A N-terminal coiled coil motif is essential for Tacaribe virus Nucleoprotein functionality

D'ANTUONO, A; FOSACALDI, S; LOUREIRO, E; LOPEZ, N

Buenos Aires

Workshop; 3rd. ICGEB WORKSHOP ON HUMAN RNA VIRUSES; 2012

Instituo Leloir

Introduction. Arenaviruses, such as Tacaribe virus (TCRV) are enveloped viruses with a negative-sense RNA genome, which encodes four proteins: the viral RNA polymerase (L protein), a matrix protein (Z), the precursor (GPC) of the envelope glycoproteins and the nucleocapsid protein (N). The N protein tightly binds to genomic and antigenomic RNAs forming nucleocapsids, which act as templates for both transcription and replication of viral genomes mediated by the virus polymerase. Besides, the ability of N to take part in multiple protein-protein interactions may be important in several steps of the viral life cycle. We have shown that the N-terminal region of N contains a coiled coil motif domain which is essential for N self-interactions. Also we have defined key hydrophobic amino acids within this motif as being essential for N-N interaction and its functional relevance. Results. Recently published crystallographic data for full-lenght nucleoprotein of Lassa virus was used as template to establish the spatial disposition of TCRV N protein residues. In this model two tight dimerization interfaces were predicted.  One of them comprising amino acids 195 to 259 and the other one residues 281 to 287. Key amino acids within the putative interfaces were replaced by the non-polar amino acid Alanine. The ability of point mutants to self-interact was evaluated by coimmunoprecipitation and their capacity to support viral RNA synthesis was assessed using a TCRV minireplicon system. Conclusiones. We predict N-N dimerization interfaces based on TCRV N protein model and Lass N crystal structure superimposition.  Functional analysis of residues within these regions revealed their relevance in N-N interaction as well as for its role in viral genome replication.