Structural Studies of Polyomavirus and Herpesvirus Attachment Processes

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URI: http://hdl.handle.net/10900/68913
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-689138
http://dx.doi.org/10.15496/publikation-10330
Dokumentart: Dissertation
Date: 2016-03-22
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biochemie
Advisor: Stehle, Thilo (Prof. Dr.)
Day of Oral Examination: 2016-02-16
DDC Classifikation: 500 - Natural sciences and mathematics
Keywords: Strukturbiologie
Other Keywords:
Polyomavirus
Herpesvirus
Structural Biology
X-ray Crystallography
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Abstract:

This dissertation addresses cell attachment processes of viruses and focuses on polyomaviruses and human herpes virus 1, commonly known as herpes simplex virus type 1. Polyomaviruses (PyVs) belong to a continuously expanding family of doublestranded DNA viruses that infect a wide spectrum of fish, mammals, and birds. While they typically remain asymptomatic in healthy hosts, they can cause serious disease in immunocompromised individuals and have been linked to respiratory ailments, neurodegenerative symptoms, nephropathy, and cancer. Most PyVs enter their host cells by binding to sialylated carbohydrate receptors that are attached to lipids, proteins, or both. This attachment process is mediated by the major capsid protein VP1, 360 copies of which form the icosahedral capsid of the virus. VP1 possesses a conserved β-barrel core structure, but the loops connecting the strands are highly divergent and define receptor specificity and virus pathogenicity; even small changes can have drastic effects on the viral life cycle. One example for this is the murine polyomavirus (MuPyV). It has long been known that single amino acid exchanges in the loops forming the receptor binding pocket of MuPyV VP1 drastically alter tumorigenicity and influence spread, but the molecular determinants are unknown. Three MuPyV strains with different pathogenicity, RA, PTA, and LID, have been investigated in this work. In addition to the already established ganglioside receptors GD1a and GT1b, a third receptor, GT1a, was identified, and its structure bound to MuPyV VP1 was solved by X-ray crystallography. Moreover, structures of GD1a as well as another glycan, DSLNT, in complex with the different MuPyV strains were elucidated in order to uncover possible strain-dependent binding modes. Interestingly, none of the introduced amino acid exchanges influence the binding mode for any of the investigated glycans. However, by employing crystallographic affinity measurements, it could be shown that the strains display altered affinities for their interaction partners, which suggests that the regulation of pathogenicity in RA, PTA, and LID is far more intricate than previously assumed. In another project, the effects of amino acid exchanges in PyV VP1 were investigated in simian virus 40 (SV40). Here, three mutations were isolated that show abolished binding to the classical SV40 receptor GM1. Additionally, one mutant also shows altered tropism. It is unknown which ganglioside these mutants utilize, but modeling of the mutated VP1 residues showed that steric clashes result in abolished GM1 binding. Finally, the VP1 structures of the newly discovered human polyomaviruses 6 and 7 (HPyV6 and HPyV7, respectively) were investigated. While they display an architecture similar to other PyV VP1 structures, their receptor binding pockets are altered so drastically that sialic acid can no longer be bound; this finding was confirmed by cell binding experiments and nuclear magnetic resonance spectroscopy. It is yet unknown which receptors are engaged by HPyV6 and HPyV7, and these two viruses have not yet been linked to disease. Due to their architecture, they have been grouped together with KI polyomavirus and WU polyomavirus in the recently established genus wukipolyomavirus. Herpes simplex virus type 1 (HSV-1) is one of the most common human pathogens. Once transmitted, it establishes a lifelong, persistent infection and frequently erupts in orolabial cold sores. In some cases, HSV-1 can migrate to the brain and cause lifethreatening encephalitis. To gain entrance to a host cell, glycoprotein C (gC) of HSV-1 attaches to heparan sulfate (HS) on the cell surface, followed by membrane fusion, which is in turn mediated by glycoproteins B, D, H, and L. The gC-HS interaction has been shown to be non-critical for HSV-1 infection, but its abolishment drastically reduces infectivity. Furthermore, gC has been found to mediate release of viral progeny from a parent cell, and it also plays a defensive role by inhibiting the C3b complement system. Structures for the other HSV-1 glycoproteins are available, but no such information exists for gC. This work introduces an expression construct that can be purified from mammalian cell culture for structure determination by X-ray crystallography. Although no crystals are yet available, the purification strategy lays an important foundation for structural studies of this multifunctional protein.

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