A novel coronavirus severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of ...infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp), also named nsp12 is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.
Nonstructural protein 14 (nsp14) of coronaviruses (CoV) is important for viral replication and transcription. The N-terminal exoribonuclease (ExoN) domain plays a proofreading role for prevention of ...lethal mutagenesis, and the C-terminal domain functions as a (guanine-N7) methyl transferase (N7-MTase) for mRNA capping. The molecular basis of both these functions is unknown. Here, we describe crystal structures of severe acute respiratory syndrome (SARS)-CoV nsp14 in complex with its activator nonstructural protein10 (nsp10) and functional ligands. One molecule of nsp10 interacts with ExoN of nsp14 to stabilize it and stimulate its activity. Although the catalytic core of nsp14 ExoN is reminiscent of proofreading exonucleases, the presence of two zinc fingers sets it apart from homologs. Mutagenesis studies indicate that both these zinc fingers are essential for the function of nsp14. We show that a DEEDh (the five catalytic amino acids) motif drives nucleotide excision. The N7-MTase domain exhibits a noncanonical MTase fold with a rare β-sheet insertion and a peripheral zinc finger. The cap-precursor guanosine-P3-adenosine-5′,5′-triphosphate and S-adenosyl methionine bind in proximity in a highly constricted pocket between two β-sheets to accomplish methyl transfer. Our studies provide the first glimpses, to our knowledge, into the architecture of the nsp14–nsp10 complex involved in RNA viral proofreading.
Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete ...mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.
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•Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed•Conformational changes in nsp8 and its interaction with the exiting RNA are observed•Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated•Transition model from primase complex to polymerase complex is proposed
Cryo-EM structures of the SARS-CoV-2 RNA polymerase in complexes with RNA, before and after RNA translocation, reveals structural rearrangements that the RNA-dependent RNA polymerase (RdRp) nsp12 and its co-factors (nsp7 and nsp8) undergo to accommodate nucleic acid binding. Further insights into how the complex is inhibited by remdesivir, and into the primase to polymerase transition, are also presented.
Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a ...SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.
Finite set model predictive torque control (FCSMPTC) of induction machines has received widespread attention in recent years due to its fast dynamic response, intuitive concept, and ability to handle ...nonlinear constraints. However, FCSMPTC essentially belongs to the open-loop control paradigm, and unmatched parameters inevitably cause electromagnetic torque tracking error. In addition, the outer loop (i.e., the speed loop) based on a proportional-integral (PI) regulator cannot achieve optimal control between speed dynamic response and torque tracking error compensation. The traditional control paradigm is abbreviated as PI-MPTC. In order to solve the aforementioned problem, this paper proposes active disturbance-rejection-based model predictive torque control (ADR-MPTC). First, the influence mechanism of mismatched parameters on torque prediction error in PI-MPTC is studied, and then the performance of a traditional PI regulator used to compensate for torque prediction error is analyzed. Second, this paper introduces several parts of the proposed ADR-MPTC, including the design of the torque prediction error observer, nonlinear prediction error compensation strategies, an enhanced predictive torque control, and a simplified full-order flux observer. Finally, PI-MPTC and ADR-MPTC are studied experimentally. The experimental results show that compared with PI-MPTC, ADR-MPTC performs better in dynamic and steady states, and has stronger robustness.
Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural ...information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15
Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins.
The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.
Evaluating how autotrophic (SRA), heterotrophic (SRH) and total soil respiration (SRTOT) respond differently to changes of environmental factors is critical to get an understanding of ecosystem ...carbon (C) cycling and its feedback processes to climate change. A field experiment was conducted to examine the responses of SRA and SRH to water and nitrogen (N) addition in a temperate steppe in northern China during two hydrologically contrasting growing seasons. Water addition stimulated SRA and SRH in both years, and their increases were significantly greater in a dry year (2007) than in a wet year (2006). N addition increased SRA in 2006 but not in 2007, while it decreased SRH in both years, leading to a positive response of SRTOT in 2006 but a negative one in 2007. The different responses of SRA and SRH indicate that it will be uncertain to predict soil C storage if SRTOT is used instead of SRH to estimate variations in soil C storage. Overall, N addition is likely to enhance soil C storage, while the impacts of water addition are determined by its relative effects on carbon input (plant growth) and SRH. Antecedent water conditions played an important role in controlling responses of SRA, SRH and the consequent SRTOT to water and N addition. Our findings highlight the predominance of hydrological conditions in regulating the responses of C cycling to global change in the semiarid temperate steppe of northern China.
The effect of resource pulses, such as rainfall events, on soil respiration plays an important role in controlling grassland carbon balance, but how shifts in long-term precipitation regime regulate ...rain pulse effect on soil respiration is still unclear. We first quantified the influence of rainfall event on soil respiration based on a two-year (2006 and 2009) continuously measured soil respiration data set in a temperate steppe in northern China. In 2006 and 2009, soil carbon release induced by rainfall events contributed about 44.5% (83.3 g C m(-2)) and 39.6% (61.7 g C m(-2)) to the growing-season total soil respiration, respectively. The pulse effect of rainfall event on soil respiration can be accurately predicted by a water status index (WSI), which is the product of rainfall event size and the ratio between antecedent soil temperature to moisture at the depth of 10 cm (r2 = 0.92, P<0.001) through the growing season. It indicates the pulse effect can be enhanced by not only larger individual rainfall event, but also higher soil temperature/moisture ratio which is usually associated with longer dry spells. We then analyzed a long-term (1953-2009) precipitation record in the experimental area. We found both the extreme heavy rainfall events (>40 mm per event) and the long dry-spells (>5 days) during the growing seasons increased from 1953-2009. It suggests the shift in precipitation regime has increased the contribution of rain pulse effect to growing-season total soil respiration in this region. These findings highlight the importance of incorporating precipitation regime shift and its impacts on the rain pulse effect into the future predictions of grassland carbon cycle under climate change.
•C:N, N:P, and C:P ratios decreased with soil depth in a mature subtropical forest.•C-N spatially declined in correlation with depth, whereas C-P and N-P increased.•Spatial variation in soil C, N, ...and P density was driven by different environmental factors at the landscape scale.
As the balance of multiple chemical substances in ecological interactions, stoichiometry of soil carbon (C), nitrogen (N) and phosphorus (P) is critical for forest sustainability. Soil depth is critical in regulating soil C-N-P stoichiometry; however, the vertical pattern of soil C-N-P stoichiometry remains unclear at the local scale in mature subtropical forests. Here, we sampled 555 soil columns from 185 grids at three soil depths (i.e., 0–20, 20–40, and 40–60 cm) in a mature subtropical evergreen forest of eastern China. We found the C:N, N:P, and C:P ratios decreased with increasing soil depth. However, the strength of bivariate correlations among C, N, and P converged from the top to the deepest soil layer. Such vertical convergence was jointly driven by decreasing C-N (R2 as 0.84, 0.80, and 0.76) and increasing C-P (R2 as 0.11, 0.26, and 0.31) correlations from 0–20 to 40–60 cm depths. Further analyses with a structural equation model showed that the spatial variations in C, N, and P were influenced by different environmental factors. For example, the spatial variations in soil organic C and total N in the top soil layer were largely influenced by soil pH, whereas the spatial variation in total P was jointly influenced by topographical, biotic, and soil factors. Our results validate the important impact of soil depth on soil C-N-P stoichiometry at the landscape scale. The converging bivariate correlations between C, N and P along the increasing soil depth indicate the depth-dependent roles of different nutrient elements in soil C cycling.
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