Characterization of a second bovine rotavirus serotype

Summary Bovine rotavirus (BRV) V 1005 was characterized by two-way cross-neutralization tests as a second serotype of BRV. Virions and inner shell particles of 65 nm and 55 nm diameter respectively, and empty capsids of 65 nm and 55 nm diameter were separated by density gradient centrifugation. Three polypeptides of molecular weight 60,000, 36,000 and 28,000 (minor protein) could be identified in the outer shell of virions and in the larger empty capsids. Inner shell particles contained three polypeptides of molecular weight 105,000, 83,000 and 43,000. Both sizes of empty capsids showed two polypeptides of molecular weight 75,000 and 55,000 not found in virions. Pulse-labelling of infected cells revealed eight major and three minor intracellular viral polypeptides. Viral polypeptide synthesis started at about 6 hours p.i. and correlated in time with double-stranded RNA synthesis. As soon as viral polypeptide synthesis was detectable, newly synthesized viral polypeptides were incorporated into intracellular viral particles. Radioactive viral polypeptides appeared without a longer lag period in extracellular viruses from 6 hours p.i. onwards.With 6 Figures     

Structural polypeptides of the hamster strain of equine herpes virus type 1: products associated with purification

Equine herpes virus purified from viremic serum of infected hamsters, either in sucrose or potassium tartrate gradients, can be further separated into an enveloped form (EF) and a membranous top component (MTC) by centrifugation in high salt-Tris-EDTA gradients. The polypeptides of sucrose purified virions, MTC, EF virions and nucleocapsids (purified in Renografin-76 density gradients) were analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EF virions were comprised of 28 polypeptides ranging in molecular weight from 276,000 to 16,000, 12 of them larger than 100,000. Sucrose purified virions and MTC were composed of a larger number of polypeptides, 50 and 38 respectively. Purified nucleocapsids contained five major structural proteins and six minor ones, these 11 nucleocapsid proteins ranged in molecular weight from 147,000 to 20,000.     

Synthesis of tacaribe viral proteins.

The synthesis of Tacaribe virus-specific proteins in infected BHK-21 cells has been analyzed by polyacrylamide slab gel electrophoresis and fluorography. The two major structural polypeptides of the virion were observed above the host cell background by pulse labeling with amino acid or sugar precursors. In addition to the virion glycoprotein (G), another major virus-specific glycoprotein (MW = 70,000) was detected in infected cells. The nucleoprotein (N) was first detected in infected cells around 24–34 hr postinfection (pi). It appeared to be synthesized as a primary gene product, since no larger precursor was detected by short pulse labeling. The rate of synthesis of N increased until 48 hr pi followed by a slight decrease by 72 hr pi. The structural and nonstructural glycoproteins were detected around 48 hr pi and their rate of synthesis increased up to 60 hr pi. The nucleocapsid protein, the minor nonglycosylated virion protein (P), as well as the structural and nonstructural glycoprotein could be specifically precipitated from infected cells using serum from rabbits hyperimmunized with Tacaribe virions. These results as well as pulse-chase experiments experiments suggest that the nonstructural glycoprotein may be a precursor of the virion glycoprotein.     

Structural proteins of HADEN virus

Purified HADEN virus was disrupted and separated into three polypeptide types in 7.5% neutral sodium dodecyl sulfate-polyacrylamide gels. The major polypeptide (HVP1) had a molecular weight of about 67,000 and accounted for 75–83% of the virion protein. The two minor components (HVP2 and HVP3) had molecular weights of about 77,000 and 85,500, respectively. When HADEN virus polypeptides were compared to the polypeptides of adenovirus-associated virus similar profiles were noted, but the polypeptides of the two viruses were distinguishable when coelectrophoresed on a single gel. Purified low density noninfectious HADEN particles were found to contain three polypeptides with the same molecular weights and concentrations as those found in the infectious virions.     

