title: Identification of two functional domains within the arenavirus nucleoprotein.
abstract: 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 (Z) protein. 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) (J. C. 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 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 to 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 to 119) as a newly recognized molecular determinant of N homotypic interactions.
authors.affiliation: Instituto de Ciencia y Tecnología Dr. Cesar Milstein, Saladillo 2468, Ciudad Autónoma de Buenos Aires C1440FFX, Argentina.
authors.forename: Jesica M
authors.initials: JM
authors.lastname: Levingston Macleod
authors.forename: Alejandra
authors.initials: A
authors.lastname: D'Antuono
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Loureiro
authors.forename: Juan Cruz
authors.initials: JC
authors.lastname: Casabona
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Gomez
authors.forename: Nora
authors.initials: N
authors.lastname: Lopez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018050:Arenavirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; C079186:Z protein, Tacaribe virus