The rockefeller university

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades(1-3). Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized(4). Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs. Here, the authors identify lymphocyte antigen 6E (LY6E) as a coronavirus (CoV) restriction factor that prevents infection of B cells and dendritic cells. LY6E inhibits both human and mouse CoV entry into cells by interfering with viral spike protein-mediated membrane fusion. It facilitates an antiviral immune response that prevents liver disease and reduces death in the mouse model of MHV-A59 CoV infection.

Allogeneic hematopoietic cell transplantation (alloHCT) for multiple myeloma (MM), with its underlying graftversus-tumor capacity, is a potentially curative approach for high-risk patients. Relapse is the main cause of treatment failure, but predictors for postrelapse survival are not well characterized. We conducted a retrospective analysis to evaluate predictors for postrelapse overall survival (OS) in 60 MM patients who progressed after myeloablative T cell-depleted alloHCT. The median patient age was 56 years, and 82% had high-risk cytogenetics. Patients received a median of 4 lines of therapy pre-HCT, and 88% achieved at least a partial response (PR) before a11oHCT. Of the 38% who received preemptive post-HCT therapy, 13 received donor lymphocyte infusions (DLIs) and 10 received other interventions. Relapse was defined as very early ( <6 months; 28%), early (6 to 24 months; 50%), or late (>24 months; 22%). At relapse, 27% presented with extramedullary disease (EMD). The median postrelapse overall survival (OS) by time to relapse was 4 months for the very early relapse group, 17 months for the early relapse group, and 72 months for the late relapse group (P = .002). Older age, relapse with EMD, a11oHCT, On multivariate analysis adjusted for age and sex, very early relapse (hazard ratio [HR], 4.37; 95% confidence interval [CI], 1.42 to 13.5), relapse with EMD (HR, 5.20; 95% CI, 2.10 to 12.9), and DLI for relapse prevention (HR, .11; 95% CI, 2.10 to 12.9) were significant predictors for postrelapse survival. Despite their shared inherent high-risk status, patients with MM have significantly disparate post-HCT relapse courses, with some demonstrating long-term survival despite relapse. (C) 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Mechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in cytoskeletal proteins and transcriptional regulators. Here, we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding as a mechanosensing mechanism by which cytoskeletal tension can govern nuclear localization.

Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G(2)/M cell cycle arrest. Vif initiates G(2)/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-beta/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis. IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G(2)/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G(2)/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

Protein S-palmitoylation, or S-fatty-acylation, regulates many fundamental cellular processes in eukaryotes. Herein, we present a chemical fatty-acylation approach that involves site-specific incorporation of cycloalkyne-containing unnatural amino acids and subsequent bioorthogonal reactions with fatty-acyl tetrazines to install fatty-acylation mimics at target protein sites, allowing gain-of-function analysis of S-palmitoylation in live cells.

We each developed from a single cell—a fertilized egg—that divided and divided and eventually gave rise to the trillions of cells, of hundreds of types, that constitute the tissues and organs of our adult bodies. Advancing our understanding of the molecular programs underlying the emergence and differentiation of these diverse cell types is of fundamental interest and will affect almost every aspect of biology and medicine.

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling similar to 4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.