Defective interfering Tacaribe virus and persistently infected cells

Persistently infected (PI) BHK 21 and Vero cell lines were established by infection with wild-type (wt) Tacaribe virus and passage of surviving cells. These cells release defective interfering (DI) Tacaribe virus particles, which caused complete inhibition of plaque formation by wt Tacaribe virions. This interference activity was reduced when the DI virus was previously uv inactivated or treated with antiviral serum, and no interferon-like activity was detected. Cells pretreated with DI virus were only partially resistant to challenge with heterotypic arenaviruses, and no interference was detected with plaque formation by vesicular stomatitis virus. PI-BHK and PI-Vero cells contained Tacaribe virus-specific polypeptides as demonstrated by immunofluorescence, and by immunoprecipitation and analysis by SDS-polyacrylamide gel electrophoresis; the major virus-specific polypeptide observed was related to the N protein. PI-Vero cells were resistant to homologous superinfection and slightly resistant to superinfection by heterotypic arenaviruses, but supported normal plaque formation by vesicular stomatitis virus. Purified DI virus had a polypeptide profile similar to wt Tacaribe virions, although the DI virus released from PI-BHK cells had a nucleoprotein which migrated slower than the nucleoprotein of wt virions. The L and S segments of standard virions were not detected in DI particles from PI-BHK cells; but five discrete smaller RNA species were observed (MW ～ 1.1?0.2 × 10⁶).     

Polypeptides associated with inclusion bodies from leaves of turnip infected with cauliflower mosaic virus

Inclusion bodies, the major intracellular site of accumulation of cauliflower mosaic virus (CaMV), have been partially purified by centrifugation onto a saturated sucrose cushion, followed by detergent treatment to lyse chloroplasts, then centrifugation on a saturated sucrose cushion again. The resulting preparation was enriched for inclusion bodies as judged by light and electron microscopic observation and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides in the preparation. Two major polypeptides of 61,000 and 43,000 apparent molecular weights were found in inclusion body preparations from leaves infected separately with each of three different isolates of CaMV. Polypeptides of apparent molecular weights 56,000, 50,000, 39,000, 37,000, and 34,000 were also detected in preparations from infected leaves, but not in comparable preparations from healthy leaves. The mobilities of all but the 61,000 and 34,000 molecular weight polypeptides were slightly different from the CM4-184 isolate, as compared with Cabbage B Davis or NY8153. The 61,000 molecular weight polypeptide did not correspond in mobility to any of the virion polypeptides. The relative intensities of polypeptide bands common to inclusion bodies and isolated virions were different in these two preparations, suggesting that considerable degradation took place during virion isolation.     

Biochemical characterization of Rift Valley fever virus

Rift Valley fever virus (RVFV) polypeptides were shown to share similar biochemical properties with members of the family Bunyaviradae. Electrophoretic analysis of RVFV revealed one nonglycosylated and two glycosylated major proteins with molecular weights of 25,000, 56,000, and 65,000, respectively. In addition, a 100,000 MW glycoprotein was found. The 25,000 MW protein was identified as the major nucleocapsid protein. The virion density in CsCl was 1.21 g/ml, while that of the nucleocapsid was 1.29 g/ml. The number and molecular weights of major structural polypeptides of several diverse RVFV isolates were identical. The presence of three RNA segments, large, medium, and small, with molecular weights of 2.7, 1.7, and 0.6 × 10⁶, was demonstrated. The close relationships of RVFV proteins and RNA with reported molecular weights of some members of the Phlebotomus fever (PHL) group viruses were compatible with the serological cross-reactivity among these viruses. These findings support the classification of RVFV with the PHL group viruses in the family Bunyaviradae.     

The structural polypeptides of RNA slow viruses

The structural polypeptides of the RNA slow viruses Visna, Maedi, Progressive Pneumonia Virus, and Zwoegerziekte have been determined by electrophoresis in polyacrylamide gels in a discontinuous SDS system. Identical patterns were obtained of 15 polypeptides of molecular weight ranging from 14,000 to 140,000 daltons. Most of the mass of protein is in three small molecular weight polypeptides; the major polypeptide species has a molecular weight of 25,000. A distinctive glycopeptide of molecular weight 140,000 is present in each virus; three additional glycopeptides were found, one of which is barely detected in radiolabeling experiments. There is one phosphorylated polypeptide. In a comparative study of RNA slow viruses and tumor viruses, 9 of 15 polypeptides of visna virus comigrated with 13 polypeptides of Rous sarcoma virus including the major 25,000 MW polypeptide of each virion. In addition, the isoelectric point of visna was 3.8 in conformity to the isoelectric point reported for Rous sarcoma virus. Distinctive small molecular weight polypeptides and a totally different pattern of glycosylation constitute the main differences of the polypeptides of these two groups of viruses. Extensive studies of the purification procedures for C-type viruses were carried out and criteria for virus purity delineated. The expectations for the relationship of the structural polypeptides to antigenic determinants are discussed.     

