Sequence of the genomic RNA of nudaurelia beta virus (Tetraviridae) defines a novel virus genome organization.

The monopartite genome of Nudaurelia beta virus, the type species of the Betatetravirus genus of the family Tetraviridae, consists of a single-stranded positive-sense RNA (ss+RNA) of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerases of ss+RNA viruses. The 3' proximal ORF of 1836 nucleotides, which encodes the 66-kDa capsid precursor protein, overlaps the replicase gene by more than 99% (1827 nucleotides) and is in the +1 reading frame relative to the replicase reading frame. This capsid precursor is expressed via a 2656-nucleotide subgenomic RNA. The 3' terminus of the genome can be folded into a tRNA-like secondary structure that has a valine anticodon; the tRNA-like structure lacks a pseudoknot in the aminoacyl stem, a feature common to both genera of tetraviruses. Comparison of the sequences of Nudaurelia beta virus and another member of the Tetraviridae, Helicoverpa armigera stunt virus, which is in the genus Omegatetravirus, shows identities of 31.6% for the replicase and 24.5% for the capsid protein. The viruses in the genera Betatetravirus and Omegatetravirus of the Tetraviridae are clearly related but show significant differences in their genome organization. It is concluded that the ancestral virus with a bipartite genome, as found in the genus Omegatetravirus, likely evolved from a virus with an unsegmented genome, as found in the genus Betatetravirus, through evolution of the subgenomic RNA into a separate genomic component, with the accompanying loss of the capsid gene from the longer genomic RNA.

Cell-free translation and identification of the replicative form of Nudaurelia beta virus RNA.

Larvae of the pine emperor moth, Nudaurelia cytherea capensis, infected with Nudaurelia beta virus (N beta V) consistently contained one species of dsRNA. This dsRNA was the correct size to be the replicative form of the N beta V genome and, in Northern blots, it hybridized with 32P-end-labelled virion RNA. Other smaller dsRNAs were obtained in a non-reproducible manner but these had no sequences in common with the genomic probe; no dsRNAs were observed in extracts from virus-free larvae. Cell-free translation of N beta V RNA resulted in the synthesis of only one major polypeptide, of Mr about 71,000, which could not be precipitated by an anti-N beta V serum.

The three-dimensional structure of frozen-hydrated Nudaurelia capensis beta virus, a T = 4 insect virus.

The three-dimensional structure of Nudaurelia capensis beta virus (N beta V) was reconstructed to 3.2-nm resolution from images of frozen-hydrated virions. The distinctly icosahedral capsid (approximately 40-nm diameter) contains 240 copies of a single 61-kDa protein subunit arranged with T = 4 lattice symmetry. The outer surface of unstained virions compares remarkably well with that previously observed in negatively stained specimens. Inspection of the density map, volume estimates, and model building experiments indicate that each subunit consists of two distinct domains. The large domain (approximately 40 kDa) has a cylindrical shape, approximately 4-nm diameter by approximately 4-nm high, and associates with two large domains of neighboring subunits to form a Y-shaped trimeric aggregate in the outer capsid surface. Four trimers make up each of the 20 planar faces of the capsid. Small domains (approximately 21 kDa) presumably associate at lower radii (approximately 13-16.5 nm) to form a contiguous, non-spherical shell. A T = 4 model, constructed from 80 trimers of the common beta-barrel core motif (approximately 20 kDa) found in many of the smaller T = 3 and pseudo T = 3 viruses, fits the dimensions and features seen in the N beta V reconstruction, suggesting that the contiguous shell of N beta V may be formed by intersubunit contacts between small domains having that motif. The small (approximately 1800 kDa), ssRNA genome is loosely packed inside the capsid with a low average density.

Biodegradable serum albumin polymers for the sustained release of virus antigens in vaccines. A preliminary report.

In an experimental vaccine, virus particles were chemically cross-linked into rabbit serum albumin beads. This vaccine was injected into rabbits without adverse reactions and the humoral antibody levels achieved were comparable to those obtained using virus emulsified in Freund's adjuvant. The antibodies produced were capable of neutralizing virus infectivity.

Titration behaviour of three strains of tobacco mosaic virus.

Hydrogen ion titration curves of the virions and proteins of three strains of tobacco mosaic virus (Y-TAMV, U2, and cowpea) were measured in the absence and the presence of Ca2+, Mg2+, or Mn2+ ions, and compared with the analogous curves for the type strain (vulgare). Extinction coefficients were also measured for all four strains' virions and proteins. Y-TAMV is very like vulgare in its cation affinities: the virion has probably three groups per protein subunit that titrate near neutral pH and significantly bind metal ions; the RNA-free protein has very little affinity for Ca2+, although moderate Ca2+ concentrations favour the existence of larger polymers. U2 and cowpea strain virions bind cations significantly more strongly than do Y-TAMV or vulgare virions: their polymerized proteins, too, have significant affinities for Ca2+ ions, which make their titration and sedimentation behaviours relatively sensitive to added calcium. These cation-binding differences correspond well with the differences between the strains' protein sequences. The features common to all four strains are that the virions are apparently structurally invariant and have at least one site per subunit with Ca2+ affinity in the region of 10(-5)M, while the RNA-free proteins lack the high-affinity sites but have weaker Ca2+ affinities in the region of 10(-3)M. Some of the cation-binding sites probably lie near the central holes of the virions.

Pigmentation and acriflavine resistance in Serratia marcescens.

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.