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Identification of novel mutations in the methyltransferase complex (Nsp10-Nsp16) of SARS-CoV-2.

ABSTRACT: A recent outburst of the pandemic caused by a member of the coronaviridae family identified as SARS-CoV-2. The highly contagious nature of the virus allows it to spread rapidly worldwide and caused severe healthcare and economic distress. So far, no proper line of treatment or vaccines has been available against SARS-CoV-2. Since, the infected people rapidly increased, causing the saturation of healthcare systems with coronavirus disease (COVID-19) patients. As the virus spread to new locations it also acquired various mutations. Here, in this study, we focused on identifying mutations in one of the crucial complex of SARS-CoV-2, the Nsp10-Nsp16 2'-O-methyltransferase complex. This complex plays indispensable role in the post-transcriptional modifications of viral RNA by its capping. We analysed 208 sequences of Nsp10-Nsp16 reported from India and compared with first reported sequence from Wuhan, China. Our analysis revealed a single mutation in Nsp10 and five mutations in Nsp16 protein. We also show that these mutations are leading to alteration in the secondary structure of Nsp10-Nsp16. Further, the protein modelling studies revealed that the mutation of both Nsp10-Nsp16 impacts the protein dynamicity and stability. Altogether, this study provides novel insights into the variations observed in the proteins of SARS-CoV-2 that might have functional consequences.

SUBMITTER: Azad GK

PROVIDER: S-EPMC7547569 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Substrate Specificity of SARS-CoV-2 Nsp10-Nsp16 Methyltransferase.

Project description:The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.

| S-EPMC8472550 | biostudies-literature

Computational Analysis of SAM Analogs as Methyltransferase Inhibitors of nsp16/nsp10 Complex from SARS-CoV-2.

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| S-EPMC9697258 | biostudies-literature

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

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| S-EPMC8216315 | biostudies-literature

Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

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| S-EPMC3102710 | biostudies-literature

The crystal structure of nsp10-nsp16 heterodimer from SARS-CoV-2 in complex with S-adenosylmethionine.

Project description:SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2'-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 Å. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

| S-EPMC7263505 | biostudies-literature

Molecular Docking Studies to Identify Promising Natural Inhibitors Targeting SARS-CoV-2 Nsp10-Nsp16 Protein Complex

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| S-EPMC8892556 | biostudies-literature

Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex.

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| S-EPMC3053173 | biostudies-literature

Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

Project description:Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV.

| S-EPMC7114426 | biostudies-literature

Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16.

Project description:Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tour-de-force and are now increasingly available, repurposing of existing drugs has been less successful. There is a clear need to develop new drugs against SARS-CoV-2 that can also be used against future coronavirus infections. Non-structural protein 10 (nsp10) is a conserved stimulator of two enzymes crucial for viral replication, nsp14 and nsp16, exhibiting exoribonuclease and methyltransferase activities. Interfering with RNA proofreading or RNA cap formation represents intervention strategies to inhibit replication. We applied fragment-based screening using nano differential scanning fluorometry and X-ray crystallography to identify ligands targeting SARS-CoV-2 nsp10. We identified four fragments located in two distinct sites: one can be modelled to where it would be located in the nsp14-nsp10 complex interface and the other in the nsp16-nsp10 complex interface. Microscale thermophoresis (MST) experiments were used to quantify fragment affinities for nsp10. Additionally, we showed by MST that the interaction by nsp14 and 10 is weak and thereby that complex formation could be disrupted by small molecules. The fragments will serve as starting points for the development of more potent analogues using fragment growing techniques and structure-based drug design.

| S-EPMC8729259 | biostudies-literature

Crystal structure of SARS-CoV-2 nsp10-nsp16 in complex with small molecule inhibitors, SS148 and WZ16.

Project description:SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.

| S-EPMC9375521 | biostudies-literature

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