Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19.

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.

The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.

Exploring the Catalytic Mechanism of the RNA Cap Modification by nsp16-nsp10 Complex of SARS-CoV-2 through a QM/MM Approach.

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2'-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2'-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

Unravelling the Immunomodulatory Effects of Viral Ion Channels, towards the Treatment of Disease.

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.

Destruction mechanisms of ozone over SARS-CoV-2.

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.

Crystal structure of SARS-CoV-2 nsp10 bound to nsp14-ExoN domain reveals an exoribonuclease with both structural and functional integrity.

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.

Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme.

Coronavirus 3'-to-5' exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10­nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle. ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog­based antivirals. Here we present cryo­electron microscopy structures of both wild-type and mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp10-nsp14 in complex with an RNA substrate bearing a 3'-end mismatch at resolutions ranging from 2.5 to 3.9 angstroms. The structures reveal the molecular determinants of ExoN substrate specificity and offer insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis. Our findings provide guidance for rational design of improved anticoronavirus therapies.

Crystal Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.

A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2'-O methylation of its first nucleotide.

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography.

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex.

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.

1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

Structural insights into SARS-CoV-2 proteins.

The unprecedented scale of the ongoing COVID-19 pandemic has catalyzed an intense effort of the global scientific community to unravel different aspects of the disease in a short time. One of the crucial aspects of these developments is the determination of more than three hundred experimental structures of SARS-CoV-2 proteins in the last few months. These include structures of viral non-structural, structural, and accessory proteins and their complexes determined by either X-ray diffraction or cryo-electron microscopy. These structures elucidate the intricate working of different components of the viral machinery at the atomic level during different steps of the viral life cycle, including attachment to the host cell, viral genome replication and transcription, and genome packaging and assembly of the virion. Some of these proteins are also potential targets for drug development against the disease. In this review, we discuss important structural features of different SARS-CoV-2 proteins with their function, and their potential as a target for therapeutic interventions.

Processing of the SARS-CoV pp1a/ab nsp7-10 region.

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

Characterization of accessory genes in coronavirus genomes.

BACKGROUND: The Covid19 infection is caused by the SARS-CoV-2 virus, a novel member of the coronavirus (CoV) family. CoV genomes code for a ORF1a / ORF1ab polyprotein and four structural proteins widely studied as major drug targets. The genomes also contain a variable number of open reading frames (ORFs) coding for accessory proteins that are not essential for virus replication, but appear to have a role in pathogenesis. The accessory proteins have been less well characterized and are difficult to predict by classical bioinformatics methods. METHODS: We propose a computational tool GOFIX to characterize potential ORFs in virus genomes. In particular, ORF coding potential is estimated by searching for enrichment in motifs of the X circular code, that is known to be over-represented in the reading frames of viral genes. RESULTS: We applied GOFIX to study the SARS-CoV-2 and related genomes including SARS-CoV and SARS-like viruses from bat, civet and pangolin hosts, focusing on the accessory proteins. Our analysis provides evidence supporting the presence of overlapping ORFs 7b, 9b and 9c in all the genomes and thus helps to resolve some differences in current genome annotations. In contrast, we predict that ORF3b is not functional in all genomes. Novel putative ORFs were also predicted, including a truncated form of the ORF10 previously identified in SARS-CoV-2 and a little known ORF overlapping the Spike protein in Civet-CoV and SARS-CoV. CONCLUSIONS: Our findings contribute to characterizing sequence properties of accessory genes of SARS coronaviruses, and especially the newly acquired genes making use of overlapping reading frames.

Structural basis of RNA cap modification by SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.

Molecular conservation and differential mutation on ORF3a gene in Indian SARS-CoV2 genomes.

A global emergency due to the COVID-19 pandemic demands various studies related to genes and genomes of the SARS-CoV2. Among other important proteins, the role of accessory proteins are of immense importance in replication, regulation of infections of the coronavirus in the hosts. The largest accessory protein in the SARS-CoV2 genome is ORF3a which modulates the host response to the virus infection and consequently it plays an important role in pathogenesis. In this study, an attempt is made to decipher the conservation of nucleotides, dimers, codons and amino acids in the ORF3a genes across thirty-two genomes of Indian patients. ORF3a gene possesses single and double point mutations in Indian SARS-CoV2 genomes suggesting the change of SARS-CoV2's virulence property in Indian patients. We find that the parental origin of the ORF3a gene over the genomes of SARS-CoV2 and Pangolin-CoV is same from the phylogenetic analysis based on conservation of nucleotides and so on. This study highlights the accumulation of mutation on ORF3a in Indian SARS-CoV2 genomes which may provide the designing therapeutic approach against SARS-CoV2.