Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

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The obligate intracellular bacterium Coxiella burnetii causes the zoonotic disease Q-fever. Coxiella pathogenesis depends on a functional type IV secretion system (T4SS). The T4SS effector AnkG inhibits pathogen-induced host cell apoptosis, which is believed to be important for the establishment of a persistent infection. However, the mode of action of AnkG is not fully understood. We have previously demonstrated that binding of AnkG to p32 is crucial for migration of AnkG into the nucleus and that nuclear localization of AnkG is essential for its anti-apoptotic activity. Here, we compared the activity of AnkG from the C. burnetii strains Nine Mile and Dugway. Although there is only a single amino acid exchange at residue 11, we observed a difference in anti-apoptotic activity and nuclear migration. Mutation of amino acid 11 to glutamic acid, threonine or valine results in AnkG mutants that had lost the anti-apoptotic activity and the ability to migrate into the nucleus. We identified Importin-α1 to bind to AnkG, but not to the mutants and concluded that binding of AnkG to p32 and Importin-α1 is essential for migration into the nucleus. Also during Coxiella infection binding of AnkG to p32 and Importin-α1 is crucial for nuclear localization of AnkG.

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.DOI: http://dx.doi.org/10.7554/eLife.01990.001