The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly

Abstract

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsidlike particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsidlike particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N-and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation. Nucleocapsids of Sendai virus, a model paramyxovirus, are large, helical, highly flexible ribonucleoprotein particles, approximately 15 nm in diameter and 1 ,um in length. They contain the 15,384-nucleotide-long negative-stranded RNA genome and three different viral proteins. The major structural component of the nucleocapsid is the nucleocapsid protein NP (58 kDa), of which about 2,600 molecules tightly encapsidate the RNA. Encapsidation renders the RNA inaccessible to RNases and is required for it to serve as a template for the viral RNA-dependent RNA polymerase complex. The two proteins necessary for polymerase functions are the large protein L (251 kDa), which is considered the core of the polymerase complex, and the phosphoprotein P (65 kDa). Both of these proteins are present in only minor amounts on nucleocapsids (4). NP also binds M protein (7). Binding of M protein to nucleocapsids seems to modify polymerase activities (12, 13) and may also be required to mediate interactions between nucleocapsids and the viral envelope proteins during virus budding. The paramyxovirus RNA polymerase is able to work in two different modes, and NP protein may be regarded as a regulatory factor that switches the polymerase from the transcriptive to the replicative mode. During transcription, intergenic start/stop signals on the viral genome are recognized, and thus a 54-nucleotide-long leader RNA and six capped, polyadenylated mRNAs are generated. Replication, as opposed to transcription, results in full-size antigenomic RNA, encapsidated by NP protein. Replication, but not

Three unique Sendai virus antigenic peptides screened from nucleocapsid protein by overlapping peptide array

“…1). The molecular weight of the 57 kDa protein was consistent with that of NP of SeV (Buchholz et al, 1993;Ogino et al, 1999;Wan et al, 1995).…”

Inducible expression of the P, V, and NP genes of the paramyxovirus simian virus 5 in cell lines and an examination of NP-P and NP-V interactions

“…Although the intracellular distribution of virus proteins will be effected by the actual process of virus replication, studies examining their intracellular localization when expressed alone or in combination have proved revealing. When expressed alone, the NP protein was present in small cytoplasmic aggregates, formed presumably through its propensity to self-aggregate (2,12,32). In contrast, the P protein had a more diffuse cytoplasmic distribution.…”

Solubility, immunogenicity and physical properties of the nucleocapsid protein of Nipah virus produced in Escherichia coli

“…capsid proteins of other paramyxoviruses: Sendai virus expressed in mammalian cells [Bunchholz et al, 1993], measles virus expressed in incest cells [Fooks et al, 1993] and in E. coli [Warnes et al, 1995], and Newcastle disease virus (NDV) expressed in insect cells [Errington and Emmerson, 1997] and in E. coli [Kho et al, 2001a[Kho et al, , 2003. It is unclear at this stage whether the NiV N protein assembled to form herringbone-like particles inside the bacteria or/and during the preparation and purification of the protein.…”