Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS-CoV-2 replication.

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

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.

Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein.

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2.

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-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-CoVs and Middle East Respiratory Syndrome coronavirus (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 two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.