Mutational analysis of positively charged amino acid residues of Uukuniemi phlebovirus nucleocapsid protein

The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA.