virulence of coronavirus.MethodNsp15 protein was selected because it shows 89% similarity to the other SARS-CoV, which caused the earlier outbreak. The assumption is that inhibition of Nsp15 slowdowns the viral replication. Phytochemicals are selected as these are present in various plant parts (seed, flower, roots, etc.), which are used in different food cuisines in different geographical regions across the globe. The molecular docking approach was performed using two different software, i.e., Autodock, and Swissdock, to study the interaction of various phytochemicals with Nsp15 protein. Hydroxychloroquine is used as a positive control as it is used by medical professionals showing some positive effects in dealing with coronavirus.ResultsThe present study demonstrated the binding potential of approximately 50 phytochemicals with Nsp15 and capable of inhibiting the viral replication, although in vitro and in vivo tests are required to confirm these findings.ConclusionsIn conclusion, the present study successfully demonstrated the binding of phytochemicals such as sarsasapogenin, ursonic acid, curcumin, ajmalicine, novobiocin, silymarin and aranotin, piperine, gingerol, rosmarinic acid, and alpha terpinyl acetate to Nsp15 viral protein and they might play a key role in inhibiting SARS-CoV-2 replication.     

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN–nsp10) complex: implications for its role in viral genome stability and inhibitor identification

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (M^pro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.     

SARS-CoV-2 Does Not Replicate in Aedes Mosquito Cells nor Present in Field-Caught Mosquitoes from Wuhan

With the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and growing fear, people have become very concerned about whether this novel coronavirus could be transmitted by mosquitoes. We evaluate the infectivity of SARS-CoV-2 in Aedes albopictus and Aedes aegypti derived cell lines. The results indicated that SARS-CoV-2 could not replicate in both C6/36, Sf9 and Aag2 cells. Further, no SARS-CoV-2 RNA was detected in the field collected Culex, and Anopheles mosquitoes during the months of April and May in Wuhan in 2020. Our findings highlight the restricted replication of SARS-CoV-2 in mosquito cells and field-caught mosquitoes.     

The tumor marker human placental protein 11 is an endoribonuclease

Human PP11 (placental protein 11) was previously described as a serine protease specifically expressed in the syncytiotrophoblast and in numerous tumor tissues. Several PP11-like proteins were annotated in distantly related organisms, such as worms and mammals, suggesting their involvement in evolutionarily conserved processes. Based on sequence similarity, human PP11 was included in a protein family whose characterized members are XendoU, a Xenopus laevis endoribonuclease involved in small nucleolar RNA processing, and Nsp15, an endoribonuclease essential for coronavirus replication. Here we show that the bacterially expressed human PP11 displays RNA binding capability and cleaves single stranded RNA in a Mn(2+)-dependent manner at uridylates, to produce molecules with 2',3'-cyclic phosphate ends. These features, together with structural and mutagenesis analyses, which identified the potential active site residues, reveal striking parallels to the amphibian XendoU and assign a ribonuclease function to PP11. This newly discovered enzymatic activity places PP11-like proteins in a completely new perspective.     

Translational control of coronaviruses

Coronaviruses represent a large family of enveloped RNA viruses that infect a large spectrum of animals. In humans, the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic and is genetically related to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which caused outbreaks in 2002 and 2012, respectively. All viruses described to date entirely rely on the protein synthesis machinery of the host cells to produce proteins required for their replication and spread. As such, virus often need to control the cellular translational apparatus to avoid the first line of the cellular defense intended to limit the viral propagation. Thus, coronaviruses have developed remarkable strategies to hijack the host translational machinery in order to favor viral protein production. In this review, we will describe some of these strategies and will highlight the role of viral proteins and RNAs in this process.