Highlights d Bat mumps virus can readily replicate in human cells d Host cell entry factors of bat mumps virus are identical to those of human viruses d Bat mumps virus can evade innate immune responses in human and in bat cells d Neurotoxic properties of bat mumps virus are comparable to human field isolates

The mumps virus (MuV) fusion protein (F) plays a crucial role for the entry process and spread of infection by mediating fusion between viral and cellular membranes as well as between infected and neighboring cells, respectively. The fusogenicity of MuV differs depending on the strains and might correlate with the virulence; however, it is unclear which mechanisms contribute to the differentiated fusogenicity. The cleavage motif of MuV F is highly conserved among all strains except the amino acid residue at position 8 (P8) that shows a certain variability with a total of four amino acid variants (leucine (L), proline (P), serine (S), threonine (T)). We demonstrate that P8 affects the proteolytic processing and the fusogenicity of MuV F. The presence of L or S at P8 position resulted in a slower proteolysis of MuV F by furin and a reduced ability to mediate cell-cell fusion. However, virus-cell fusion was more efficient for F proteins harboring L or S at P8, suggesting that P8 contributes to the mechanism of viral spread: P and T enable a rapid spread of infection by cell-to-cell fusion, whereas viruses harboring L or S at P8 spread preferentially by the release of infectious viral particles. Our study provides novel insights into the fusogenicity of MuV and its influence on the mechanisms of virus spread within infected tissues. Assuming a correlation between MuV fusogenicity and virulence, sequence information on the amino acid residue at P8 might be helpful to estimate the virulence of circulating and emerging strains. IMPORTANCE Mumps virus (MuV) is the causative agent of the highly infectious disease mumps. Mumps is mainly associated with mild symptoms, but severe complications such as encephalitis, meningitis or orchitis can also occur. There is evidence that the virulence of different MuV strains and variants might correlate with the ability of the fusion protein (F) to mediate cell-to-cell fusion. However, the relation between virulence and fusogenicity or the mechanisms responsible for the varying fusogenicity of different MuV strains are incompletely understood. Here we focused on the amino acid residue 8 (P8) of the proteolytic cleavage site of MuV F, because this amino acid residue shows a striking variability depending on the genotypes of MuV. The P8 residue has a significant effect on the proteolytic processing and fusogenicity of MuV F and might thereby determine the route of viral spread within infected tissues.