Immunological dysfunction persists for 8 months following initial mild-to-moderate SARS-CoV-2 infection.

A proportion of patients surviving acute coronavirus disease 2019 (COVID-19) infection develop post-acute COVID syndrome (long COVID (LC)) lasting longer than 12 weeks. Here, we studied individuals with LC compared to age- and gender-matched recovered individuals without LC, unexposed donors and individuals infected with other coronaviruses. Patients with LC had highly activated innate immune cells, lacked naive T and B cells and showed elevated expression of type I IFN (IFN-ß) and type III IFN (IFN-λ1) that remained persistently high at 8 months after infection. Using a log-linear classification model, we defined an optimal set of analytes that had the strongest association with LC among the 28 analytes measured. Combinations of the inflammatory mediators IFN-ß, PTX3, IFN-γ, IFN-λ2/3 and IL-6 associated with LC with 78.5-81.6% accuracy. This work defines immunological parameters associated with LC and suggests future opportunities for prevention and treatment.

Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control.

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.

Ready, steady, go: Rapid ribosomal scanning to reach start codons.

Wang et al. (2022)1 employ real-time single-molecule fluorescence spectroscopy to monitor eukaryotic translation initiation events, revealing that, while mRNA engagement by ribosomal 43S subunits is slow, the subsequent mRNA scanning process is rapid- ∼10 times faster than translation.

Structural basis of ribosomal 30S subunit degradation by RNase R.

Protein synthesis is a major energy-consuming process of the cell that requires the controlled production1-3 and turnover4,5 of ribosomes. Although the past few years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3' to 5' exonuclease ribonuclease R (RNase R). The structures reveal that RNase R binds at first to the 30S platform to facilitate the degradation of the functionally important anti-Shine-Dalgarno sequence and the decoding-site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S subunit, and this is overcome by a major structural rearrangement of the 30S head, involving the loss of ribosomal proteins. RNase R parallels this movement and relocates to the decoding site by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also indicate that the enzyme alone is sufficient for complete degradation of 30S subunits. Collectively, our results provide a mechanistic basis for the degradation of 30S mediated by RNase R, and reveal that RNase R targets orphaned 30S subunits using a dynamic mechanism involving an anchored switching of binding sites.

Synaptic wiring motifs in posterior parietal cortex support decision-making.

The posterior parietal cortex exhibits choice-selective activity during perceptual decision-making tasks1-10. However, it is not known how this selective activity arises from the underlying synaptic connectivity. Here we combined virtual-reality behaviour, two-photon calcium imaging, high-throughput electron microscopy and circuit modelling to analyse how synaptic connectivity between neurons in the posterior parietal cortex relates to their selective activity. We found that excitatory pyramidal neurons preferentially target inhibitory interneurons with the same selectivity. In turn, inhibitory interneurons preferentially target pyramidal neurons with opposite selectivity, forming an opponent inhibition motif. This motif was present even between neurons with activity peaks in different task epochs. We developed neural-circuit models of the computations performed by these motifs, and found that opponent inhibition between neural populations with opposite selectivity amplifies selective inputs, thereby improving the encoding of trial-type information. The models also predict that opponent inhibition between neurons with activity peaks in different task epochs contributes to creating choice-specific sequential activity. These results provide evidence for how synaptic connectivity in cortical circuits supports a learned decision-making task.

Ribosome rescue, nearing the end.

In eukaryotes, Dom34 is involved in the rescue of ribosomes that stall on mRNAs during protein synthesis. Using ribosome profiling, Guydosh and Green reveal that, in addition to rescue of ribosomes stalled on truncated mRNAs, Dom34 also recycles ribosomes that are unexpectedly found in the 3' untranslated regions of many cellular mRNAs.

A cell-type-specific error-correction signal in the posterior parietal cortex.

Neurons in the posterior parietal cortex contribute to the execution of goal-directed navigation1 and other decision-making tasks2-4. Although molecular studies have catalogued more than 50 cortical cell types5, it remains unclear what distinct functions they have in this area. Here we identified a molecularly defined subset of somatostatin (Sst) inhibitory neurons that, in the mouse posterior parietal cortex, carry a cell-type-specific error-correction signal for navigation. We obtained repeatable experimental access to these cells using an adeno-associated virus in which gene expression is driven by an enhancer that functions specifically in a subset of Sst cells6. We found that during goal-directed navigation in a virtual environment, this subset of Sst neurons activates in a synchronous pattern that is distinct from the activity of surrounding neurons, including other Sst neurons. Using in vivo two-photon photostimulation and ex vivo paired patch-clamp recordings, we show that nearby cells of this Sst subtype excite each other through gap junctions, revealing a self-excitation circuit motif that contributes to the synchronous activity of this cell type. These cells selectively activate as mice execute course corrections for deviations in their virtual heading during navigation towards a reward location, for both self-induced and experimentally induced deviations. We propose that this subtype of Sst neurons provides a self-reinforcing and cell-type-specific error-correction signal in the posterior parietal cortex that may help with the execution and learning of accurate goal-directed navigation trajectories.

Structural Basis for Bacterial Ribosome-Associated Quality Control by RqcH and RqcP.

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

Paenilamicins are context-specific translocation inhibitors of protein synthesis.

The paenilamicins are a group of hybrid nonribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here we determine structures of paenilamicin PamB2-stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site transfer RNAs (tRNAs). In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms used by P. larvae. We further demonstrate that PamB2 interferes with the translocation of messenger RNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a class of context-specific translocation inhibitors.

Evolving precision: rRNA expansion segment 7S modulates translation velocity and accuracy in eukaryal ribosomes.

Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

A role for the S4-domain containing protein YlmH in ribosome-associated quality control in Bacillus subtilis.

Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation.

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states, as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.

Structural basis for translation inhibition by the glycosylated drosocin peptide.

The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation not only influences cellular uptake of the peptide but also interacts with its intracellular target, the ribosome. Cryogenic electron microscopy structures of glycosylated drosocin on the ribosome at 2.0-2.8-Å resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base pair with A752. Collectively, our study reveals novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provide a structural basis for future development of this class of antimicrobials.

Challenges of COVID-19 Case Forecasting in the US, 2020-2021.

During the COVID-19 pandemic, forecasting COVID-19 trends to support planning and response was a priority for scientists and decision makers alike. In the United States, COVID-19 forecasting was coordinated by a large group of universities, companies, and government entities led by the Centers for Disease Control and Prevention and the US COVID-19 Forecast Hub (https://covid19forecasthub.org). We evaluated approximately 9.7 million forecasts of weekly state-level COVID-19 cases for predictions 1-4 weeks into the future submitted by 24 teams from August 2020 to December 2021. We assessed coverage of central prediction intervals and weighted interval scores (WIS), adjusting for missing forecasts relative to a baseline forecast, and used a Gaussian generalized estimating equation (GEE) model to evaluate differences in skill across epidemic phases that were defined by the effective reproduction number. Overall, we found high variation in skill across individual models, with ensemble-based forecasts outperforming other approaches. Forecast skill relative to the baseline was generally higher for larger jurisdictions (e.g., states compared to counties). Over time, forecasts generally performed worst in periods of rapid changes in reported cases (either in increasing or decreasing epidemic phases) with 95% prediction interval coverage dropping below 50% during the growth phases of the winter 2020, Delta, and Omicron waves. Ideally, case forecasts could serve as a leading indicator of changes in transmission dynamics. However, while most COVID-19 case forecasts outperformed a naïve baseline model, even the most accurate case forecasts were unreliable in key phases. Further research could improve forecasts of leading indicators, like COVID-19 cases, by leveraging additional real-time data, addressing performance across phases, improving the characterization of forecast confidence, and ensuring that forecasts were coherent across spatial scales. In the meantime, it is critical for forecast users to appreciate current limitations and use a broad set of indicators to inform pandemic-related decision making.

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P.

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

The cyclic octapeptide antibiotic argyrin B inhibits translation by trapping EF-G on the ribosome during translocation.

Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.

Three-Coordinate Nickel and Metal-Metal Interactions in a Heterometallic Iron-Sulfur Cluster.

Biological multielectron reactions often are performed by metalloenzymes with heterometallic sites, such as anaerobic carbon monoxide dehydrogenase (CODH), which has a nickel-iron-sulfide cubane with a possible three-coordinate nickel site. Here, we isolate the first synthetic iron-sulfur clusters having a nickel atom with only three donors, showing that this structural feature is feasible. These have a core with two tetrahedral irons, one octahedral tungsten, and a three-coordinate nickel connected by sulfide and thiolate bridges. Electron paramagnetic resonance (EPR), Mössbauer, and superconducting quantum interference device (SQUID) data are combined with density functional theory (DFT) computations to show how the electronic structure of the cluster arises from strong magnetic coupling between the Ni, Fe, and W sites. X-ray absorption spectroscopy, together with spectroscopically validated DFT analysis, suggests that the electronic structure can be described with a formal Ni1+ atom participating in a nonpolar Ni-W σ-bond. This metal-metal bond, which minimizes spin density at Ni1+, is conserved in two cluster oxidation states. Fe-W bonding is found in all clusters, in one case stabilizing a local non-Hund state at tungsten. Based on these results, we compare different M-M interactions and speculate that other heterometallic clusters, including metalloenzyme active sites, could likewise store redox equivalents and stabilize low-valent metal centers through metal-metal bonding.

Mixed Valence {Ni2+Ni1+} Clusters as Models of Acetyl Coenzyme A Synthase Intermediates.

Acetyl coenzyme A synthase (ACS) catalyzes the formation and deconstruction of the key biological metabolite, acetyl coenzyme A (acetyl-CoA). The active site of ACS features a {NiNi} cluster bridged to a [Fe4S4]n+ cubane known as the A-cluster. The mechanism by which the A-cluster functions is debated, with few model complexes able to replicate the oxidation states, coordination features, or reactivity proposed in the catalytic cycle. In this work, we isolate the first bimetallic models of two hypothesized intermediates on the paramagnetic pathway of the ACS function. The heteroligated {Ni2+Ni1+} cluster, [K(12-crown-4)2][1], effectively replicates the coordination number and oxidation state of the proposed "Ared" state of the A-cluster. Addition of carbon monoxide to [1]- allows for isolation of a dinuclear {Ni2+Ni1+(CO)} complex, [K(12-crown-2)n][2] (n = 1-2), which bears similarity to the "ANiFeC" enzyme intermediate. Structural and electronic properties of each cluster are elucidated by X-ray diffraction, nuclear magnetic resonance, cyclic voltammetry, and UV/vis and electron paramagnetic resonance spectroscopies, which are supplemented by density functional theory (DFT) calculations. Calculations indicate that the pseudo-T-shaped geometry of the three-coordinate nickel in [1]- is more stable than the Y-conformation by 22 kcal mol-1, and that binding of CO to Ni1+ is barrierless and exergonic by 6 kcal mol-1. UV/vis absorption spectroscopy on [2]- in conjunction with time-dependent DFT calculations indicates that the square-planar nickel site is involved in electron transfer to the CO π*-orbital. Further, we demonstrate that [2]- promotes thioester synthesis in a reaction analogous to the production of acetyl coenzyme A by ACS.