Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography (preprint)

The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5' terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2'-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2'-O Cap-1, where 2'OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors (preprint)

The number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60-2.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2 (preprint)

ABSTRACTSARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (-) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase (ADRP): from the apo form to ligand complexes (preprint)

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

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

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 [A]. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report the high-resolution crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 - a virus causing current world-wide epidemics. We compare this structure with previously reported models of Nsp15 from SARS and MERS coronaviruses.