A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

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.

A viral protein disrupts vacuolar acidification to facilitate virus infection in plants.

Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.

Drought shortens subtropical understory growing season by advancing leaf senescence.

Subtropical forests, recognized for their intricate vertical canopy stratification, exhibit high resistance to extreme drought. However, the response of leaf phenology to drought in the species-rich understory remains poorly understood. In this study, we constructed a digital camera system, amassing over 360,000 images through a 70% throughfall exclusion experiment, to explore the drought response of understory leaf phenology. The results revealed a significant advancement in understory leaf senescence phenology under drought, with 11.75 and 15.76 days for the start and end of the leaf-falling event, respectively. Pre-season temperature primarily regulated leaf development phenology, whereas soil water dominated the variability in leaf senescence phenology. Under drought conditions, temperature sensitivities for the end of leaf emergence decreased from -13.72 to -11.06 days °C-1, with insignificance observed for the start of leaf emergence. Consequently, drought treatment shortened both the length of the growing season (15.69 days) and the peak growth season (9.80 days) for understory plants. Moreover, this study identified diverse responses among intraspecies and interspecies to drought, particularly during the leaf development phase. These findings underscore the pivotal role of water availability in shaping understory phenology patterns, especially in subtropical forests.

Astaxanthin attenuates cigarette smoke-induced small airway remodeling via the AKT1 signaling pathway.

BACKGROUND: Astaxanthin (AXT) is a keto-carotenoid with a variety of biological functions, including antioxidant and antifibrotic effects. Small airway remodeling is the main pathology of chronic obstructive pulmonary disease (COPD) and is caused by epithelial-to-mesenchymal transition (EMT) and fibroblast differentiation and proliferation. Effective therapies are still lacking. This study aimed to investigate the role of AXT in small airway remodeling in COPD and its underlying mechanisms. METHODS: First, the model of COPD mice was established by cigarette smoke (CS) exposure combined with intraperitoneal injection of cigarette smoke extract (CSE). The effects of AXT on the morphology of CS combined with CSE -induced emphysema, EMT, and small airway remodeling by using Hematoxylin-eosin (H&E) staining, immunohistochemical staining, and western blot. In addition, in vitro experiments, the effects of AXT on CSE induced-EMT and fibroblast function were further explored. Next, to explore the specific mechanisms underlying the protective effects of AXT in COPD, potential targets of AXT in COPD were analyzed using network pharmacology. Finally, the possible mechanism was verified through molecular docking and in vitro experiments. RESULTS: AXT alleviated pulmonary emphysema, EMT, and small airway remodeling in a CS combined with CSE -induced mouse model. In addition, AXT inhibited the EMT process in airway cells and the differentiation and proliferation of fibroblasts. Mechanistically, AXT inhibited myofibroblast activation by directly binding to and suppressing the phosphorylation of AKT1. Therefore, our results show that AXT protects against small airway remodeling by inhibiting AKT1. CONCLUSIONS: The present study identified and illustrated a new food function of AXT, indicating that AXT could be used in the therapy of COPD-induced small airway remodeling.

HDAC6-selective inhibitor CAY10603 ameliorates cigarette smoke-induced small airway remodeling by regulating epithelial barrier dysfunction and reversing.

