Structure of the Vesicular Stomatitis Virus Nucleoprotein-RNA Complex
ABSTRACT Vesicular stomatitis virus is a negative-stranded RNA virus. Its nucleoprotein (N) binds the viral genomic RNA and is involved
in multiple functions including transcription, replication, and assembly. We have determined a 2.9 angstrom structure of a
complex containing 10 molecules of the N protein and 90 bases of RNA. The RNA is tightly sequestered in a cavity at the interface
between two lobes of the N protein. This serves to protect the RNA in the absence of polynucleotide synthesis. For the RNA
to be accessed, some conformational change in the N protein should be necessary.
- SourceAvailable from: Nikos Vasilakis
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- "The N ORFs each encode 434 aa mildly acidic polypeptides (ABTV, 49.7 kDa; PTAMV, 49.9 kDa) that share 38.6 % overall amino acid sequence identity. A CLUSTAL_X alignment of their N proteins with that of Vesicular stomatitis Indiana virus (VSIV) indicated preservation of several known conserved motifs, as well as each of the eight basic residues located in the RNA-binding cavity that are known to coordinate binding to viral genomic RNA in the ribonucleoprotein (RNP) (Green et al., 2006; Luo et al., 2007) (Fig. S2). "
ABSTRACT: Arboretum (ABTV) and Puerto Almendras (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11,482 nt (ABTV) and 11,876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.Journal of General Virology 01/2014; 95(Pt_4). DOI:10.1099/vir.0.058685-0 · 3.18 Impact Factor
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- "These viruses assemble their RNA replication/transcription complexes in close association with membranous cell compartments that provide an effective shield against dsRNA sensors . -ssRNA viruses carry their own RdRp and prevent the exposure of duplex RNA by protecting their genomic RNA with nucleocapsid proteins, and by assembling transcriptionally active ribonucleoprotein (RNP) complexes . Finally, prototypical dsRNA viruses enclose their genomes in a highly conserved structure, the so-called T = 2 capsid or transcriptional core, that contains all the enzymatic machinery required to synthesize and extrude translatable mRNAs into the cytoplasm of the infected cell. "
ABSTRACT: Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed, results presented here also support the previously proposed evolutionary connection between birnaviruses and positive-strand single-stranded RNA viruses.PLoS ONE 01/2014; 9(1):e87790. DOI:10.1371/journal.pone.0087790 · 3.23 Impact Factor
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- "The ring-shaped RNPs of respiratory syncytial, rabies, and vesicular stomatitis viruses contain 10–11 N protein molecules (Albertini et al., 2006; Green et al., 2006; Tawar et al., 2009), whereas the N protein of Borna disease virus was crystallized as a tetramer (Rudolph et al., 2003). In most viruses, RNA was observed inside the N ring-structures. "
ABSTRACT: The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA.Virus Research 07/2012; 167(1):118-23. DOI:10.1016/j.virusres.2012.04.003 · 2.32 Impact Factor