SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron.

The coronavirus nucleocapsid (N) protein is known to bind to nucleic acids and facilitate viral genome encapsulation. Here we report that the N protein can mediate RNA or DNA entering neighboring cells through ACE2-independent, receptor (STEAP2)-mediated endocytosis, and achieve gene expression. The effect is more pronounced for the N protein of wild-type SARS-CoV-2 than that of the Omicron variant and other human coronaviruses. This effect is enhanced by RANTES (CCL5), a chemokine induced by N protein, and lactate, a metabolite produced in hypoxia, to cause more damage. These findings might explain the clinical observations in SARS-CoV-2-infected cases. Moreover, the N protein-mediated function can be inhibited by N protein-specific monoclonal antibodies or p38 mitogen-activated protein kinase inhibitors. Since the N-protein-mediated nucleic acid endocytosis involves a receptor commonly expressed in many types of cells, our findings suggest that N protein may have an additional role in SARS-CoV-2 pathogenesis.

Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Application of B-cell receptor (BCR) pathway inhibitor ibrutinib for chronic lymphocytic leukemia (CLL) is a major breakthrough, yet the downstream effects following inhibition of BCR signaling and during relapse await further clarification. By comparative phosphoproteomic profiling of B cells from patients with CLL and healthy donors, as well as CLL B cells collected at multiple time points during the course of ibrutinib treatment, we provided the landscape of dysregulated phosphoproteome in CLL and its dynamic alterations associated with ibrutinib treatment. Particularly, differential phosphorylation events associated with several signaling pathways, including BCR pathway, were enriched in patient CLL cells. A constitutively elevated phosphorylation level of KAP1 at serine 473 (S473) was found in the majority of CLL samples prior to treatment. Further verification showed that BCR activation promoted KAP1 S473 phosphorylation, whereas ibrutinib treatment abolished it. Depletion of KAP1 in primary CLL cells decelerated cell-cycle progression and ectopic expression of a KAP1 S473 phospho-mimicking mutant accelerated G2-M cell-cycle transition of CLL cells. Moreover, temporal phosphoproteomic profiles using a series of CLL cells isolated from one patient during the ibrutinib treatment revealed the dynamic changes of several molecules associated with BCR signaling in the ibrutinib responsive and recurrent stages. IMPLICATIONS: This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

O-GlcNAcylation is required for B cell homeostasis and antibody responses.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) catalyzes O-GlcNAc modification. O-GlcNAcylation is increased after cross-linking of the B-cell receptor (BCR), but the physiological function of this reaction is unknown. Here we show that lack of Ogt in B-cell development not only causes severe defects in the activation of BCR signaling, but also perturbs B-cell homeostasis by enhancing apoptosis of mature B cells, partly as a result of impaired response to B-cell activating factor. O-GlcNAcylation of Lyn at serine 19 is crucial for efficient Lyn activation and Syk interaction in BCR-mediated B-cell activation and expansion. Ogt deficiency in germinal center (GC) B cells also results in enhanced apoptosis of GC B cells and memory B cells in an immune response, consequently causing a reduction of antibody levels. Together, these results demonstrate that B cells rely on O-GlcNAcylation to maintain homeostasis, transduce BCR-mediated activation signals and activate humoral immunity.

Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells.

Crosslinking of B-cell receptor (BCR) sets off an apoptosis programme, but the underlying pathways remain obscure. Here we decipher the molecular mechanisms bridging B-cell activation and apoptosis mediated by post-translational modification (PTM). We find that O-GlcNAcase inhibition enhances B-cell activation and apoptosis induced by BCR crosslinking. This proteome-scale analysis of the functional interplay between protein O-GlcNAcylation and phosphorylation in stimulated mouse primary B cells identifies 313 O-GlcNAcylation-dependent phosphosites on 224 phosphoproteins. Among these phosphoproteins, temporal regulation of the O-GlcNAcylation and phosphorylation of lymphocyte-specific protein-1 (Lsp1) is a key switch that triggers apoptosis in activated B cells. O-GlcNAcylation at S209 of Lsp1 is a prerequisite for the recruitment of its kinase, PKC-ß1, to induce S243 phosphorylation, leading to ERK activation and downregulation of BCL-2 and BCL-xL. Thus, we demonstrate the critical PTM interplay of Lsp1 that transmits signals for initiating apoptosis after BCR ligation.