High resolution two-dimensional gel electrophoresis of structural proteins of baculoviruses of Autographa californica and Porthetria (lymantria) dispar

The structural polypeptides of baculoviruses of Autographa californica (AcMNPV) and Porthetria dispar (PdMNPV) were analyzed by two-dimensional (2-D) gel electrophoresis. Purified proteins were solubilized in urea-NP40 mix and separated by isoelectric focusing in the first dimension; electrophoresis in the presence of sodium dodecyl sulfate (SDS) separated proteins by molecular weight in the second dimension. Eighty-one acidic polypeptides ranging in molecular weight from 13,500 to 86,000 Da were resolved in AcMNPV enveloped virions. The predominant polypeptide had a molecular weight of 41,500 and was considered to be the major capsid protein. Nucleocapsids from AcMNPV were resolved into 64 polypeptides. At least 11 of the polypeptides, including most of the high molecular weight proteins, that were not resolved in nucleocapsids were considered to be envelope proteins. For PdMNPV enveloped virions, there were 95 acidic polypeptides ranging in molecular weight from 13,500 to 85,500. The predominant polypeptide had a molecular weight of 46,500 Da. Polyhedral proteins (polyhedrin) isolated from protease-inactivated polyhedra and separable into a single major polypeptide (approx. 31,000) on one-dimensional SDS-polyacrylamide gel electrophoresis were resolved into six polypeptides for both viruses. All six polyhedrin polypeptides had the same molecular weight, but their isoelectric points ranged from pH 5.3 to 5.9 for AcMNPV and from pH 5.7 to 6.2 for PdMNPV. These six polypeptides were also detected when protease-inactivated or noninactivated whole polyhedra were analyzed directly by 2-D electrophoresis. It is assumed that not all the observed baculovirus polypeptides were unique species. Some proteins, especially the polyhedrin polypeptides, appeared to be related and had altered mobilities as a consequence of post-translational modifications.     

Japanese encephalitis virus glycoproteins

Mature Japanese encephalitis (JE) virus, or N-form virus, contained three structural proteins: V-1, V-2, and V-3. The large membrane protein V-3 was glycosylated, whereas both V-1 (the small membrane protein) and V-2 (the nucleocapsid protein) were not. Intracellular (I-form), immature virions from infected chick embryo cells did not contain V-1 but a larger protein NV-2, which was glycosylated. T-form virions, released by LLC-MK₂ cells incubated with tris(hydroxymethyl)amino-methane (Tris), also contained the glycoprotein NV-2 instead of the nonglycosylated and smaller V-1. We therefore concluded that JE contained two structural membrane glycoproteins, at least one of which is modified during morphogenesis. The NV-2 polypeptide was heterogeneous, and slight differences in electrophoretic mobility were detected among the NV-2 polypeptide peaks from glucosamine-labeled I-form and T-form virions, glucosamine-labeled cell extracts, and amino acid-labeled cell extracts. The significance of these differences is not clear, but they may indicate that NV-2 is composed of several proteins of similar molecular weight. By analyzing extracts of infected cells labeled with glucosamine or amino acids, we tentatively classified the intracellular polypeptide NV-3 as a virus-specified nonstructural glycoprotein; this polypeptide may be a proteolytic fragment of V-3. The virus-specified polypeptides NV-5, NV-4, and NV-1 were classified as nonglycosylated, nonstructural proteins.     

Characterization of the structural proteins of hemorrhagic enteritis virus

Summary The structural proteins of hemorrhagic enteritis (HEV), a turkey adenovirus, were analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using polyspecific, monospecific and monoclonal antibodies for detection. In purified HEV preparations, eleven polypeptides with apparent molecular weights ranging from 96,000 to 9,500 (96k to 9.5k), were specifically recognized by convalescent turkey serum. Six of these polypeptides were further characterized by PAGE, Western blotting, ELISA, sucrose gradient centrifugation and electron microscopy. The 96k polypeptide was identified as the hexon polypeptide which is a monomer of the major outer capsid or hexon protein. The 51/52k and 29k polypeptides, identified as the penton base and fiber polypeptides respectively, were the components of the vertex or penton protein. The 57k polypeptide was identified as a homologue of the human adenovirus type 2 (Ad 2) IIIa protein with which it shares a common epitope. Two core proteins with molecular weights of 12.5 and 9.5k were present in purified HEV nucleoprotein cores. The proteins of two HEV isolates, one apathogenic (HEV-A) and one virulent (HEV-V), resembled each other in most respects. However, differences between HEV-A and HEV-V were found in electrophoretic migration of the penton base protein both under native and denatured conditions, and in the electrophoretic migration of the 43/44k polypeptide. Moreover, homologous antiserum against the fiber protein reacted stronger than heterologous antiserum in an ELISA. Single fibers were detected by electron microscopy attached to the penton base proteins of HEV virions and in isolated pentons. The feature of having single fibers is shared with the mammalian adenoviruses and the avian egg drop syndrome 1976 virus (EDS 76 V), but not with the fowl adenoviruses which have double fibers attached to their penton base proteins.     

