Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.

Cellular signalling cascades regulate the activity of transcription factors that convert extracellular information into gene regulation. C/EBPβ is a ras/MAPkinase signal-sensitive transcription factor that regulates genes involved in metabolism, proliferation, differentiation, immunity, senescence, and tumourigenesis. The protein arginine methyltransferase 4 PRMT4/CARM1 interacts with C/EBPβ and dimethylates a conserved arginine residue (R3) in the C/EBPβ N-terminal transactivation domain, as identified by mass spectrometry of cell-derived C/EBPβ. Phosphorylation of the C/EBPβ regulatory domain by ras/MAPkinase signalling abrogates the interaction between C/EBPβ and PRMT4/CARM1. Differential proteomic screening, protein interaction studies, and mutational analysis revealed that methylation of R3 constraines interaction with SWI/SNF and Mediator complexes. Mutation of the R3 methylation site alters endogenous myeloid gene expression and adipogenic differentiation. Thus, phosphorylation of the transcription factor C/EBPβ couples ras signalling to arginine methylation and regulates the interaction of C/EBPβ with epigenetic gene regulatory protein complexes during cell differentiation.

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The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-␤ (C/EBP␤) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBP␤ transactivation domain (TAD). Binding between G9a and C/EBP␤ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBP␤ is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBP␤ TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBP␤. A C/EBP␤ TAD mutant that contained a lysine-toalanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBP␤ target gene. Our data identify C/EBP␤ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBP␤ during gene regulation.