Search

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.

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.

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.

Cocultivation of cells derived from embryos of golden pheasants or Amherst pheasants with chicken embryo cells infected with Bryan strain of Rous sarcoma virus resulted in the detection of viruses which appear to be endogenous in these pheasant cells. The pheasant viruses (PV) were similar to avian leukosis-sarcoma viruses (ALSV) in their gross morphology, in the size of their RNA, in the presence of a virion-associated RNA-dependent DNA polymerase (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7), and in their growth characteristics. PV also serves as a helper for the glycoprotein-defective Rous sarcoma virus. However, PV was shown to be different from both ALSV and reticuloendotheliosis virus in the following properties: (i) PV does not have ALSV group specific antigens; (ii) the protein composition of PV is different from those of the other two groups of viruses; (iii) PV fails to complement the defective polymerase of alpha type Rous sarcoma virus; and (iv) PV RNA shows no detectable homology with nucleic acids of the other two groups of viruses. Thus, PV appears to be a new class of RNA viruses which contain RNA-dependent DNA polymerase.

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.

Rotavirus was purified from nine patients hospitalized with acute gastroenteritis from October to December, 1978, in Mexico City. Analysis of their ribonucleic acids by gel electrophoresis showed the presence of two distinct patterns (2s and 22) which had been observed in 1977, but which now were found in a very different proportion: the pattern called 2s, observed in only 11% (6 of 52) of the patients in 1977, was found in 90% (8 of 9) of the patients in 1978. Improvements in the electrophoretic method allowed us to observe differences in the migration of up to seven segments between the two patterns and to distinguish small differences in one or two segments within either of the two ribonucleic acid patterns.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.

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.

A previously healthy middle aged man died following a 6 month illness which presented with middle ear symptoms, apparently resolved, and then 2 months later manifested as encephalitis. The illness was characterized initially by depression and intellectual deterioration. No family member or working associate was affected. The clinical diagnosis of viral encephalitis was confirmed by brain biopsy but no virus was isolated in the laboratory. Numerous intracisternal toroidal virus-like particles were demonstrated by electron microscopy in the perikarya and dendrites but not in glia. The particles resemble, but are not identical to, the oncornaviruses associated with spontaneous and induced murine neoplasms. The resemblance of these structures to the intracisternal toroidal type "A" virus of murine leukemia is noted and other possible causes for this atypical meningoencephalitis are discussed.

RNA extracted from Tilligerry virus, a member of the Eubenangee group of orbiviruses, separated into ten segments after electrophoresis in acrylamide gels. The molecular weights of most segments differed from the corresponding segments of Eubenangee virus.

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.

Two distinct types of rotavirus particle were isolated in caesium chloride density gradients. The higher density particle banded at 1-38 g/ml and measured 55 +/- 0-4 nm in diameter while the less dense particle banded at 1-36 g/ml, measured 66 +/- 0-4 nm in diameter and appeared to possess an extra outer capsid layer. Some forms intermediate between the two types were seen. Since the two particles had a similar but not identical morphology and polypeptide composition, they were considered to be different forms of the same virus. Infectivity was associated with the larger particle which contained one more polypeptide than the smaller particle.

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.

Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.

Procedures were developed for purification of virus-like particles (VLPs) from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. Morphologically similar spherical VLPs measuring 40 nm in diameter were extracted from all three phenotypes. The particles were partially purified by high-speed centrifugation through a layer of CsCl (1.26 g/cm3) and sucrose density gradient centrifugation. Gradient-purified preparations contained three centrifugal species that sedimented at approximately 43, 102, and 162S. The 43S component is considered to be an artifact. Preparations from killer strains contained three double-stranded RNA (ds-RNA) components with molecular weights of 1.19 x 10(6), 1.29 x 10(6) and 2.54 x 10(6). VLPs from neutral and sensitive strains contained only the largest ds-RNA species. VLP preparations were subsequently separated into two major density components by CsCl equilibrium gradient centrifugation. The light component banding at 1.28 to 1.30 g/cm3 was void of nucleic acid, and the heavy component banding at 1.40 g/cm3 contained only the largest ds-RNA species.

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 purification of coliphage MS2 dinitrophenol (DNP) conjugates provided a system for localization of the single molecule of A-protein in the capsid of the MS2 phage particle. Three A-protein preparations isolated from unconjugated MS2, overconjugated DNP-MS2, and purified 78S DNP-MS2 were tested for the presence of covalently bound DNP. The binding characteristics to Dowex 1-X8 and rabbit anti-DNP bovine serum albumin (DNP-BSA) immunoglobulin G of the 78S DNP-MS2 and overconjugated DNP-MS2 A-protein preparations indicate that the A-protein is located on the surface of the phage particle where it can be covalently conjugated with hapten. Extensive enzymatic iodination of the A-protein of intact unconjugated MS2 substantiates this conclusion.

A method for preparation of single-stranded RNA for electron microscopy determination of molecular weight is reported. The method uses treatment with formaldehyde at elevated temperatures to remove secondary structure and spreading in a protein monolayer from 50% formamide onto a 50% formamide hypophase. Molecular weights were determined for some bacterial and animal viruses, for which conflicting values had been reported earlier. Molecular weights determined by the method, using Escherichia coli large subunit rRNA for a standard (1.1 x 10(6)), are as follows: E. coli small subunit rRNA, 0.53 x 10(6); coliphage f2-RNA, 1.3 x 10(6); Qbeta-RNA, 1.55 x 10(6); and Newcastle disease virus RNA, 5.78 x 10(6).

