Another triple-spanning envelope protein among intracellularly budding RNA viruses: the torovirus E protein.

The nucleotide sequence of the Berne virus envelope (E) protein gene was determined and its 26.5K translation product was identified by in vitro transcription and translation. Computer analysis of the protein sequence revealed the characteristics of a class III membrane protein lacking a cleaved signal sequence but containing three successive transmembrane alpha-helices in the N-terminal half, much the same as the coronavirus membrane (M) protein. The disposition of the E protein in the membrane was studied by in vitro translation in the presence of microsomes and by subsequent proteinase K digestion. Only small portions of either end of the polypeptide were found to be exposed on opposite sides of the vesicle membranes. Experiments with a hybrid E protein (EM) containing the C-terminal tail of a coronavirus M protein, to which an anti-peptide serum was available, showed that this C-terminus was present at the cytoplasmic side of the membrane, which is another similarity to the coronavirus M protein. Immunofluorescence experiments indicated that the EM protein, expressed by a recombinant vaccinia virus, accumulated in intracellular membranes, predominantly those of the endoplasmic reticulum. The common features of the torovirus E and the coronavirus M protein support our hypothesis that an evolutionary relationship exists between these groups of intracellularly budding viruses.

Identification and characterization of the virus causing rabbit hemorrhagic disease.

Liver tissue from animals that died of rabbit hemorrhagic disease (RHD) was used to identify the causative agent. After extraction of liver homogenates and sucrose density gradient ultracentrifugation, distinct bands were obtained. The respective gradient fractions reacted positively in an enzyme-linked immunosorbent assay as well as in hemagglutination assays and were infective for rabbits. These fractions contained virions which had a diameter of 40 nm and resembled morphologically those of the family Caliciviridae. By immunoblotting, a major structural protein with a molecular weight of 60,000 was identified. Highly pure RNA of about 8 kilobases was isolated from virions. Labeled cDNA synthesized from virion RNA detected two RNAs of 8 and 2 kilobases in Northern (RNA) blots of liver RNA from animals infected with RHD virus. Finally, isolated virion RNA injected into the liver of rabbits produced a disease with clinical symptoms and pathological findings typical of RHD. We conclude that a calicivirus represents the causative agent of RHD.

Isolation and characterization of a RNA-virus like particle from Candida curvata.

A virus-like particle (VLP) of 35 nm diameter has been isolated from the lipolytic yeast Candida curvata. The VLP contains a linear, double stranded RNA molecule of 1.55 microns in length.

LR1: a candidate RNA virus of Leishmania.

Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately 6000 nucleotides, is single-stranded, and is largely, if not exclusively, located in the cytoplasm. No homologous LR1 sequences are detected in genomic DNA. The candidate viral RNA is associated with a spherical particle 32 nm in diameter that has a sedimentation coefficient of approximately 130 S. There is as yet no evident effect of this potential virus on parasite physiology or the disease caused by the parasite.

The capsid polypeptides of the 190S virus of Helminthosporium victoriae.

SDS-PAGE of the 190S virus of Helminthosporium victoriae, using a discontinuous buffer system, revealed two major capsid polypeptides of mol. wt. 88K and 83K (p88 and p83) and a minor polypeptide, p78. Peptide mapping by both limited proteolysis and selective chemical cleavage showed p83 and p78 to be closely related to p88. The origin of p83/p78 could not be explained by proteolysis of p88 during virus preparation and storage. In rabbit reticulocyte lysates, denatured dsRNA directed the synthesis of a single major translation product which was identical to capsid polypeptide p88 on the basis of coelectrophoresis, immunoprecipitation and peptide mapping. No translation products comparable in size to p83 or p78 were detected in vitro. These data indicated that the capsid of the 190S virus is encoded by a single gene and verified the classification of the virus as a member of the family Totiviridae. Radioiodination of intact virus under conditions considered optimum for surface-specific iodination showed p88 to be more readily available for labelling than p83 or p78. Furthermore, when Western blots of capsid polypeptides were reacted with an antiserum to glutaraldehyde-stabilized virus (190S-G), p88 was more reactive to 190S-G antibodies than was p83/p78. These results suggest p88 is external to p83/p78 in the capsid.

