The family of bacterial SidE enzymes catalyzes phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophilia , a pathogenic bacterium causing Legionnaires’ disease 1 , 2 , 3 . SidEs share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes their toxicity in yeast and mammalian cells, through an unknown mechanism 4 – 6 . Deletion of SidJ leads to a significant defect in the growth of Legionella in both its natural host amoeba and in murine macrophages 4 , 5 . Here, we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADPribosyl transferase (mART) domain of SdeA thus blocking its ubiquitin (Ub) ligase activity. SidJ glutamylation activity requires interaction with Calmodulin (CaM), a eukaryotic specific co-factor, and can be regulated by intracellular changes in Ca 2+ concentrations. The cryo-EM structure of SidJ/human apo-CaM complex revealed the architecture of this unique heterodimeric glutamylase. In infected cells, we show that SidJ mediates glutamylation of SidEs on the surface of Legionella- containing vacuoles (LCVs). Using quantitative proteomics, we also uncovered multiple host proteins as putative targets of SidJ-mediated glutamylation. Collectively, this study reveals the mechanism of SidE ligases inhibition by a SidJ/CaM glutamylase and opens new avenues for studying protein glutamylation, an understudied protein modification in higher eukaryotes.

Summary The Integrator is a specialized 3′ end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs). We provide evidence of the modular nature of the Integrator complex by biochemically characterizing its two subcomplexes, INTS5/8 and INTS10/13/14. Using cryoelectron microscopy (cryo-EM), we determined a 3.5-Å-resolution structure of the INTS4/9/11 ternary complex, which constitutes Integrator’s catalytic core. Our structure reveals the spatial organization of the catalytic nuclease INTS11, bound to its catalytically impaired homolog INTS9 via several interdependent interfaces. INTS4, a helical repeat protein, plays a key role in stabilizing nuclease domains and other components. In this assembly, all three proteins form a composite electropositive groove, suggesting a putative RNA binding path within the complex. Comparison with other 3′ end-processing machineries points to distinct features and a unique architecture of the Integrator’s catalytic module.

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry. IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

27Merkel Cell Polyomavirus (MCPyV) is a small, non-enveloped tumor virus associated 28 with an aggressive form of skin cancer, the Merkel cell carcinoma (MCC). MCPyV 29 infections are highly prevalent in the human population with MCPyV virions being 30 continuously shed from human skin. However, the precise host cell tropism(s) of 31MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal 32fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. 33However, MCPyV appears different from other polyomaviruses as it requires sulfated 34 polysaccharides such as heparan sulfates and/or chondroitin sulfates for initial 35 attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co-36 )receptor. To explore the infectious entry process of MCPyV, we analyzed the cell 37 biological determinants of MCPyV entry into A549 cells, a highly transducible lung 38 carcinoma cell line, in comparison to well-studied simian virus 40 and a number of 39 other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-40 mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis or 41 glycosphingolipid-enriched carriers. The viruses internalized in small endocytic pits 42 that led the virus to endosomes and from there to the endoplasmic reticulum (ER). 43Similar to other polyomaviruses, trafficking required microtubular transport, 44 acidification of endosomes, and a functional redox environment. To our surprise, the 45 virus was found to acquire a membrane envelope within endosomes, a phenomenon 46 not reported for other viruses. Only minor amounts of viruses reached the ER, while 47 the majority was retained in endosomal compartments suggesting that endosome-to-48 ER trafficking is a bottleneck during infectious entry. 49 50 51 52 3 Importance 53 MCPyV is the first polyomavirus directly implicated in the development of an 54 aggressive human cancer, the Merkel Cell Carcinoma (MCC). Although MCPyV is 55 constantly shed from healthy skin, MCC incidence increases among aging and 56 immunocompromised individuals. To date, the events connecting initial MCPyV 57 infection and subsequent transformation still remain elusive. MCPyV differs from 58 other known polyomaviruses concerning its cell tropism, entry receptor requirements, 59 and infection kinetics. In this study, we examined the cellular requirements for 60 endocytic entry as well as the subcellular localization of incoming virus particles. A 61 thorough understanding of the determinants of the infectious entry pathway and the 62 specific biological niche will benefit prevention of virus-derived cancers such as 63 MCC. 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 Polyomaviruses (PyV) are small, non-enveloped dsDNA viruses with a diameter of 80 45-50 nm. The icosahedral (T=7) capids consist of 72 homopentameric capsomers of 81 the major capid protein VP1 with minor capsid proteins VP2/VP3 located within a 82 cavity underneath the VP1 pentamers. The PyV capsid harbors a chromatinized, 83 circu...

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Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

The Integrator was originally discovered as a specialized 3’-end processing endonuclease complex required for maturation of RNA polymerase II (RNAPII)-dependent small nuclear RNAs (snRNAs). Since its discovery, Integrator’s spectrum of substrates was significantly expanded to include non-polyadenylated long noncoding RNAs (lncRNA), enhancer RNAs (eRNAs), telomerase RNA (tertRNA), several Herpesvirus transcripts, and messenger RNAs (mRNAs). Recently emerging transcriptome-wide studies reveled an important role of the Integrator in protein-coding genes, where it contributes to gene expression regulation through promoter-proximal transcription attenuation. These new functional data are complemented by several structures of Integrator modules and higher-order complexes, providing mechanistic insights into Integrator-mediated processing events. In this work, we summarize recent progress in our understanding of the structure and function of the Integrator complex.

Heparin is a polymeric carbohydrate with a variety of biomedical applications that is particularly challenging from a synthetic point of view. Here, we present the synthesis of carbohydrate-polymer based hybrid structures by combining defined heparin fragments with monodisperse, sequence-controlled glycooligo(amidoamines) suitable as glycan mimetic model compounds of heparin as demonstrated by STD-NMR binding studies with viral capsids.