M. HERREN^1,2, F. BRINGOLF^1,2, M. WYSS¹, P. PLATTET<sup>1

1</sup>Division of Neurological Sciences, Department of Clinical Research and Veterinary Public Health (DCRVPH),Vetsuisse Faculty, University of Bern, Bern, Switzerland, ²Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

The fusion machinery of Morbillivirus (i.e. canine distemper [CDV], measles [MeV]), consisting of tightly interacting attachment (H) and fusion (F) protein, is crucial for host cell invasion. The H-ectodomain is composed of an F-activating stalk region and a cuboidal receptor-binding head domain, which are separated by a short “connector” segment. The current model of F-activation assumes that the H-heads move away from the stalk after receptor engagement; a motion enabled by putative structural flexibility of the connectors. Subsequently, H-stalk rearranges and activates F, which in turn undergoes drastic conformational changes ultimately leading to membrane merging. We aim to unravel the role of the connector microdomain of the Morbillivirus attachment protein H (residues 142-170) in fusion activation. The role of specific amino acids in the H-connector domain is examined by site-directed mutagenesis. The mutants’ surface expression, fusion promoting activity, interaction with F and receptor binding capability are investigated in receptor negative and positive cells using established methods. We found that the isoleucine residue at position 146 can be mutated into various amino acids while keeping wildtype-like surface expression. Interestingly, the fusion promotion of those H-I146 mutants was modulated when substituted into hydrophobic amino acids, while their bioactivity was severely impaired when exchanged into non-conserved polar/charged amino acids. Our preliminary data suggests that, while irrelevant for transport-competence, hydrophobicity of residue I146 of the H-connector is crucial for F-triggering. Whether the H-I146 mutants impacted fusion-promotion by locking H in a “pre-receptor-binding” conformation or induced premature conformational change is under current investigations.