title: Molecular determinants of arenavirus Z protein homo-oligomerization and L polymerase binding.
abstract: The arenavirus Z is a zinc-binding RING protein that has been implicated in multiple functions during the viral life cycle. These roles of Z involve interactions with viral and cellular proteins that remain incompletely understood. In this regard, Z inhibits viral RNA transcription and replication through direct interaction with the viral L polymerase. Here, we defined the L-binding domain of Tacaribe virus (TCRV) Z protein and the structural requirements mediating Z homo-oligomerization. By using site-directed mutagenesis, coimmunoprecipitation, and functional assays, we showed that residues R37, N39, W44, L50, and Y57, located around the zinc coordination site I, play a critical role in the Z-L interaction. We also found that Z protein from either TCRV or the pathogenic Junin virus (JUNV) self-associates into oligomeric forms in mammalian cells. Importantly, mutation of the myristoylation site, the strictly conserved residue G at position 2, severely impaired the ability of both TCRV Z and JUNV Z to self-interact as well as their capacity to accumulate at the plasma membrane, strongly suggesting that Z homo-oligomerization is associated with its myristoylation and cell membrane targeting. In contrast, disruption of the RING structure or substitution of W44 or N39, which are critical for L protein recognition, did not affect Z self-binding. Overall, the data presented here indicate that homo-oligomerization is not a requirement for Z-L interaction or Z-mediated polymerase activity inhibition.
authors.affiliation: CEVAN, Instituto de Ciencia y Tecnología Dr. Cesar Milstein, CONICET, Saladillo 2468, Buenos Aires C1440FFX, Argentina.
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Loureiro
authors.forename: Maximiliano
authors.initials: M
authors.lastname: Wilda
authors.forename: Jesica M
authors.initials: JM
authors.lastname: Levingston Macleod
authors.forename: Alejandra
authors.initials: A
authors.lastname: D'Antuono
authors.forename: Sabrina
authors.initials: S
authors.lastname: Foscaldi
authors.forename: Cristina
authors.initials: C
authors.lastname: Marino Buslje
authors.forename: Nora
authors.initials: N
authors.lastname: Lopez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018050:Arenavirus / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D012367:RNA, Viral / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D012367:RNA, Viral; D014764:Viral Proteins; C079186:Z protein, Tacaribe virus; D012321:DNA-Directed RNA Polymerases; D015032:Zinc