Machupo virus polypeptides: Identification by immunoprecipitation

Summary The most abundant protein in purified Machupo virions (Corvallo strain) labelled with¹⁴C-Protein hydrolysate is a 64K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78K polypeptide and a minor 50K polypeptide have been detected.In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64K, NP), and the others were glycosylated polypeptides (78K and 37K). The synthesis of NP and 78K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78K is a precursor for structural glycoproteins of Machupo virions.With 4 Figures     

Structural polypeptides of Machupo virus.

The structural proteins of an arenavirus pathogen, Machupo virus, were compared to the structural proteins of two previously characterized non-pathogenic arenaviruses, Pichinde and Tacaribe, in SDS-polyacrylamide gels. Similarities in mol. wt. of the major structural proteins from both pathogenic and non-pathogenic viruses were apparent; however, some differences in the number of glycosylation properties of minor proteins were observed. Machupo virions contain two major protein species. The most prominent is a non-glycosylated protein with a mol. wt. of 68000, while the other was glycosylated protein with a mol. wt. of 41000. Minor amounts of other proteins (mol. wt. 84000, 74000, 50000 and 15000) and a glycolipid were also observed.     

Characterization of structural and non-structural proteins of hog cholera virus by means of monoclonal antibodies

Summary A panel of 15 monoclonal antibodies, produced against the hog cholera virus, were characterized by radioimmunoprecipitation assays. Using this panel, we were able to identify 4 sets of monoclonal antibodies precipitating each a different viral protein with relative molecular weight of 40, 46, 120 kDa, respectively, and a protein complex containing 15, 16, 27, and 55 kDa polypeptides which were further characterized. One monoclonal antibody recognized an antigenic determinant at the C-terminal cleavage product of the non-structural p 125 of BVDV. The 40 kDa protein was precipitated from the pelleted virions, indicating its structural importance. On the contrary the 46 kDa protein could only be precipitated from the cell lysate and not from the pelleted virions. The glycosylated 15/16 kDa-55 kDa proteins form a disulfide linked heterodimer on the virus particle with a relative molecular weight of 65 kDa.     

Identification of the structural proteins of turkey enteric coronavirus

Summary Coronaviruses in the intestinal contents of turkey poults from Quebec flocks with outbreaks of enteritis were detected by electron microscopy. Five isolates were serially propagated in embryonic turkey eggs by inoculation of clarified intestinal contents into the amniotic cavity. Viral particles contained in the intestinal contents of infected embryos were purified by differential and isopycnic centrifugation in sucrose gradients. Intact virions present in fractions of densities 1.18 to 1.20 g/ml were precipitated with trichloroacetic acid and the protein content was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least seven polypeptides with molecular weights ranging from 27,000 to 180,000 were regularly detected after electrophoresis of SDS-solubilized virions. Homologous rabbit antisera to turkey coronaviruses and pooled sera from convalescent turkeys immunochemically stained five polypeptides of apparent molecular weights of 95,000, 72–75,000, 66,000, 52,000 and 27,000. A further polypeptide with a molecular weight of 140,000 appeared in the absence of 2-mercaptoethanol and probably represents a dimer of the 66,000 polypeptide. One Quebec isolate differed from the Minnesota strain by two of its polypeptides, but no antigenic variations could be demonstrated amongst the various isolates either by immuno-electron microscopy, hemagglutination inhibition, or enzyme immuno assays on nitrocellulose.This study was partly supported by the Medical Research Council of Canada and the Quebec Federation of Poultry Producers.     

The group-specific antigen and other structural proteins of hamster and mouse C-type viruses

The major group-specific antigen of hamster C-type viruses was purified by isoelectric focusing and used to prepare specific antibody in guinea pigs. The isoelectric point of this antigen was 6.9. The patterns of hamster and mouse virus polypeptides in SDS-polyacrylamide gels were similar and showed three major components ranging from 13,000 to 35,000 in molecular weight. The group-specific protein was eluted from SDS-polyacrylamide gels in antigenically active form and was found to correspond to the third fastest migrating component for each virus; molecular weight calculations relative to standards gave average values of 31,000 for the mouse protein and 35,000 for the hamster protein. These slight differences were maintained in coelectrophoresis experiments as were the differences for the second fastest migrating component, 17,500 for the mouse virus, 23,000 for the hamster virus. The fastest migrating polypeptides had molecular weights of 13,500–14,000 for each virus. The major gs antigens of hamster and mouse viruses did not cross-react in complement-fixation and gel diffusion tests when tested with their respective monospecific guinea pig antisera.     

