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.
Highlights
The New World group of arenaviruses comprises three phylogenetically distinct clades
In order to map the region of Tacaribe virus (TCRV) N protein required for its interaction with the matrix Z protein, six deletions were introduced throughout the N gene either at the 5Ј end, at the 3Ј end, or at internal positions
Both wildtype and truncated N proteins were expressed as fusion proteins with the Flag tag at their N-terminal end, since we have previously shown that the Flag epitope-tagged version of full-length N is totally functional in a minireplicon-based virus-like particles (VLPs) system [8]
Summary
The New World group of arenaviruses comprises three phylogenetically distinct clades. The small (S) segment encodes two major structural proteins, the glycoprotein precursor (GPC) and the nucleoprotein (N). In both S and L RNAs, the genes are arranged in opposite orientations and are separated by noncoding intergenic sequences with the potential to form stable secondary structures. N tightly binds to genomic and antigenomic RNAs, forming nucleocapsids, which were earlier thought to act as templates for viral genome transcription and replication, both mediated by the virus polymerase [6]. Evidence of an in vivo transcriptional antitermination activity has been reported for JUNV N protein [59]
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