Einfluss gezielter Mutationen auf die biologische Aktivität des Oberflächen-Glykoproteins eines afrikanischen Henipavirus

Hendra virus and Nipah virus are the two highly pathogenic representatives of the genus henipavirus. Fruit bats of the genus Pteropus serve as their natural reservoir. While bats do not show any clinical symptoms upon infection, henipaviruses can cause severe illness after natural spillover to humans, pigs or horses. Due to their zoonotic potential and high pathogenicity, henipaviruses are classified as BSL-4 pathogens. Initially, henipaviruses were believed to be restricted to Southeast Asia and Australia. But in recent years, there is increasing evidence of a much broaden geographic distribution because henipaviral RNA has been identified also in Africa. One of these African bat henipaviruses, Kumasi virus (KV), isolated from an Eidolon helvum fruit bat in Ghana, could be fully sequenced. Since isolation of infectious viruses was not successful so far, evaluation of the zoonotic potential of new viruses is based on functional analyses of individual viral surface proteins in comparison to their pathogenic homologues from Nipah or Hendra viruses. The receptor-binding G protein and the fusion protein F are central determinants for virus entry and cell-to-cell spread. Efficient binding to cellular receptors and fusion-helper function of henipavirus G proteins are essentially required to support F-mediated viruscell and cell-cell-fusion processes. It has been shown earlier that surface expression and fusion-helper activity of KV-G is clearly reduced compared to the G protein of pathogenic NiV (NiV-G). To gain insight into the molecular determinants affecting protein transport and function, several KV-G mutants resulting in changes in N glycosylation, oligomerization and endocytosis pattern were generated. Western Blot analysis, metabolic labeling and functional fusion assays revealed that, similar to NiV-G, KV-G contains six N glycans which are all required for surface transport and functionality. However, KV-G differs in its oligomerization by almost exclusively forming stable tetramers, while NiV-G is expressed in a well-balanced dimer-tetramer ratio. Although cysteine mutations in the stalk domain of KV-G altered the oligomerization pattern, neither surface transport nor fusion-helper activity was improved. However, when mutating a non-conserved cysteine in the globular head domain of KV-G some gain of function was observed. This was further increased when we additionally disrupted an endocytosis motive in the cytoplasmic domain. This work shows for the first time an African henipavirus glycoprotein with enhanced functionality. Although the bioactivity is still far below that of highly pathogenic henipavirus G proteins, this indicates that African henipaviruses with improved fusion-helper function and perhaps an increased zoonotic potential might evolve by just a few adaptive mutations.