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Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

From a designed library of indolyl pyrimidinamines we identified a highly potent and cell-active chemical probe (analog 17) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of inhibitor selectivity confirmed that this PIKfyve probe demonstrates excellent kinome-wide selectivity. A structurally related indolyl pyrimidinamine (analog 30) was characterized as a suitable negative control analog that lacks PIKfyve inhibitory activity and exhibits exquisite selectivity when profiled against the screenable human kinome. Our chemical probe disrupts multiple phases of the life cycle of β-coronaviruses. We observed potent inhibition of viral replication, reduced viral entry, and impacts on a mediator of viral transmission (lysosomes). Our scaffold is a distinct chemotype versus published PIKfyve inhibitors and lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors. Our chemical probe set can be used by the community to characterize the role of PIKfyve in virology and beyond.

There is significant interest in T-cell mediated immunity against SARS-CoV-2. Both vaccination and infection have been observed to elicit durable T-cell responses against the virus. The classical role of CD4+ T-cell responses in coordinating humoral immunity is well understood but it is less clear to what degree, if any, T-cell responses play a direct protective role against infection In this study we vaccinated BALB/c mice with peptides derived from the SARS-CoV-2 proteome designed to either elicit T-cell responses or B-cell responses against linear epitopes. These peptides were administered in combination with either of two adjuvants, poly(I:C) and the STING agonist BI-1387466. Both adjuvants consistently elicited responses against the same peptides, preferentially from the group selected for predicted T-cell immunogenicity. The magnitude of T-cell responses was, however, significantly higher with BI-1387466 compared with poly(I:C). Neither adjuvant group, however, provided any protection against infection with the murine adapted virus SARS-CoV-2-MA10 or from disease following infection.

Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19.One-Sentence SummaryCovid-19 is associated with targeted inhibition of mitochondrial gene transcription.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine β-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in β-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-β-coronavirus drugs.GRAPHICAL ABSTRACT

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine β-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in β-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-β-coronavirus drugs., GRAPHICAL ABSTRACT

The epsilon subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3'-->5' exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core. To gain further insight into how epsilon performs these joint structural and catalytic functions, we have investigated a set of 20 newly isolated dnaQ mutator mutants. The mutator effects ranged from strong (700-8000-fold enhancement) to moderate (6-20-fold enhancement), reflecting the range of proofreading deficiencies. Complementation assays revealed most mutators to be partially or fully dominant, suggesting that they carried an exonucleolytic defect but retained binding to the pol III core subunits. One allele, containing a stop codon 3 amino acids from the C-terminal end of the protein, was fully recessive. Sequence analysis of the mutants revealed mutations in the Exo I, Exo II and recently proposed Exo IIIepsilon motifs, as well as in the intervening regions. Together, the data support the functional significance of the proposed motifs, presumably in catalysis, and suggest that the C-terminus of straightepsilon may be specifically involved in binding to the alpha (polymerase) subunit.

The Escherichia coli dnaQ gene encodes the 3*35* exonucleolytic proofreading («) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that « might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (a) subunit. We have created truncated forms of dnaQ resulting in « subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain « subunits with the a and u subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the a subunit while retaining normal binding to the u subunit. In contrast, the ND186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the a subunit but was defective in binding to the u subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective a binding mutant. The data are consistent with the existence of two functional domains in «, with the C-terminal domain responsible for polymerase binding. The Escherichia coli DNA polymerase III (Pol III) holoenzyme (HE) is responsible for the efficient and accurate replication of the bacterial chromosome (for a review, see references 9, 10, and 16). This multisubunit enzyme contains a core polymerase with both polymerizing and exonucleolytic activities. Unlike those of most other proofreading polymerases, these two activities of Pol III are contained in two separate polypeptides. The polymerase activity is contained in the a subunit (dnaE gene product), while the 39359 exonuclease (Exo) activity, which serves as a proofreader for replication errors, is contained in the e subunit (dnaQ gene product). The Pol III core also contains the u subunit (holE gene product), but the function of this subunit is unclear (4, 20, 21). The subunits are arranged in a linear fashion, a-e-u, with e binding both a and u (21). The precise interactions, both functionally and structurally, between the core subunits and of the core subunits with the additional accessory factors of the HE are the subject of active current interest (2, 7, 14). Previously, in an effort to better understand the roles of the e subunit, we undertook a genetic analysis of a large collection of dnaQ mutator mutants (23). These studies confirmed and extended the importance of three conserved exonuclease motifs in the catalytic activity of the DnaQ enzyme (see Fig. 1) and also suggested that the C terminus of e might be required for interaction with the a subunit. dnaQ932, a mutant containing a stop codon 3 amino acids from the C-terminal end of e, was recessive in the presence of a single copy of dnaQ 1 , suggesting that the DnaQ932 protein does not compete efficiently with the wild-type protein for binding to a. Furthermore, the region between residues 190 and 212 has been identified as a so-called Q-linker (24). In certain proteins, Q-linkers act as a hinge to link domains that have different functions (22, 24). In e, this linker may tether the N-terminal exonuclease domain to the

