Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

Morphogenetic pathway of bacteriophage φ6: A flow analysis of subviral and viral particles in infected cells

Three types of virus-specific particles of double-stranded RNA bacteriophage φ6 were isolated and characterized by pulse-label and pulse-chase experiments on φ6-infected Pseudomonas phaseolicola. The first particle was “previrion I”, which consisted of early proteins P1, P2, P4 and P7, and had no RNA. It was detected immediately after labeling of proteins and the radioactivity was chased into the second structure, designated previrion II, after ten minutes. Previrion II contained three segments of double-stranded RNA in addition to the component of previrion I, and had RNA polymerase activity that produced messenger RNA species coding for late proteins. The RNA polymerase activity in the cell extract emerged nearly in parallel with the synthesis of late proteins, and this activity of previrion II was supposed to be responsible for late protein synthesis in infected cells. Via previrions I and II, the third radioactive particle was observed in infected cells after late protein synthesis started. This particle was identified as the intact virion, because it had infectivity as well as all of the viral components, including lipids. This intact virion was accumulated in the infected cell before bursting the cell.     

Peptide map comparison of the proteins of infectious bursal disease virus.

The genome of infectious bursal disease virus consists of two segments of double-stranded RNA of 2.5 X 10(6) and 2.2 X 10(6) molecular weight. Polyacrylamide gel electrophoresis of purified virus resolved four structural polypeptides: VP-1 (90,000), VP-2 (41,000), VP-3 (35,000), and VP-4 (28,000). Peptide map comparisons of radioiodinated virion proteins indicated no precursor-product relationship between them. The possible relationship between the size of the virus genome and the number and sizes of different viral proteins is discussed.     

Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.     

Coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins.

We examined the synthesis of viral structural proteins in cultured cells infected with the avian coronavirus infectious bronchitis virus. Tryptic peptide mapping was used to determine the structural relationships of the intracellular proteins to the virion polypeptides. Pulse-chase experiments were performed to identify precursors to the virus-specific proteins. We found that the nucleocapsid protein, P51, and the small viral membrane proteins GP31, GP28, and P23 do not undergo post-translational proteolytic processing. In contrast, GP90 and GP84, the two large virion membrane proteins, were found to be produced by cleavage of a single precursor, GP155. This demonstrated that at least one coronavirus mRNA specifies two virion proteins.     

Bacteriophage ϕ6 nucleocapsid surface protein 8 interacts with virus-specific membrane vesicles containing major envelope protein 9

Enveloped double-stranded RNA (dsRNA) bacterial virus Pseudomonas phage ϕ6 has been developed into an advanced assembly system where purified virion proteins and genome segments self-assemble into infectious viral particles, inferring the assembly pathway. The most intriguing step is the membrane assembly occurring inside the bacterial cell. Here, we demonstrate that the middle virion shell, made of protein 8, associates with the expanded viral core particle and the virus-specific membrane vesicle.     

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.     

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus)

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.     

Processing of Adenovirus 2-Induced Proteins

Analysis of (35)S-methionine-labeled extracts of adenovirus 2-infected KB cells revealed 22 virus-induced polypeptide components. Most proteins of the virion were easily detected in extracts of whole cells labeled for short periods between 15 and 30 h after infection; however, several virion components were conspicuously absent. Radioactivity appeared in two of these virion components during a chase in nonradioactive medium, and this appearance was paralleled by a decrease in the radioactivity associated with two nonvirion adenovirus-induced proteins, results which imply precursor-product relationships for these components. Comparison of one of the chasable adenovirus-induced components (designated P-VII; mass of 20,000 daltons) and the major core protein (VII; mass of 18,500 daltons) of the virion showed that they have four common methionine-containing tryptic peptides; P-VII has an additional methionine residue which is not found in the major core protein. We propose that at least two of the adenovirus 2 virion components are derived by the cleavage of higher molecular weight precursor polypeptides.     

Regulation of Herpesvirus Macromolecular Synthesis I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins

Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp-2 cells in the absence of protein synthesis we examined the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide- or puromycin-mediated inhibition of protein synthesis. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated alpha, beta, and gamma. Specifically: (i) The alpha group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 h postinfection in untreated cells and at diminishing rates thereafter. The beta group, also containing minor structural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 h and at decreasing rates thereafter. The gamma group containing major structural polypeptides was synthesized at increasing rates until at least 12 h postinfection. (ii) The synthesis of alpha polypeptides did not require prior infected cell protein synthesis. In contrast, the synthesis of beta polypeptides required both prior alpha polypeptide synthesis as well as new RNA synthesis, since the addition of actinomycin D immediately after removal of cycloheximide precluded beta polypeptide synthesis. The function supplied by the alpha polypeptides was stable since interruption of protein synthesis after alpha polypeptide synthesis began and before beta polypeptides were made did not prevent the immediate synthesis of beta polypeptides once the drug was withdrawn. The requirement of gamma polypeptide synthesis for prior synthesis of beta polypeptides seemed to be similar to that of beta polypeptides for prior synthesis of the alpha group. (iii) The rates of synthesis of alpha polypeptides were highest immediately after removal of cycloheximide and declined thereafter concomitant with the initiation of beta polypeptide synthesis; this decline in alpha polypeptide synthesis was less rapid in the presence of actinomycin D which prevented the appearance of beta and gamma polypeptides. The decrease in rates of synthesis of beta polypeptides normally occurring after 7 h postinfection was also less rapid in the presence of actinomycin D than in its absence, whereas ongoing synthesis of gamma polypeptides at this time was rapidly reduced by actinomycin D. (iv) Inhibitors of DNA synthesis (cytosine arabinoside or hydroxyurea) did not prevent the synthesis of alpha, beta, or gamma polypeptides, but did reduce the amounts of gamma polypeptides made.     

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes.

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.     

Synthesis and Cleavage of Influenza Virus Proteins

The NWS strain of influenza virus grows rapidly in and kills the MDCK dog kidney cell strain. Within 1 to 2 hr, the virus inhibits host cell protein synthesis and for 3 to 4 hr more it directs the synthesis of influenza virus proteins at a rate about twice that of uninfected cell synthesis. The rates of virus ribonucleic acid (RNA) and protein synthesis reach a maximum within the first few hours after infection and then drop. Plaque assays exhibit a linear dose-response, indicating that only one virion is necessary for productive infection. We have confirmed earlier reports regarding the fragmented nature of the RNA genome of purified influenza virions. However, high resolution gel electrophoresis indicated that each size class of viral RNA is heterogenous, so that there are at least 10 and probably more fragment sizes of RNA in these virions. Repeated attempts to detect infectivity in preparations of extracted viral RNA were completely negative (over a 10(8)-fold loss of infectivity after extraction). Even infection of the "infectious" RNA-treated cells with intact, related, influenza viruses failed to support infectivity of the isolated RNA or to rescue a host range genetic marker of the RNA. Purified influenza virions exhibit only three major protein peaks based on separation according to molecular weights. These three major virion proteins are the only major virion proteins synthesized in infected cells. This is true throughout the infectious cycle from several hours after infection until the cells are dying. However, the molecular weight of these virion proteins differs slightly depending upon the cell type in which the virus is grown. No host membrane proteins are incorporated into the virions as they bud through the cell membrane. Pulse-chase labeling early after infection or prolonged chase experiments indicate that influenza virus proteins are cleaved from one or more precursor polypeptides. In fact, each of the three major peaks seems to be a heterogeneous mixture of polypeptides in various stages of cleavage. Peptide analysis confirms that the three major peaks share common peptides, but the exact precursor product relationships are not clear. There may be one or several precursor proteins. Also there could be overlapping messenger RNA molecules of varying length giving rise to polypeptides of various sizes and overlapping sequences. Late in infection, amino acid labeling shows a preponderance of internal nucleocapsid protein synthesis, indicating that either this protein is much more stable to cleavage in infection or it is made from a more stable messenger. There is no obvious relationship between virion RNA fragments and viral protein sizes, so these fragments may be artifacts.     

Rabies virus protein synthesis in infected BHK-21 cells.

Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions.     

Cell-free translation of murine coronavirus RNA.

The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These studies have shown that RNA 7 codes for the nucleocapsid protein, RNA 6 codes for the E1 protein, RNA 3 codes for the E2 protein, and RNA 2 codes for a 35,000-dalton nonstructural protein. Genomic RNA directs the cell-free synthesis of three structurally related polypeptides of greater than 200,000 in molecular weight.     

Polypeptides encoded by polyadenylated and non-polyadenylated messenger RNAs from normal and heat shocked HeLa cells

There is an increased synthesis of proteins in the molecular weight region of 100,000 72,000-74,000 and 37,000 two hours after treatment of HeLa cells for 10 min at 45 degrees C. In vitro translation, using a rabbit reticulocyte cell-free protein synthesising system, of HeLa cell cytoplasmic RNA shows that the prominent 72,000-74,000 Mr heat shock protein band comprises seven polypeptide species (namely alpha d beta gamma delta epsilon zeta) and these polypeptides are directly encoded by both polyadenylated and nonpolyadenylated mRNA.