In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses

The messenger RNAs of SV5 and two strains of Sendai virus were isolated from infected cells and translated in a wheat germ cell-free system. Comparison of the peptide maps of the polypeptides synthesizedin vivo andin vitro established that the nonglycosylated polypeptides P, NP, and M of both SV5 and Sendai virus had been synthesizedin vitro. In immunoprecipitation studies of the putative SV5 polypeptides synthesizedin vitro, antiserum against whole virions precipitated NP, P, and M, and other polypeptides which did not comigrate with mature virion polypeptides. Monospecific antisera against the HN and F glycoproteins precipitated two of the latter polypeptides with molecular weights of ～55,000 and 50,000, respectively, suggesting that the nonglycosylated forms of these polypeptides had been synthesizedin vitro. Polypeptide C, previously found in Sendai virus-infected cells and proposed to be a virus-specific nonstructural polypeptide, has been found to exhibit strain-specific differences in migration in polyacrylamide gels. Polypeptides with the appropriate strain-specific migration have been synthesizedin vitro with mRNAs from cells infected with the different Sendai virus strains, and shown by peptide mapping to be the same polypeptides as those synthesizedin vivo. The results have thus provided further evidence that C is a virus-coded nonstructural protein. Another polypeptide (C′), with migrates slightly slower than C, has been found in infected cells and synthesizedin vitro. This polypeptide also exhibits strain-specific differences in migration in polyacrylamide gel electrophoresis, and has been shown by peptide mapping to be similar to C. The explanation for the difference in migration between C and C′ is as yet unknown.     

Characterisation of murine pneumonia virus proteins.

Twelve [³⁵S]methionine-labelled polypeptides ranging in molecular weight from 12,000 to 191,000, which were not present in mock-infected BS-C-1 cells, were detected in cells infected with murine pneumonia virus (PVM). All but two of these polypeptides were present in cell-released virus concentrated by polyethylene glycol precipitation and partially purified by equilibrium centrifugation. Two major glycopolypeptides, VPII (79,000 mw) and VPIII (68,000 mw) were present in partially purified virus but only VPII was prominent intracellularly. Nucleocapsids were isolated from lysates of infected cells by centrifugation in a gradient of 15–50% metrizamide and contained a major (VPIV) and a minor (VPVI) polypeptide with molecular weights of 42,300 and 35,700, respectively. Polypeptides with the electrophoretic mobilities of six of the PVM polypeptides (VPIV, VPV, VPVII, VPIX, VPX, and VPXI) were detected by in vitro translation of cytoplasmic RNA from PVM-infected cells in a cell-free system prepared from rabbit reticulocytes. PVM resembled RS virus, the other member of the pneumovirus genus, in its ability to multiply in enucleate cells. It is distinguished, however, by its four low molecular weight polypeptides (VPIX, VPX, VPXI, and VPXII), which appear to be primary gene products, and by the different electrophoretic mobility of the glycopolypeptides (VPII and VPIII).     

Mechanism of endotoxin-type interferon production in mice

Encephalomyocarditis (EMC) virus, human rhinovirus-1A (HRV-1A), and poliovirus are each representatives of a different, major subgroup of the picornaviruses. In this comparative study the patterns of biosynthesis of the virus-specific polypeptides of these three viruses were examined in detail. The molecular weight and molar ratio of each polypeptide and the genetic map of each virus were determined, and the kinetics of cleavage of the analogous precursor polypeptides were contrasted. Previous work has shown that there are three main families of EMC virus-specific polypeptides. Translation of the EMC viral ribonucleic acid generates at least three primary products, polypeptides A, F, and C. Polypeptide A undergoes successive cleavages to produce the four major capsid polypeptides. Polypeptide F is stable. Polypeptide C cleaves to form polypeptide D, which in turn cleaves to form polypeptide E. Evolutionary pressures on these viruses have resulted in extensive differences in the antigenicity of the virions. In addition, it was found that each profile had polypeptides that were unique to that particular virus, and the kinetic studies revealed that analogous cleavages proceeded at extensively different rates. However, as judged by these studies with EMC virus, HRV-1A, and poliovirus, the general mechanism of synthesis, size, and genetic order of the three main families of polypeptides remain the same.