Die Rolle des Signalpeptids für die Reifung des Lassavirus Glykoproteins

The surface glycoprotein of Lassa virus is a type-1 membrane protein. It is synthesized as precursor preGP-C and posttranslationally modified with N-glycans. After proteolytic cleavage of the signal peptide GP-C is processed into its subunits GP1 and GP2 by the host cell protease SKI-1/S1P. After computer analysis the signal protein of the glycoprotein exhibits an unusual lenght. The topic of this work was the characterization of this signal peptide. 1. First, the exact length of the signal peptide of 58 amino acid residues was determined experimentally by N-terminal sequencing of the GP1-subunit and mutational analysis of the potential signal peptide cleavage site. 2. Topological studies have shown that the signal peptide comprises an unusual structure with two distinct hydrophobic domains. As a result, several models of the topology of the signal peptide in the ER-membrane are possible. It was shown in this work that only the N-terminal of both hydrophobic domains spans the ER-membrane leaving an extraordinary long tail, including the second hydrophobic region, ER-luminal. 3. The signal peptide was studied concerning additonal functions apart from its translocation function. To do so, the native signal peptide was replaced by other signal peptides. This replacement abolished the maturation cleavage of the glycoprotein GP-C into its subunits GP1 and GP2. Cleavage was restored upon coexpression of the solitary native signal peptide. Using mutational analysis the region of the signal peptide which is essential for proteolytic activation of the glycoprotein was mapped to the ER-luminal part of the signal peptide. Furthermore, especially the region between both hydrophobic domains is essential for maturation of the glycoprotein. Using coprecipitation experiments a direct interaction between the glycoprotein and its signal peptide was shown. This interaction is mediated by the GP2 subunit of the glycoprotein. 4. It was shown in this work that the signal peptide is a structural component of the Lassa virion. Furthermore, the incorporation of the signal peptide depends on incorporation of the glycoprotein. The signal peptide itself is not transport competent. 5. The signal peptides of the arenaviral glycoproteins are homologous, the interchangeability of the signal peptides between arenaviral glycoproteins was confirmed in this work. The presented data therefore can be transferred to the signal peptides of other arenaviral glycoproteins.