Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

Dysregulation of M segment gene expression contributes to influenza A virus host restriction.

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.

Direct Comparison of B Cell Surface Receptors as Therapeutic Targets for Nanoparticle Delivery of BTK Inhibitors.

Targeting different cell surface receptors with nanoparticle (NP)-based platforms can result in differential particle binding properties that may impact their localization, bioavailability, and, ultimately, the therapeutic efficacy of an encapsulated payload. Conventional in vitro assays comparing the efficacy of targeted NPs often do not adequately control for these differences in particle-receptor binding, potentially confounding their therapeutic readouts and possibly even limiting their experimental value. In this work, we characterize the conditions under which NPs loaded with Bruton's Tyrosine Kinase (BTK) inhibitor differentially suppress primary B cell activation when targeting either CD19 (internalizing) or B220 (noninternalizing) surface receptors. Surface binding of fluorescently labeled CD19- and B220-targeted NPs was analyzed and quantitatively correlated with the number of bound particles at given treatment concentrations. Using this binding data, suppression of B cell activation was directly compared for differentially targeted (CD19 vs B220) NPs loaded with a BTK inhibitor at a range of particle drug loading concentrations. When NPs were loaded with lower amounts of drug, CD19-mediated internalization demonstrated increased inhibition of B cell proliferation compared with B220 NPs. However, these differences were mitigated when particles were loaded with higher concentrations of BTK inhibitor and B220-mediated "paracrine-like" delivery demonstrated superior suppression of cellular activation when cells were bound to lower overall numbers of NPs. Taken together, these results demonstrate that inhibition of B cell activation can be optimized for NPs targeting either internalizing or noninternalizing surface receptors and that particle internalization is likely not a requisite endpoint when designing particles for delivery of BTK inhibitor to B cells.

Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing intercellular PC gap formation. For the first time, an EC-specific MIF knockout mouse was used to investigate the effects of selective deletion of EC MIF. In a model of acute lung injury, selective deletion of EC MIF decreases neutrophil infiltration to the bronchoalveolar lavage and tissue and simultaneously decreases PC relaxation by increasing myosin light-chain phosphorylation. We conclude that paracrine signals from EC via MIF decrease PC contraction and enhance PC-regulated neutrophil transmigration.-Pellowe, A. S., Sauler, M., Hou, Y., Merola, J., Liu, R., Calderon, B., Lauridsen, H. M., Harris, M. R., Leng, L., Zhang, Y., Tilstam, P. V., Pober, J. S., Bucala, R., Lee, P. J., Gonzalez, A. L. Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

A human cellular noncoding RNA activates the antiviral protein 2'-5'-oligoadenylate synthetase 1.

The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes sense cytosolic dsRNA, a potent signal of viral infection. In response to dsRNA binding, OAS proteins synthesize the second messenger 2'-5'-linked oligoadenylate that activates the latent ribonuclease L (RNase L). RNase L-mediated degradation of viral and cellular RNAs effectively halts viral replication and further stimulates innate immune responses by inducing type I interferon. The OAS/RNase L pathway is therefore central in innate immune recognition and promotion of antiviral host responses. However, the potential for specific RNA sequences or structures to drive OAS1 activation and the molecular mechanisms by which they act are not currently fully understood. Moreover, the cellular regulators of OAS activity are not well defined. Here, we demonstrate that the human cellular noncoding RNA 886 (nc886) activates OAS1 both in vitro and in human A549 cells. We show that a unique structure present only in one of the two structural conformers adopted by nc886 drives potent OAS1 activation. In contrast, the conformer lacking this unique structure activated OAS1 only very weakly. We also found that formation of this OAS1-activating structural motif depends on the nucleotides in the apical-most loop of nc886 and the adjacent helix. These findings identify a cellular RNA capable of activating the OAS/RNase L pathway in human cells and illustrate the importance of structural elements, and their context, in potentiating OAS1 activity.

Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) senses dsRNA produced during viral infection and halts cellular protein synthesis to block viral replication. How basal PKR activity is controlled in the absence of infection was unclear until the recent identification of a potential endogenous regulator, the cellular noncoding RNA 886 (nc886). However, nc886 adopts two distinct conformations for which the structural details and potential functional differences remain unclear. Here, we isolated and separately dissected the function of each form of nc886 to more clearly define the molecular mechanism of nc886-mediated PKR inhibition. We show that nc886 adopts two stable, noninterconverting RNA conformers that are functionally nonequivalent using complementary RNA structure probing and mutational analyses combined with PKR binding and activity assays. One conformer acts as a potent inhibitor, while the other is a pseudoinhibitor capable of weakly activating the kinase. We mapped the nc886 region necessary for high affinity binding and potent inhibition of PKR to an apical stem-loop structure present in only one conformer of the RNA. This structural feature is not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, the eukaryotic translation initiation factor 2α subunit. The identification of different activities of the nc886 conformers suggests a potential mechanism for producing a gradient of PKR regulation within the cell and reveals a way by which a cellular noncoding RNA can mask or present a structural feature to PKR for inhibition.

A novel RNA molecular signature for activation of 2'-5' oligoadenylate synthetase-1.

Human 2'-5' oligoadenylate synthetase-1 (OAS1) is central in innate immune system detection of cytoplasmic double-stranded RNA (dsRNA) and promotion of host antiviral responses. However, the molecular signatures that promote OAS1 activation are currently poorly defined. We show that the 3'-end polyuridine sequence of viral and cellular RNA polymerase III non-coding transcripts is critical for their optimal activation of OAS1. Potentiation of OAS1 activity was also observed with a model dsRNA duplex containing an OAS1 activation consensus sequence. We determined that the effect is attributable to a single appended 3'-end residue, is dependent upon its single-stranded nature with strong preference for pyrimidine residues and is mediated by a highly conserved OAS1 residue adjacent to the dsRNA binding surface. These findings represent discovery of a novel signature for OAS1 activation, the 3'-single-stranded pyrimidine (3'-ssPy) motif, with potential functional implications for OAS1 activity in its antiviral and other anti-proliferative roles.

Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.

Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency.

Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon-induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2'-5'-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell-derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L-mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.