Mutations- und Funktionsanalyse des Lassa-Virus Fusionsproteins GP-2

An important step of Lassa virus entry is the fusion of viral and cellular membranes after endosomal uptake. In the present work it was examined if the Lassa virus envelope protein GP-2 is the functional fusion protein of the virus, and additionally it was tested which amino acids within the N-terminus of the protein are crucially involved in the fusion process. To examine these questions a quantitative GP-2-expression-based recombinant cell-cell fusion assay (RCCFA) was developed, which identified GP-2 as the fusion protein of the virus. Additionally it was shown that the cleavage of the GP-2-precursor protein GP-C as well as activation by unusually low pH are prerequisites for fusogenicity. Experiments with other members of the same viral family Arenaviridae revealed differences between the fusion proteins of old world arenaviruses (Lassa virus, Lymphocytic choriomeningitis virus) and new world arenaviruses (Junín virus) in regard to the pH-optimum of the fusion process. In order to map the region responsible for fusion in the N-terminus of GP-2 twenty-two individual amino acids were exchanged to alanine and the recombinant constructs were tested for cleavage, cell surface expression and activity in the RCCFA. It was found that – in contrast to fusion proteins of other viruses – the entire N-terminus of Lassa virus GP-2 is involved in the fusion process. To additionally test the infectivity of the constructs a retroviral pseudotype assay was developed in which particles are generated consisting of a retroviral capsid and a Lassa virus envelope. Examination of the GP-2 mutants revealed that fusogenicity strongly correlated with infectivity. Based on the observed results a membrane interaction model of Lassa virus GP-2 with the endosomal target membrane was developed. This model showed similarities with the presumed fusion process of Ebola virus. Attempts to inhibit fusion with short synthetic peptides failed, whereas several sera of convalescent Lassa fever patients showed a strong inhibitory effect.