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Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins’ target location and provide translocation fidelity. In this review, we follow the signal peptide into the ER and discuss the recent insights that structural biology has provided on the governing principles of those interactions.

Polyoma- and adenoviruses are important human pathogens with worldwide significance. Both are non-enveloped DNA viruses, but differ significantly in terms of how they infect humans: Polyomaviruses (PyVs) cause persistent and occasionally fatal diseases involving multiple organs, while adenoviruses (HAdVs) are ubiquitous among societies worldwide and generally cause self-limiting diseases of mucous epithelial tissues that range from common cold symptoms to gastroenteritis and eye infections. The main focus of this dissertation is placed on the fundamental processes underlying the earliest steps of the viral life cycles of both viruses: cell attachment and entry. Most of the work is dedicated to the unraveling of the attachment and entry strategies of specific strains or serotypes, and a comparison of the factors that govern the differences between them. The cell entry of polyomaviruses is mediated by the interaction of the major capsid protein VP1 with ubiquitous surface glycolipids called gangliosides. I have investigated three well-known strains of the murine polyomavirus (MuPyV) that show remarkable differences in terms of pathogenicity and tissue tropism, but only differ by singular amino acid exchanges located within their ganglioside binding cavities. This work establishes the ganglioside GT1a as a novel functional receptor and discovers minimal changes in the receptor binding affinities among the three strains that presumably lead to the drastically altered in vivo behavior. Unlike PyVs, HAdVs generally employ a two-step mechanism mediated by distinct sets of capsid proteins to enter their target cells. The interaction of the C-terminal knob domain of the so-called fiber with a cellular primary attachment factor selects and tethers the viral particles to the host cell and facilitates cell entry mediated by the interaction of the viral penton base with a secondary entry receptor. These processes are assumed to contribute to the manifestation of viral tropism and host range. Here, the discovery and functional and structural characterization of novel primary attachment factors for two unrelated HAdV types is reported. The first type, HAdV-G52, is a rare and unique serotype that possesses two distinct fibers, which use completely different receptors: the long fiber recognizes the tight junction protein coxsackie- and adenovirus receptor (CAR), while the short fiber shows a specific preference for the glycan polysialic acid using a novel binding site on its fiber knob. The second type, HAdV-D36, is associated with obesity and has the unique ability to infect animals. This work demonstrates that HAdV D36 uses a yet unidentified protein for attachment, and at the same time possesses a specificity for CAR and a sialic acid variant that is presumably only found in animals. Both projects have implications for the infectious routes of the two viruses. An additional, third project presents the purification of the penton base protein of HAdV-D09 with the aim of structurally characterizing the interactions with its receptor counterpart, the integrin αvβ3, and unraveling the factors that dominate later phases of cell entry. The fourth part of this study addresses ways to interfere with adenoviral infections and to use adenoviruses as vectors for highly specific therapeutic applications. To this end, the development and evaluation of a series of second-generation inhibitors for the ocular pathogen HAdV-D37 is reported, whose design was inspired by its natural receptor, the sialic acid-containing glycan GD1a. Furthermore, this work sets the stage for the development of an HAdV-G52-based viral vector for the oncolytic treatment of somatic cancers expressing the tumor antigen polysialic acid. The last part of this dissertation describes ongoing work for the structural characterization of the adenoviral early gene product E4ORF1, a viral powerhouse protein that deregulates cell metabolism and polarity through various host interactions. The findings presented in this dissertation have implications for our general understanding of how the differences among virus entry strategies emerge on a structural level, and provide valuable information for the development of efficient antiviral strategies and safer virus-based drugs.