The death of a cell is an inevitable part of its biology. During homeostasis, most cells die through apoptosis. If homeostasis is disturbed, cell death can switch to proinflammatory forms of death, such as necroptosis, pyroptosis, or NETosis. We demonstrate that the formation of neutrophil extracellular traps (NETs), a special form of neutrophil cell death that releases chromatin structures to the extracellular space, is dependent on gasdermin D (GSDMD). GSDMD is a pore-forming protein and an executor of pyroptosis. We screened a chemical library and found a small molecule based on the pyrazolo-oxazepine scaffold that efficiently blocks NET formation and GSDMD-mediated pyroptotic cell death in human cells. During NETosis, GSDMD is proteolytically activated by neutrophil proteases and, in turn, affects protease activation and nuclear expansion in a feed-forward loop. In addition to the central role of GSDMD in pyroptosis, we propose that GSDMD also plays an essential function in NETosis.

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Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich Cterminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.Hepatitis B virus (HBV), a small DNA virus belonging to the family Hepadnaviridae, causes acute and chronic hepatitis in humans. Worldwide, an estimated 350 million persons are persistently infected with HBV (4). A significant subset of these HBV carriers progresses to severe liver disease, such as hepatocellular carcinoma, which is assumed to cause up to one million deaths per year (30). Current treatment of chronic HBV infections by the approved therapeutics alpha interferon and lamivudine has its limitations, and there is a clear medical need for new therapeutic strategies (33).The mature HBV virion consists of an enveloped, spherical nucleocapsid which contains the viral DNA genome and is assembled from dimers of a single capsid protein, the 21-kDa HBV core protein (43). During the assembly process, viral polymerase mediates the specific encapsidation of pregenomic RNA and subsequently converts the pregenomic RNA to viral genomic DNA (3, 19; for reviews, see references 13 and 33).Numerous studies have shown that the HBV core protein is phosphorylated in intact cells (27,32,41). The serine residues of three repeated SPRRR motifs in its arginine-rich C-terminal region were identified as phosphoacceptor sites in vivo (S155, S162, and S170 in strain ayw) (25). Core protein becomes phosphorylated prior to nucleocapsid assembly, and mutational analysis strongly suggests that phosphorylation of serines 162 and 170 is critical for subsequent pregenomic RNA packaging to occur (14,24). As none of the viral proteins possesses intrinsic protein kinase activit...

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