BACKGROUND: Small airway remodelling is a vital characteristic of chronic obstructive pulmonary disease (COPD), which is mainly caused by epithelial barrier dysfunction and epithelial-mesenchymal transition (EMT). Recent studies have indicated that histone deacetylase 6 (HDAC6) plays an important role in the dysregulation of epithelial function. In this study, we investigated the therapeutic effects and underlying mechanisms of an inhibitor with high selectivity for HDAC6 in COPD. METHODS: Cigarette smoke (CS) exposure was used to establish a CS-induced COPD mouse model. CAY10603 at doses of 2.5 and 10 mg/kg was injected intraperitoneally on alternate days. The protective effects of CAY10603 against CS-induced emphysema, epithelial barrier function and small airway remodeling were evaluated using hematoxylin and eosin (H&E) staining, Masson's trichrome staining, immunohistochemical staining, and western blot. The human lung bronchial epithelial cell line (HBE) was used to elucidate the underlying molecular mechanism of action of CAY10603. RESULTS: HDAC6 levels in the lung homogenates of CS-exposed mice were higher than that those in control mice. Compared to the CS group, the mean linear intercept (MLI) of the CAY10603 treatment group decreased and the mean alveolar number (MAN)increased. Collagen deposition was reduced in groups treated with CAY10603. The expression of α-SMA was markedly upregulated in the CS group, which was reversed by CAY10603 treatment. Conversely, E-cadherin expression in the CS group was further downregulated, which was reversed by CAY10603 treatment. CAY10603 affects the tight junction protein expression of ZO-1 and occludin. ZO-1 and occludin expression were markedly downregulated in the CS group. After CAY10603treatment, the protein expression level of ZO-1 and occludin increased significantly. In HBE cells, Cigarette smoke extract (CSE) increased HDAC6 levels. CAY10603 significantly attenuated the release of TGF-ß1 induced by CSE. CAY10603 significantly increased the E-cadherin levels in TGF-ß1 treated HBE cells, while concurrently attenuated α-SMA expression. This effect was achieved through the suppression of Smad2 and Smad3 phosphorylation. CAY10603 also inhibited TGF-ß1 induced cell migration. CONCLUSIONS: These findings suggested that CAY10603 inhibited CS induced small airway remodelling by regulating epithelial barrier dysfunction and reversing EMT via the TGF-ß1/Smad2/3 signalling pathway.

Rice-produced classical swine fever virus glycoprotein E2 with herringbone-dimer design to enhance immune responses.

Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone-dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 µg provided effective protection in pigs without interference from pre-existing antibodies. Crystal structure and small-angle X-ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Efficacy of high-flow nasal cannula in patients with acute heart failure: a systematic review and meta-analysis.

BACKGROUND: Acute heart failure (AHF) is often associated with diffuse insufficiency and arterial hypoxemia, requiring respiratory support for rapid and effective correction. We aimed to compare the effects of high-flow nasal cannula(HFNC) with those of conventional oxygen therapy(COT) or non-invasive ventilation(NIV) on the prognosis of patients with AHF. METHODS: We performed the search using PubMed, Embase, Web of Science, MEDLINE, the Cochrane Library, CNKI, Wanfang, and VIP databases from the inception to August 31, 2023 for relevant studies in English and Chinese. We included controlled studies comparing HFNC with COT or NIV in patients with AHF. Primary outcomes included the intubation rate, respiratory rate (RR), heart rate (HR), and oxygenation status. RESULTS: From the 1288 original papers identified, 16 studies met the inclusion criteria, and 1333 patients were included. Compared with COT, HFNC reduced the intubation rate (odds ratio [OR]: 0.29, 95% CI: 0.14-0.58, P = 0.0005), RR (standardized mean difference [SMD]: -0.73 95% CI: -0.99 - -0.47, P < 0.00001) and HR (SMD: -0.88, 95% CI: -1.07 - -0.69, P < 0.00001), and hospital stay (SMD: -0.94, 95% CI: -1.76 - -0.12, P = 0.03), and increase arterial oxygen partial pressure (PaO2), (SMD: 0.88, 95% CI: 0.70-1.06, P < 0.00001) and oxygen saturation (SpO2 [%], SMD: 0.70, 95% CI: 0.34-1.06, P = 0.0001). CONCLUSIONS: There were no significant differences in intubation rate, RR, HR, arterial blood gas parameters, and dyspnea scores between the HFNC and NIV groups. Compared with COT, HFNC effectively reduced the intubation rate and provided greater clinical benefits to patients with AHF. However, there was no significant difference in the clinical prognosis of patients with AHF between the HFNC and NIV groups. TRIAL REGISTRATION: PROSPERO (identifier: CRD42022365611).

Structural analysis of a trimeric assembly of the mitochondrial dynamin-like GTPase Mgm1.

