Substructures and Polypeptides of Visna Virus

The protein of Visna virus, disrupted by 8 M guanidine hydrochloride and heating, was resolved into 10 polypeptides by agarose gel column chromatography in 6 M guanidine hydrochloride. Two of the peaks contained glycopolypeptides. Nonidet-disrupted virions were resolved into two fractions by potassium tartrate gradient centrifugation, with densities of 1.08 and 1.24 g/ml, respectively. About 70% of the viral DNA polymerase directed by added template was released into the light fraction, in which very little endogenous enzyme activity was detected. Also released into the light fraction were all of the glycopolypeptides, 50% of the viral RNA, and a part of each of the other viral protein components. The data indicate that extensive degradation of subviral structures occurred, even under mild conditions for virion disruption. The 1.24-g/ml fraction was composed of 50% of the viral RNA, most of the endogenous DNA polymerase activity (80%), and a major internal polypeptide (GuHCl6) with an estimated mol wt of 28,000. Two other polypeptides were also consistently detected in the heavy fraction, but they constituted less than 25% of the ribonucleoprotein complex, compared with 75% for GuHCl6.     

Characterization of supercoiled nucleoprotein complexes released from detergent-treated vaccinia virions.

Treatment of vaccinia virions with 1% sodium dodecyl sulfate in the absence of reducing agents resulted in the release of subviral particles termed "subnucleoids," which contained viral DNA in combination with four polypeptides with molecular weights of 90,000, 68,000, 58,000 and 10,000. Biochemical and electron microscopic studies showed that viral DNA in combination with these polypeptides was maintained in a superhelical configuration. When subnucleoids were "fixed" with glutaraldehyde and formaldehyde and then examined by electron microscopy, spherical particles were observed, in which the supercoiled DNA was folded into globular structures that were 20 to 60 nm in diameter and were interconnected by DNA-protein fibers resembling the nucleosome structures described for eucaryotic chromatin.     

Influenza virus proteins: preparation of a soluble M1 polypeptide by means of a stepwise deproteination of virions

Layer by layer uncoating of influenza A and B viruses with non-ionic detergent (NP-40) at fixed pH was developed. Treatment of virions with NP-40 at neutral or alkaline pH solubilized the lipoprotein envelope and the surface glycopolypeptides HA1 and HA2, but the internal core structures containing matrix protein M1 remained. Exposition of the cores in acidic media (pH 4,5 and lower) selectively solubilized protein M1 and released viral ribonucleoprotein (RNP). The resulting M1 sedimented in a glycerol gradient with a coefficient of 2.8 S and most probably exists as a monomer of 27,000 Da polypeptide. Neutralization of protein M1 with Tris-HC1 at pH 7.0 did not cause aggregation of M1 polypeptides. The described method of viron layer by layer uncoating with non-ionic detergent at fixed pH is suitable for isolation of subvirus structures and individual viral proteins.     

DNA-Dependent RNA Polymerase Activity Associated with Subviral Particles of Polyhedral Cytoplasmic Dexoyribovirus

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.     

SDS-acrylamide gel electrophoresis and its application to the proteins of poliovirus- and adenovirus-infected human cells

Sensitive techniques for acrylamide gel electrophoretic analysis have been applied to animal virus systems and have proven generally useful. Estimates of the number of kinds, molecular weights and number of molecules of proteins in almost any biological sample have been made with ease. As applied to the poliovirus-HeLa cell system they reveal four major proteins in the virion and at least ten additional proteins in the infected cell. Some of the intracellular and particulate proteins undergo cleavage reactions following a unique translation in which the genome is apparently translated in toto as one large polypeptide of molecular weight greater than 200,000 daltons. The splits occur at three levels: (a) during synthesis; (b) at intermediate stages; and (c) co-incident with maturation. In vitro studies on protein synthesis, RNA synthesis and virus assembly have substantiated and extended the in vivo observations. The structure of the adenovirion has been established in detail. Hexon, penton base, fiber and core polypeptides and certain relevant subviral structures have been identified. Nearly all of the proteins synthesized in the infected cells after 20 hours are viral. The major structural antigens (hexon and penton) predominate and are made in 10 to 50 fold excess but the internal core polypeptides are not produced in great excess. Studies on the synthesis of polypeptides and their assembly into morphological subunits and virions show that hexon and penton polypeptides are made in about four and two minutes respectively on cytoplasmic polyribosomes, that morphological subunits are formed within five minutes of synthesis of protein, and that there is a delay of greater than one half hour for entry of hexons into virions.     

Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

Iodination of reovirus, cytoplasmic polyhedrosis virus (CPV), and wound tumor virus (WTV), and their respective subviral forms, followed by analysis of the labeled polypeptides by using polyacrylamide gel electrophoresis, has been used to compare the protein contents of these three diplornaviruses. This approach, when combined with electron microscopy and buoyant density determinations, appears capable of localizing individual polypeptides in some of the viral and subviral forms. CPV (p = 1.435 g/cm(3)) seems to resemble reovirus cores (p = 1.440 g/cm(3)) in both ultrastructure and polypeptide composition. CPV is composed of five polypeptides with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000. The polyhedral matrix, which in nature encapsulates the virions, is, in turn, composed mainly of two polypeptide species with molecular weights of about 30,000 and 20,000, and several minor proteins. The proteins of WTV consist mainly of four species of polypeptide with molecular weights of about 156,000, 122,000, 63,000, and 44,000, and several minor components. These molecular weight determinations are consistent with the hypothesis that, as has been suggested for reovirus, the viral proteins of CPV and WTV seem to be coded for by monocistronic mes senger RNA molecules transcribed from distinct segments of the double-stranded RNA viral genomes.     

Protein composition of the chromosomal scaffold and interphase nuclear matrix

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.     

Time Course of Synthesis and Assembly of Influenza Virus Proteins

The synthesis of viral polypeptides was analyzed in BHK-21-F cells infected with the WSN strain of influenza virus at various times in the growth cycle. The relative amounts of polypeptides P, HA, NP, and NS did not change markedly between early and late times in the growth cycle; however, there was a progressive increase in the relative amount of the M polypeptide at later time points. In cell fractionation experiments, the patterns of newly synthesized polypeptides associated with various cytoplasmic fractions remained similar throughout the growth cycle except for an increase in polypeptide M in all fractions late in the growth cycle. The HA polypeptide was chased out of cytoplasmic membranes completely 6 h after synthesis, whereas the M polypeptide was not chased effectively from such membranes. Marked differences were found in the incorporation into mature virions of polypeptides synthesized at different times in the growth cycle. Polypeptides P and NP synthesized at early times were incorporated preferentially, whereas M was synthesized and incorporated predominantly late in the growth cycle. The fact that the rates of incorporation of polypeptides into virions differed significantly from their rates of synthesis indicates that different polypeptides were assembled into virions by distinct pathways.     

Intermediates in adenovirus assembly.

Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII.     

Subcellular fractions and the refringent granules of the spermatozoa of Ascaris suum (Nematoda)

Summary Six subcellular fractions were isolated by differential centrifugation of the homogenate of spermatozoa of Ascaris suum. The cellular constituents of pelleted fractions, as identified by electron microscopy, were membranes and membranous organelles (fraction A1), microsomal (A2), cytoplasmic (A3), large refringent granules (B1), small refringent granules (B2) and a detergent-soluble fraction (B3).Polypeptide analysis by SDS-PAGE showed that the 18,400-dalton band, one of the major spermatozoan proteins, is detectable in all of the fractions. However, the cytoplasmic (A1) and refringent-granule (B1) fractions contained the highest level.The isolated refringent granules consisted of 2–6 % lipid while the nonlipid fraction formed an insoluble matrix with a fibrillar network morphology. This fibrillar matrix contained three polypeptides of small molecular weight (7,000–14,000) in addition to the 18,400-dalton polypeptide. These small polypeptides (7,000–14,000 MW) are detectable only in fractions of the refringent granules and are therefore called the refringent-granule proteins (RGP). These RGP are sensitive to tryptic hydrolysis and have solubility properties similar to the protein, ascaridine.Adult Ascaris suum were generously provided by Wilson and Company, Cedar Rapids, Iowa. This study was supported by postdoctoral fellowship 5F32AI05646 from NIH. The assistance of Mr. Douglas Wood is gratefully acknowledged     