The frequency of oligonucleotides obtained from simian sarcoma virus RNA by digestion with ribonuclease T1 was compared with the frequency expected of an RNA molecule in which nucleotides are arranged in random distribution. Oligonucleotides containing C-residue attached to 3'-Gp were found significantly less in simian sarcoma virus 70S RNA than expected by random distribution.

Temperature-sensitive mutants of the related orbiviruses, Wallal and Mudjinbarry, recombine with high frequency when grown in pairs in cell culture. The genome of each virus consists of discrete segments of double-stranded RNA and high frequency recombination suggests that reassortment of genome segments occurs rather than classical recombination. Electrophoresis in acrylamide gels of RNA extracted from the progeny of a cross between Wallal ts 101 and Mudjinbarry ts 3 mutants, revealed that three plaque isolates of 60 tested differed in RNA pattern from each of the parent viruses and from each other. Further analysis of the electrophoretic profiles suggested that the isolates were recombinants with RNA segments derived from each of the parent viruses.

Proteins from four fish rhabdoviruses have been studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viruses were: trout viral hemorrhagic septicemia (VHS), infectious hematopoietic necrosis virus (IHN), spring viremia virus of carp (SVC), and the pike fry rhabdovirus (PFR). For the two salmonid viruses (VHS-IHN), gel electrophoresis indicated the proteins, with molecular weights estimated to be 190,000, 80,000, 38,000, 25,000, and 19,000, respectively. The electrophoretic profile of the two other viruses (SVC-PFR) revealed four major proteins with molecular weights of 190,000 80,000 42,000 and 21,000, respectively. In this case a minor component with 50,000 daltons was found. For each virus only one protein was found to be glycosylated, i.e., the one with a molecular weight of 80,000. A major protein (molecular weight between 38,000 and 42,000) was found to be associated with the nucleocapsid. All these results revealed marked similarities in protein structure between the four fish rhabdoviruses and the previously well-characterized members of rhabdovirus group. However, one can distinguish two groups of viruses: the first one is composed of salmonid viruses (VHS and IHN) with a protein structure comparable to that of rabies virus and potato yellow dwarf virus; the second one is composed of carp and pike viruses, having a protein structure very similar to that of vesicular stomatitis virus.

The RNAs of 4 avian rotavirus isolates were analysed by polyacrylamide gel electrophoresis. One of these isolates was made from chickens, the other three were from turkeys. The RNAs were separated into 8 or 9 bands consisting of 11 segments, ranging in molecular weight from 0.24 X 10(6) to 2.09 X 10(6). In contrast to calf rotavirus, segments 10 and 11 of avian rotavirus RNA had very similar or identical electrophoretic mobilities. This may be a unique feature of avian rotavirus RNA. Co-electrophoresis experiments showed that the RNAs of 2 of the turkey isolates were electrophoretically identical. However, differences existed between these 2 isolates, the chicken isolate and the remaining turkey isolate with respect to segments 5, 6, the 7, 8, 9 complex and 10. Segment 5 of the chicken isolate was sometimes resolved into 2 components, and was further anomalous in that it was absent in some (analyses).

The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T(1) digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosine-labeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CCl(3)COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.

Radiolabelled bovine viral diarrhoea/mucosal disease virus (BVDV) strains NADL and Oregon C24V were purified by different steps. Following immunoprecipitation, electrophoresis in SDS-polyacrylamide gels revealed three BVDV structural polypeptides with molecular weights of 57 (VP1), 44 (VP2), and 34 (VP3) kd. The two larger BVDV polypeptides VP1 and VP2 were found to be glycosylated (gp57, gp44). The data obtained on BVDV structural proteins demonstrate common features with hog cholera virus and indicate a common grouping with the family Togaviridae.

Potato spindle tuber viroid (PSTV), a small infectios RNA, has been completely digested with RNase T1 and RNase A, and the resulting oligonucleotides have been sequenced using 5'-terminal 32p-labelling with gamma-32p ATP and T4 polynucleotide kinase, fingerprinting and controlled nuclease P1 digestion. Modified nucleotides have not been detected in 5'-positions of these oligonucleotides. PSTV consists of about 359 nucleotides and contains a remarkable stretch of 18 purines, mainly adenosines; there is no AUG initiation triplet present. The established oligonucleotide sequences preclude a perfect intramolecular base complementarity within the covalently closed viroid circle. Therefore, the rigid, rod-like native secondary structure of PSTV, as seen in the electron microscope, must be based on a defective rather than on a homogeneous RNA helix. The detailed analysis of the bisulfite-catalized modification of cytidine to uridine in PSTV revealed a higher reactivity for the majority of the cytidines than would be expected for a perfect helix. Since only cytidines in single-stranded regions are knonw to be fully reactive, this finding provides additional evidence for defects in the helical secondary structure of PSTV.

Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10(6)-10(7) have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.

Pike fry rhabdovirus (pfr) rna has been characterized as a single-stranded non-segmented molecule of about 4 X 10(6) daltons. In sucrose gradients in 0-1 M-NaC1 it had a sedimentation coefficient of 40 to 45S, somewhat lower than that Of Semliki Forest virus RNA. The sedimentation velocity of the RNA is strongly influenced by the salt concentration and divalent cations. It shows a buoyant density of 1-65 g/ml in caesium sulphate. The base composition of the RNA is 21-6% G, 25-I% A, 22-4% C and 30-9% U. It is thus comparable to the RNAs of other rhabdoviruses.

Media from cells producing RNA tumor viruses, when treated with sodium dodecyl sulfate and polyvinyl sulfate, yield 70S RNA as the major species binding oligo(dT)-cellulose. The procedure described for purifying 70S RNA requires no special equipment and is suitable for rapidly processing large quantities of media or for purifying RNA from commercially avialable virus, with a 5- to 10-fold higher yield than was obtained using existing methods.