The fusion of inner mitochondrial membranes requires dynamin-like GTPases, Mgm1 in yeast and OPA1 in mammals, but how they mediate membrane fusion is poorly understood. Here, we determined the crystal structure of Saccharomyces cerevisiae short Mgm1 (s-Mgm1) in complex with GDP. It revealed an N-terminal GTPase (G) domain followed by two helix bundles (HB1 and HB2) and a unique C-terminal lipid-interacting stalk (LIS). Dimers can form through antiparallel HB interactions. Head-to-tail trimers are built by intermolecular interactions between the G domain and HB2-LIS. Biochemical and in vivo analyses support the idea that the assembly interfaces observed here are native and critical for Mgm1 function. We also found that s-Mgm1 interacts with negatively charged lipids via both the G domain and LIS. Based on these observations, we propose that membrane targeting via the G domain and LIS facilitates the in cis assembly of Mgm1, potentially generating a highly curved membrane tip to allow inner membrane fusion.

Plant photosynthetic overcompensation under nocturnal warming: lack of evidence in subtropical evergreen trees.

BACKGROUND AND AIMS: Increased plant photosynthesis under nocturnal warming is a negative feedback mechanism to overcompensate for night-time carbon loss to mitigate climate warming. This photosynthetic overcompensation effect has been observed in dry deciduous ecosystems but whether it exists in subtropical wet forest trees is unclear. METHODS: Two subtropical evergreen tree species (Schima superba and Castanopsis sclerophylla) were grown in a greenhouse and exposed to ambient and elevated night-time temperature. The occurrence of the photosynthetic overcompensation effect was determined by measuring daytime and night-time leaf gas exchange and non-structural carbohydrate (NSC) concentration. KEY RESULTS: A reduction in leaf photosynthesis for both species and an absence of persistent photosynthetic overcompensation were observed. The photosynthetic overcompensation effect was transient in S. superba due to respiratory acclimation and stomatal limitation. For S. superba, nocturnal warming resulted in insufficient changes in night-time respiration and NSC concentration to stimulate overcompensation and inhibited leaf stomatal conductance by increasing the leaf-to-air vapour pressure deficit. CONCLUSIONS: The results indicate that leaf stomatal conductance is important for the photosynthetic overcompensation effect in different tree species. The photosynthetic overcompensation effect under nocturnal warming may be a transient occurrence rather than a persistent mechanism in subtropical forest ecosystems.

The root-specific NtR12 promoter-based expression of RIP increased the resistance against bacterial wilt disease in tobacco.

BACKGROUND: Tobacco is an important economic crop, but the quality and yield have been severely impaired by bacterial wilt disease (BWD) caused by Ralstonia solanacearum. METHODS AND RESULTS: Here, we describe a transgenic approach to prevent BWD in tobacco plants. A new root-specific promoter of an NtR12 gene was successfully cloned. The NtR12 promoter drove GUS reporter gene expression to a high level in roots but to less extent in stems, and no significant expression was detected in leaves. The Ribosome-inactivating proteins (RIP) gene from Momordica charantia was also cloned, and its ability to inhibit Ralstonia solanacearum was evaluated using RIP protein produced by the prokaryotic expression system. The RIP gene was constructed downstream of the NtR12 promoter and transformed into the tobacco cultivar "Cuibi No. 1" (CB-1), resulting in many descendants. The resistance against BWD was significantly improved in transgenic tobacco lines expressing NtR12::RIP. CONCLUSION: This study confirms that the RIP gene confers resistance to BWD and the NtR12 as a new promoter for its specific expression in root and stem. Our findings pave a novel avenue for transgenic engineering to prevent the harmful impact of diseases and pests in roots and stems.

DWARF14 is a non-canonical hormone receptor for strigolactone.

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/ß hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.

Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis.