Quantitation of the glycoprotein spike area on the surface of enveloped viruses

The density of glycoprotein (GP) distribution on the virion surface substantially influences the virus infectivity and pathogenicity. A method to quantitatively determine the area occupied by surface GP spikes was proposed for influenza virus (Flu) strain A/PR/8/34 on the basis of data of tritium bombardment and dynamic light scattering. The latter was used to measure the diameter of intact virions and subviral particles (Flu virions lacking GP spikes after bromelain digestion). Intact virions and subviral particles were bombarded with a hot tritium atom flux, and the specific radioactivity of the matrix M1 protein was analyzed. The tritium label was incorporated into the amino acid residues of a thin exposed protein layer and partly penetrated through the lipid bilayer of the viral envelope, labeling M1, located under the lipid bilayer. The tritium label distribution among different amino acid residues was the same in M1 isolated from subviral particles and M1 isolated from intact virions, demonstrating that the M1 spatial structure remained unchanged during proteolysis of GP spikes. The difference in specific radioactivity between the M1 proteins isolated from intact virions and subviral particles was used to calculate the GP-free portion of the viral surface. Approximating the Flu virion as a sphere, the GP-covered area was estimated at 1.4 × 10⁴ nm², about 40% of the total virion surface. This was consistent with the cryoelectron tomography data published for Flu strain A/X-31. The approach can be applied for other enveloped high pathogenic viruses, such as HIV and the Ebola virus.     

Mutants of Sindbis virus. III. Host polypeptides present in purified HR and ts103 virus particles.

The amounts of host-encoded protein present in purified Sindbis virions of both the HR strain and of a mutant (ts103) which makes multicored particles were examined. Cells were labeled with [35S]methionine before infection and with [3H]methionine postinfection. Virions were purified by velocity sedimentation and isopycnic banding, and their polypeptides were examined by polyacrylamide gels in a sodium dodecyl sulfate-containing discontinuous buffer system. Host prelabeled material was found principally in a small number of discrete polypeptides in HR virions, which contained as little as 0.2% host-encoded protein. Virus-sized particles of mutant ts103 contained significantly more host material, and multiploid particles from ts103 infection contained up to 12% host prelabeled protein.     

Structure of adenovirus chromatin

The structure of adenovirus type 2 chromatin isolated from wild-type and ts1 virions was investigated by micrococcal nuclease digestion and electron microscopy. Partial digestion of wild-type and ts1 chromatin with micrococcal nuclease generated a multimeric DNA smear devoid of the 200 basepair nucleosome repeating pattern characteristic of cellular chromatin. However, 11 S monomer cores of 150 basepairs were detectable. The chromatin of ts1 (39°C) was more resistant to digestion by micrococcal nuclease. Two-dimensional electrophoresis of the monomer core showed that wild-type core contained protein VII while ts1 (39°C) core contained PVI and PVII. Protein V appears to be located on the variable-length intermonomer region. Crosslinking studies suggest that proteins PVII and VII exist in dimeric form within the monomer core. Electron microscopy revealed a 5.5-fold-condensed two-micron-long beaded structure with about 200 monomer particles spaced irregularly. Based on these observations, a model for adenovirus prochromatin and chromatin is proposed that differs in important aspects from the model proposed previously (Corden, J., Engelking, H.M. and Pearson, G.D. (1976) Proc. Natl. Acad. Sci. USA 73, 401–404).     

Entry of adenovirus 2 into HeLa cells

Adenovirus 2 (Ad2) uncoating was analyzed as the destabilization of virions which renders the parental genome sensitive to DNase treatment. This event demonstrated a strong temperature dependence, and an Arrhenius plot of initial uncoating rates revealed an inflection point at around 16 degrees C. Activation energies of 331 kJ/mol below and 88 kJ/mol above this temperature were obtained for the uncoating process. Penetration of Ad2 through the plasma membrane was completely inhibited by sodium azide, whereas uncoating was only slightly influenced. This indicated that uncoating had already taken place at the outside of the plasma membrane. Incubations of Ad2 with isolated plasma membranes and cell homogenates showed that intact and metabolizing cells were required for uncoating. We further suggest, based on the inhibitory patterns of EDTA, EGTA, dansylcadaverine, and dithiothreitol, that this destabilization of virions follows upon reorganization in the plasma membrane. In the electron microscope the involvement of coated vesicles was shown for the initial uptake of virions, possibly followed by the engagement of acidic vesicles as judged from the effects of lysosomotropic agents on gene expression. The vectorial transport of virions from the plasma membrane to the nucleus was not affected by reagents interfering with the cytoskeletal system. Consequently, we propose that Ad2 virions are internalized by adsorptive endocytosis.     

The herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells.

Earlier studies have shown that the UL11 gene of herpes simplex virus encodes a myristylated virion protein and that the UL11 gene enables efficient virion envelopment and export from infected cells. A rabbit polyclonal antibody directed against an affinity-purified UL11-glutathione-S-transferase fusion protein was made and used to study the properties of the UL11 protein and its distribution in infected cells. We report the following: (i) UL11 protein formed up to five bands (apparent M(r)s, 17,000 to 22,000) in denaturing polyacrylamide gels; (ii) fluorescent-antibody studies revealed the presence of UL11 protein in the perinuclear space and in sites within the nucleus; (iii) immune electron microscopic studies indicated that the UL11 gene products were associated with the inner nuclear membrane, with cytoplasmic membranes and ribbon-like cytoplasmic structures resembling membranous organelles, with nuclear bodies shown by fluorescence microscopy to be different from nucleoli in which US11 protein accumulates, and with enveloped virions but not with nuclear capsids; and (iv) the nuclear bodies containing UL11 protein were reminiscent both of type IV morphotypes consisting of an electron-dense core containing the UL11 proteins surrounded by a more electron-transluscent core and of type V morphotypes consisting of material homogenous in electron opacity. We conclude that (i) the UL11 protein is processed after synthesis; (ii) the localization of UL11 protein with virions and membranes is consistent with the hypothesis that UL11 plays a role in the transport of virions to the extracellular space; and (iii) although the significance of the association of UL11 proteins with nuclear bodies is unknown, the results indicate that nuclear bodies differ with respect to their morphologies and contents of viral protein and suggest that UL11 protein may have more than one function in the infected cell.     

Characterization of two cell-envelope fractions from chemotrophically grownRhodospirillum rubrum

Two cell-envelope fractions were isolated from chemotrophically grown cells ofRhodospirillum rubrum. On the basis of electron-microscopic investigations, chemical analysis, distribution of components involved in respiration, and polyacrylamide gel electrophoresis, the heavy fraction (ρ²⁰=1.246 g per cm³) was identified as cell-wall, and the light fraction (ρ²⁰=1.145 g per cm³) as cytoplasmic-membrane fragments. Electron micrographs showed cell-wall fragments as open structures while cytoplasmic-membrane preparations were composed of closed membrane vesicles. With respect to the main classes of chemical compounds, cell wall could be distinguished from cytoplasmic membranes by a rather low ratio of phospholipids per protein and a high ratio of carbohydrates per protein. The relative proportion of individual neutral sugars as well as phospholipids (except for lysophosphatidyl ethanolamine) revealed no significant differences between both envelope fractions. Fatty acid analysis demonstrated a higher proportion of saturated fatty acids in cell-wall than in cytoplasmic-membrane fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions showed distinct protein compositions. While in cell-wall preparations polypeptides of 43000 and 14000 daltons predominated, 56000- and 52000-dalton polypeptides were the main protein subunits of cytoplasmic membranes. Cross contaminations of both cell-envelope fractions were defined.     

Phosphorylation of Animal Virus Proteins by a Virion Protein Kinase

Compared with several other enveloped viruses, purified virions of frog virus 3 contained a relatively high activity of a protein kinase which catalyzed the phosphorylation of endogenous polypeptides or added substrate proteins. Virions also contained a phosphoprotein phosphatase activity which released phosphate covalently linked to proteins. It was possible to select reaction conditions where turnover of protein phosphoesters was minimal, as the phosphatase required Mn(2+) ions for activity whereas the protein kinase was active in the presence of Mg(2+) ions. Electrophoretic studies in polyacrylamide gels containing sodium dodecyl sulfate indicated that at least 10 of the virion polypeptides were phosphorylated in the in vitro protein kinase reaction. Characterization of these phosphoproteins demonstrated that the phosphate was incorporated predominantly in a phosphoester linkage with serine residues. The protein kinase was solubilized by disrupting purified virions with a nonionic detergent in a high-ionic-strength buffer and was separated from many of the virion substrate proteins by zonal centrifugation in glycerol gradients. The partially purified protein kinase would phosphorylate polypeptides of many different animal viruses, and maximal activity was not dependent on added cyclic nucleotides. These properties distinguished the virion protein kinase from a well characterized cyclic AMP-dependent protein kinase which phosphorylated viral proteins only to a small extent.