The peplomers of Berne virus.

Using [3H]glucosamine and [3H]mannose labels, two virus-specific glycosylated polypeptide species with Mr values of about 200,000 (200K) and in the 75K to 100K range, respectively, were recognized in Berne virus-infected embryonic mule skin cells. In purified virions only the latter glycoprotein occurred. Concanavalin A was bound to the virion as evidenced by reduction in infectivity. Analyses using SDS-PAGE, blotting and glycoprotein identification with concanavalin A and horseradish peroxidase showed coincidence of the virion glycoprotein signals with the maximum infectivity and haemagglutinating activity in an isokinetic sucrose gradient. Polyclonal rabbit immune serum and a neutralizing and haemagglutination-inhibiting monoclonal antibody raised against Berne virus recognized both the 75K to 100K and the '200K' glycoproteins. Using tunicamycin, a concentration-dependent inhibition of infectivity was noted; however, non-infectious particles containing the two major polypeptides (20K and 22K) were released from the cells in small quantities. The glycoproteins were absent from cytoplasmic extracts and a novel polypeptide of about 150K was identified instead. Translation of poly(A)-selected intracellular RNA from infected cells in a rabbit reticulocyte cell-free system also resulted in the appearance of a new high Mr polypeptide (about 170K). Using pulse-chase labelling and radioimmunoprecipitation, suggestive evidence for a precursor-product relationship between the intracellular '200K' and the virion glycoproteins has been obtained. These experiments identify the N-glycosylated proteins in the 75K to 100K range as constituents of the peplomeric envelope projection of Berne virus; they probably arise by post-translational processing of a 150K to 170K precursor molecule involving glycosylation and subsequent cleavage.

Structure and assembly of turnip crinkle virus. I. X-ray crystallographic structure analysis at 3.2 A resolution.

The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic symmetry. The structures are very closely related, especially in the subunit arm and S domain, where only small insertions and deletions and small co-ordinate shifts relate one chain to another. The P domains, although quite similar in fold, are oriented somewhat differently with respect to the S domains. Understanding of the structure of turnip crinkle virus has been important for analyzing its assembly, as described in an accompanying paper.

Sequence homology between the coat proteins of DNA and RNA plant viruses.

The coat proteins of the leafhopper transmitted gemini viruses, viz., maize streak virus and wheat dwarf virus that contain a single circle of DNA in their genomes are shown to bear good amino acid sequence homology with the coat protein of an RNA plant virus, the satellite tobacco necrosis virus. It is suggested that for icosahedral assemblage, the coat protein architecture transcends genomic preferences.

The nucleocapsid of Berne virus.

In Berne virus-infected cells and in gradient-purified virions two major proteins with mol. wt. of 20K and 22K were detected. The 22K species is thought to represent the main envelope polypeptide; in infectious culture media it was present in a low envelope polypeptide; in infectious culture media it was present in a low density substructure which could be quantitatively converted into slowly sedimenting material by detergent treatment. The 20K polypeptide (accounting for about 80% of the 14C-amino acid label in the virion) was phosphorylated, occurred in an intracellular substructure of higher density than the virion (rho = 1.36 g/ml in CsCl) and was the only viral protein possessing RNA-binding properties; it was recognized preferentially by heterologous animal sera in immune precipitation. The 20K species is therefore identified as the main capsid protein. Two additional polypeptides (19K and 17K) were regularly detected in extracts of infected cells; they appeared to share oligopeptides with the 20K protein and are interpreted as being proteolytic cleavage products. The nucleocapsid of Berne virus was visualized after ether treatment as a flexible bacilliform structure with conspicuous transverse striation. Demonstration of a 20K nucleocapsid protein further supports the authors' proposal that Berne virus is a representative of a new family of enveloped RNA viruses (Toroviridae).

Identity between infectious pancreatic necrosis virus VR-299 and a Chilean isolate.

Nucleic acid and protein electrophoretic analyses of the Chilean isolate of infectious pancreatic necrosis virus showed that genomic RNA, as well as the major proteins, were indistinguishable from those of the VR-299 serotype. The data are in good agreement with the theory that the virus was introduced into Chile from North America.