To date, an effective therapeutic treatment that confers strong attenuation toward coronaviruses (CoVs) remains elusive. Of all the potential drug targets, the helicase of CoVs is considered to be one of the most important. Here, we first present the structure of the full-length Nsp13 helicase of SARS-CoV (SARS-Nsp13) and investigate the structural coordination of its five domains and how these contribute to its translocation and unwinding activity. A translocation model is proposed for the Upf1-like helicase members according to three different structural conditions in solution characterized through H/D exchange assay, including substrate state (SARS-Nsp13-dsDNA bound with AMPPNP), transition state (bound with ADP-AlF4-) and product state (bound with ADP). We observed that the ß19-ß20 loop on the 1A domain is involved in unwinding process directly. Furthermore, we have shown that the RNA dependent RNA polymerase (RdRp), SARS-Nsp12, can enhance the helicase activity of SARS-Nsp13 through interacting with it directly. The interacting regions were identified and can be considered common across CoVs, which provides new insights into the Replication and Transcription Complex (RTC) of CoVs.

Crystal Structure of the Epo1-Bem3 Complex for Bud Growth.

Tubules of the endoplasmic reticulum (ER) spread into the buds of yeast by an actin-based mechanism and, upon entry, become attached to the polarisome, a proteinaceous micro-compartment below the tip of the bud. The minimal tether between polarisome and cortical ER is formed by a protein complex consisting of Epo1, a member of the polarisome, Scs2, a membrane protein of the ER and Cdc42 guanosine triphosphatase-activating protein Bem3. Here, we report the crystal structure of a complex between Epo1 and Bem3. In addition, we characterize through the hydrogen/deuterium (H/D) exchange assay the interface between Scs2 and Epo1. Our findings provide a first structural insight into the molecular architecture of the link between cortical ER and the polarisome.

Increasing soluble dietary fiber content and antioxidant activity of wheat bran through twin-screw extrusion pretreatment.

As a by-product during flour production, wheat bran is mainly used as raw material for fodder or fermentation. In the present work, wheat bran was extruded with different moisture conditions and the consequently chemical component, absorption capacity, and antioxidant activity of treated wheat bran were analyzed. Results showed that extrusion decreased the particle size and crystallinity of wheat bran, but increased the soluble dietary fiber content of which from 3.08% to 11.78%. Meanwhile, water holding capacity, oil holding capacity for peanut oil and lard, and swelling capacity of WB-W-G-Na reached 5.67 g/g, 3.34 g/g, 3.58 g/g and 4.3 mL/g, respectively. Moreover, DPPH radical scavenging activity of WB-W-G-Na increased from 6.8% to 18.4% and hydroxyl radical scavenging activity increased from 5.3% to 15.9%. Overall, this work provides an excellent pretreatment method for increasing the functional activities of wheat bran in the food industry.

Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus.

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 in vitro 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.IMPORTANCE 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.

Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex.

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 ß-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 ß-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.

Joint control of terrestrial gross primary productivity by plant phenology and physiology.

Terrestrial gross primary productivity (GPP) varies greatly over time and space. A better understanding of this variability is necessary for more accurate predictions of the future climate-carbon cycle feedback. Recent studies have suggested that variability in GPP is driven by a broad range of biotic and abiotic factors operating mainly through changes in vegetation phenology and physiological processes. However, it is still unclear how plant phenology and physiology can be integrated to explain the spatiotemporal variability of terrestrial GPP. Based on analyses of eddy-covariance and satellite-derived data, we decomposed annual terrestrial GPP into the length of the CO2 uptake period (CUP) and the seasonal maximal capacity of CO2 uptake (GPPmax). The product of CUP and GPPmax explained >90% of the temporal GPP variability in most areas of North America during 2000-2010 and the spatial GPP variation among globally distributed eddy flux tower sites. It also explained GPP response to the European heatwave in 2003 (r(2) = 0.90) and GPP recovery after a fire disturbance in South Dakota (r(2) = 0.88). Additional analysis of the eddy-covariance flux data shows that the interbiome variation in annual GPP is better explained by that in GPPmax than CUP. These findings indicate that terrestrial GPP is jointly controlled by ecosystem-level plant phenology and photosynthetic capacity, and greater understanding of GPPmax and CUP responses to environmental and biological variations will, thus, improve predictions of GPP over time and space.