Further Physicochemical Characterization of Nodamura Virus. Evidence that the Divided Genome Occurs in a Single Component

Tang

et al. 2004

J Virol

The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T‫3؍‬ particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Å resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.Analysis of the three-dimensional structure of icosahedral viruses has provided insights into the function and biology of virus particles. The techniques used rely on the symmetry of the particles, and therefore less is known about the structure of the encapsidated genome. The nucleic acid is seen only where it is arranged with symmetry that correlates with that of the icosahedrally ordered protein shell. Nevertheless, a portion of the genome has been visualized in several icosahedral viruses with positive-sense RNA genomes (reviewed in reference 22). Although these viruses contain single-stranded genomes, the RNA visualized by X-ray crystallography in both Tϭ1 and Tϭ3 virus particles predominantly resembles A-form duplex RNA, suggesting that the conformation of the RNA encapsidated in these particles is influenced by factors other than the genome sequence. In the 3-Å X-ray crystallographic structure of Pariacoto virus (PaV), a Tϭ3 nodavirus, an unprecedented 1,500 nucleotides (nt) of the 4,322-nt genome were visible (27), providing an opportunity to examine the factors involved in ordering of the RNA.The structure and assembly of viruses from the Alphanodavirus genus of the Nodaviridae family have been studied extensively (reviewed in references 10 and 25). Nodaviruses are positive-sense RNA viruses that have 30-nm nonenveloped particles with Tϭ3 icosahedral symmetry. Each particle is assembled from 180 copies of a capsid protein precursor (alpha), which is autocatalytically cleaved following assembly into the two mature capsid proteins beta and gamma (7). A copy of each of the two genome segments, RNA1 (3 kb) and RNA2 (1.3 kb), are coencapsidated in each virus particle (16,21). Authentic nodavirus particles do not encap...

mentioning

confidence: 99%

Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions

Tang

et al. 2004

J Virol

“…NoV is unique among alphanodaviruses in its ability to lethally infect both insects and mammals, including the mosquitoes Aedes aegypti, Aedes albopictus, and Toxorhynchites amboinensis (4, 36, 41), suckling mice, and suckling hamsters (17,35,36). NoV infects cultured mosquito cells from Aedes pseudoscutellaris, A. aegypti, and A. albopictus (1, 3, 41) and cultured baby hamster kidney BHK21 cells (3,23,30). When NoV genomic RNAs are introduced by transfection, they can replicate in a wide range of cultured cells (6).…”

mentioning

confidence: 99%

“…These two genomic RNAs are copackaged into the same virion, and both are required for infectivity (24,30,38). RNA1 encodes protein A, the RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of both genome segments.…”

mentioning

confidence: 99%

Nodamura Virus Nonstructural Protein B2 Can Enhance Viral RNA Accumulation in both Mammalian and Insect Cells

Price

Eckerle

et al. 2004

J Virol

During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.Nodamura virus (NoV) is the type species of the genus Alphanodavirus of the Nodaviridae, a family of small riboviruses with bipartite, positive-strand RNA genomes that also includes Flock House virus (FHV). NoV is unique among alphanodaviruses in its ability to lethally infect both insects and mammals, including the mosquitoes Aedes aegypti, Aedes albopictus, and Toxorhynchites amboinensis (4, 36, 41), suckling mice, and suckling hamsters (17,35,36). NoV infects cultured mosquito cells from Aedes pseudoscutellaris, A. aegypti, and A. albopictus (1, 3, 41) and cultured baby hamster kidney BHK21 cells (3,23,30). When NoV genomic RNAs are introduced by transfection, they can replicate in a wide range of cultured cells (6).The divided nodavirus genome naturally separates the replicative and packaging functions onto two different positivesense RNA molecules, RNA1 and RNA2, respectively. These two genomic RNAs are copackaged into the same virion, and both are required for infectivity (24,30,38). RNA1 encodes protein A, the RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of both genome segments. RNA2 encodes the viral capsid precursor protein, ␣. RNA2 and protein ␣ are dispensable for RNA1 replication. Protein A also catalyzes the synthesis of a single subgenomic RNA3 from an RNA1 template. RNA3 is not packaged into virus particles. For FHV, RNA3 encodes two small proteins, B1 and B2, in overlapping reading frames. Protein B1 is in the same reading frame as protein A and thus represents its C-terminal fragment, whereas protein B2 is in the ϩ1 reading frame relative to protein A (11). For NoV, the first and second AUG codons of RNA3 initiate the translation of two forms of B2 (B2-137 and B2-134) that differ only at the N terminus, whereas B1 initiates at the third AUG codon. As for FHV, the B2 proteins are in the ϩ1 reading frame relative to the A/B1 open reading frame (ORF) (23). All three proteins are detected in cells transfected with NoV RNA1 (NoV1), which replicates autonomously and leads to the synthesis of RNA3 (23).The functions of the nodavirus B1 and B2 prote...

Journal of General Virology

“…Secondly, attempts to cross-link the strands by u.v. irradiation, using a procedure (Newman & Brown, 1978) similar to that used for a nodavirus, failed. Clearly, further work is needed to resolve the question of how the strands of the HaSV genome are encapsidated.…”

Section: Presence O F Another F O R M O F H a S Vmentioning

confidence: 99%

“…The molar ratio of HaSV strands was determined by quantitative densitometry using a Hoefer GS 300 densitometer on negatives of fluorograms of varying amounts ofHaSV RNA resolved on denaturing gels. Cross-linking of RNAs to assess possible co-encapsidation was attempted using the methodology of Newman & Brown (1978).…”

Section: T N Hanzlik and Othersmentioning

confidence: 99%

A novel small RNA virus isolated from the cotton bollworm, Helicoverpa armigera

Hanzlik

Dorrian

Gordon

et al. 1993

A small RNA virus with novel characteristics has been isolated from laboratory-bred larvae of Helicoverpa armigera. Infection by the H. armigera stunt virus causes severe retardation of larval development and subsequent death. Its particles are isometric, 38 nm in diameter, and have a buoyant density of 1.296 g/ml in caesium chloride.The viral capsid has two major non-glycosylated protein components with Mrs of 65 000 and 6000, and contains