Polyacrylamide gel electrophoresis of the Kemerovo virus RNA.

Ribonuclease (RNase)-resistant RNA was isolated from partially purified Kemerovo virus by gel chromatography and or sucrose density gradient centrifugation. Double-stranded (ds) RNA only was found in the purified viral cores. The RNAs from both sources exhibited the same pattern of distribution in polyacrylamide gels. Ten dsRNA segments were identified. According to the results of coelectrophoresis of the Kemerovo virus and reovirus dsRNAs, the size of Kemerovo virus genome was estimated to be of about 11.7 X 10(6). The grouping of Kemerovo virus double-stranded segments according to their size in polyacrylamide gels corresponded to the 2:4:3:1 pattern.

The capsid polypeptides of the yeast viruses.

The yeast virus is a double-stranded RNA virus with a large genomic dsRNA and one major viral capsid polypeptide. Most strains of yeast have a major and a minor species of the large genomic dsRNA present. The major species has previously been shown to encode a capsid polypeptide with a Mr of about 88,000. We show that the minor species also encodes its capsid polypeptide with a Mr of about 80,000. Unlike all the dsRNA viruses of procaryotes and higher eucaryotes, the yeast virus appears to have only one major polypeptide in its virions. There are some 60 molecules of this capsid polypeptide per particle, consistent with a simple icosohedron of T=1.

Characterization of Hantaan virions, the prototype virus of hemorrhagic fever with renal syndrome.

Hantaan virus, strain 76-118, was propagated to high titer in a clone of Vero cells, and infectious virions were successfully concentrated and purified. Infectivity and virus antigenic activity were closely associated with a virus particle that exhibited a sedimentation rate indistinguishable from a representative member of the Bunyaviridae. Purified virions sedimented to a density of 1.16-1.17 in sucrose and 1.20-1.21 in cesium chloride. Detergent disruption of virions resulted in a nucleocapsid structure (density, 1.18 in sucrose and 1.25 in cesium chloride) and soluble protein antigens. Three separate nucleocapsids were resolved by rate-zonal centrifugation and contained a single but common polypeptide of 50,000 daltons. Electrophoresis of radiolabeled RNA extracted from purified virions yielded a profile of three RNA species with apparent molecular weights of 2.7, 1.2, and 0.6 X 10(6). These data support earlier electron microscopy reports which suggested that Hantaan virus has characteristics similar to some members of the virus family Bunyaviridae.

Codon usage and mistranslation. In vivo basal level misreading of the MS2 coat protein message.

The coat protein of the small RNA virus MS2 shows charge heterogeneity in vivo. In most strains there is a basic satellite of the native protein. We have shown that this basic satellite is greatly diminished or absent in strains with the streptomycin-resistant allele, rpsL, a mutation which leads to increased translational accuracy. Further, the satellite is present in cells where the coat protein is encoded by duplex DNA. Tryptic digests of the satellite show that it contains new lysine-containing peptides which appear to be the same as those found in derivatives of coat protein which have a lysine for asparagine substitution. Sequencing of the NH2-terminal 19 amino acids of the satellite protein shows that the asparagine codon AAU at amino acid 12 is misread approximately 8 times more frequently than the AAC at amino acid 3. We conclude that the satellite species is the result of basal level lysine for asparagine substitution. These substitutions are most likely caused by preferential misreading of AAU codons at a frequency of approximately 5 X 10(-3), 10-fold higher than the average error frequency.

Bluegill virus is a ribovirus of positive-strand polarity.

RNA was extracted from purified Bluegill virus (BGV) and fractionated onto a poly (U)-Sepharose-4 B column. More than 70 per cent of this RNA became bound and could be subsequently eluted from the column. By polynucleotide phosphorylase digestion, the poly (A) sequences were located at the 3'-terminus of the RNA. This RNA and purified BGV RNA were infectious as shown by plaque assay titration of the virus produced. Furthermore, we were unable to detect RNA polymerase activity in preparations of BGV. These results indicate that the genome in the BGV particle is a positive-strand RNA.