F. BRINGOLF^1,2, M. HERREN^1,2, M. WYSS¹, A. ZURBRIGGEN¹, 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

Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Recent structural data from different paramyxovirus M-proteins revealed a monomeric unit composed of N- and C-terminal modules (NTD and CTD, respectively) that are connected via a flexible linker and which can self-assemble into inverted “head-to-tail” dimers. Because growing evidences support the notion that M-dimerization and/or higher-order oligomerisation play a critical role in virus particle formation, we investigated the role of the asparagine 138 (N138) of CDV M, which putatively locates at the core of the dimeric interface. While intracellular expression of M-mutants carrying N138 substitutions into alanine (N138A) or arginine (N138R) remained unaltered, the overall steady state levels of M-N138 mutated into phenylalanine (N138F), methionine (N138M) or glutamate (N138E) amino acids were impaired. Surprisingly, while cross-linking analyses indicated that all M-mutants displayed proper dimeric-like assembly, M-M co-immunoprecipitation experiments conversely revealed that, except for M-N138A, all mutants displayed a modulated affinity of interaction with another M-unit. Remarkably, all M-mutants remained well transported to the cell periphery and could properly relocate N proteins from the cytosol to the cell periphery, thereby indicating some preservation of the overall M-monomeric/dimeric structure. Furthermore, the expression-deficient M-mutants N138F, N138M and N138E were additionally impaired in triggering the formation of cell protrusions and virus-like particles (VLP). Taken together, while our data suggest that residue N138 of CDV M contributes to the regulation of dimer stabilization, expression and VLP formation, M may self-oligomerize and interact with N through additional discrete microdomains.