Identification of Two Functional Domains within the Arenavirus Nucleoprotein.

LEVINGSTON JM; D´ANTUONO A; LOUREIRO ME; CASABONA JC; GÓMEZ G; LÓPEZ N

JOURNAL OF VIROLOGY

AMER SOC MICROBIOLOGY

Lugar: Washington DC. ; Año: 2010 vol. 85 p. 2012 - 2023

0022-538X

Tacaribe virus (TCRV) belongs to the Arenaviridae family. Its bisegmented negative-stranded RNA genome encodes the nucleoprotein (N), the precursor of the envelope glycoproteins, the polymerase (L) and a RING finger matrix protein (Z). The 570-amino-acid N protein binds to viral RNA, forming nucleocapsids, which are the template for transcription and replication by the viral polymerase. We have previously shown that the interaction between N and Z is required for assembly of infectious virus-like particles (VLPs) (Casabona et al., J Virol. 83 ;7029-7039, 2009). Here, we examine the functional organization of TCRV N protein. A series of deletions and point mutations were introduced into the N coding sequence and the ability of the mutants to sustain heterotypic (N-Z) or homotypic (N-N) interactions was analyzed. We found that the N protein displays two functional domains. By using coimmunoprecipitation studies, VLP-incorporation assays, and double immunofluorescence staining, the carboxy-terminal region of N was found to be required for N-Z interaction and also necessary for incorporation of N protein into VLPs. Moreover, further analysis of this region showed that the integrity of a putative zinc-finger motif, as well as its amino-flanking sequence (residues 461-489), are critical for Z binding and N incorporation into VLPs. In addition, we provide evidence of an essential role of the amino-terminal region of N protein for N-N interaction. In this regard, using reciprocal coimmunoprecipitation analysis, we identified a 28-residue region predicted to form a coiled-coil domain (residues 92-119) as a newly recognized molecular determinant of N-homotypic interactions.