The DNA polymerase III (Pol III) holoenzyme (HE) is the major chromosomal replication enzyme in Escherichia coli (19, 22, 30, 31). The enzyme is composed of 17 subunits, 10 of which are distinct (19, 31). HE contains two polymerase core molecules, each consisting of an α, ɛ, and θ subunit arranged in the linear order α-ɛ-θ. The two cores are connected through a dimer of the τ subunit, establishing the basic arrangement of a dimeric polymerase that simultaneously replicates the leading and lagging strands (14). In addition, the HE contains, for each core, a β-clamp (β2) tethering the polymerase to the DNA to ensure high processivity and the five-subunit γ-complex (γδδ′χψ) responsible for loading and unloading the β-clamp. The precise functioning of the HE complex in chromosomal replication is under active investigation (5, 24, 26, 31). Within the Pol III core, α (the dnaE gene product) is the DNA polymerase, while ɛ (the dnaQ gene product) is the 3′→5′ proofreading exonuclease. The function of θ (the holE gene product), which is tightly bound to ɛ, is unclear. Genetic analysis of the Pol III core constituents has provided insight into the role of its constituents. For example, dnaE(Ts) mutants, encoding temperature-sensitive polymerase subunits, are conditionally lethal, as expected in view of the essential nature of the Pol III replication function. Several dnaE mutants display mutator or antimutator effects (9, 28), indicating the important fidelity role of this enzyme. Many dnaQ mutants exhibit strong mutator phenotypes, indicating the importance of the 3′-exonuclease activity for replication fidelity (45). Deletion mutants of dnaQ (23, 27) or mutants lacking the domain necessary for interaction with the polymerase (46) have been generated, but these mutants proved essentially inviable unless accompanied by a suppressing mutation in dnaE. Based on these studies, ɛ is assigned at least two functions: a fidelity function through its 3′-exonuclease activity and a structural function based on its tight, and presumably stabilizing, interaction with the polymerase (23, 27, 45, 46). In contrast, the role of the θ subunit of the Pol III core is unknown. Loss of θ (ΔholE) results in healthy cells with no morphology changes and little or no change in mutant frequencies (42). Based on these studies, it was suggested that θ is not necessary or important for proper functioning of the Pol III core. θ does not affect DNA synthesis by α or α-ɛ (43); however, gel filtration (44), coexpression (1), and yeast two-hybrid experiments (18) have demonstrated a tight interaction between θ and ɛ, but none between θ and α. Interestingly, θ was shown to moderately affect the 3′→5′ proofreading exonuclease activity, as addition of θ to an exonuclease assay measuring the removal of a G · T mispair increased ɛ-mediated excision of the terminal T residue by about 2.5-fold (44). The above findings suggest that θ, while not essential, could play a role in DNA replication and its fidelity, presumably indirectly through its interaction with the ɛ subunit. The precise nature of this interaction is being pursued structurally by both nuclear magnetic resonance (NMR) and crystallography studies. Structures of both ɛ and θ have been reported (6, 15, 20), as well as the ɛ-θ interaction surface on ɛ (7). Here, we report on a series of genetic experiments on the ɛ-θ interaction. Specifically, we have studied (i) in greater detail, the possible mutator effect resulting from a holE deletion, (ii) the effect of the holE deletion on dnaQ mutator mutants, and (iii) the effect of θ on the α-ɛ interaction as measured by yeast two- and three-hybrid assays. The results suggest that θ may be a stabilizing factor for the ɛ subunit, which has been shown to be intrinsically unstable (11).