Synthesis of glycoproteins in cells infected by the flavivirus Kunjin

The synthesis of glycoproteins in Vero cells infected by Kunjin virus was studied by labeling infected cells with radioactive mannose labeled glycoproteins by gel electrophoresis. In infected cells glycoproteins with approximate molecular weights of 53,000 (gp53), 59,000 (gp59), and 66,000 (gp66) were observed. Limited proteolytic digestion of gp53, gp59, and gp66 demonstrated that gp53 and gp59 contain similar amino acid sequences, and are probably related to gp66. Antiserum raised against the envelope protein V3 of purified virions selectively precipitated gp53, gp59, and the ³H-amino acid-labeled labeled protein of 51,000 daltons (p51), from extracts of infected cells; p51 is known to be almost identical with V3 by tryptic peptide mapping. Thus p51, gp53, gp59, and probably gp66 are intracellular forms of the envelope protein V3 of Kunjin virus. When infected cells were labeled with [³H]mannose for short time periods, gp59 was labeled within 5 min, gp66 within 10 min and gp53 within 20 min. After pulse labeling of infected cells with [³H]mannose followed by incubation in medium containing excess glucose, label was lost from gp66 and accumulated in gp53, demonstrating a possible precursor-product relationship between gp66 and gp53. The size of the glycopeptides derived from gp53, gp59, and gp66 by ehaustive digestion with Pronase were identical.     

Characterization of hog cholera virus II. Determination of sedimentation coefficient

Summary The sedimentation coefficient of hog cholera virus was determined by ultracentrifugation of unpurified and unconcentrated tissue culture virus material using a Spinco preparative ultracentrifuge and following the technique ofPolson andVan Regenmortel. The mean value calculated from the experiments was 108±24 Svedberg units. From these data, a particle size for the hog cholera virion of 28 to 39 nm. was estimated.     

Solubilized RNA replication complex from Semliki Forest virus-infected cells

The postmitochondrial pellet from SFV-infected cells was incubated under in vitro RNA-synthesizing conditions in the presence of [³H]UTP to label nascent chains in the RNA replication complex. The solubilized complex was purified by centrifugation in a glycerol gradient followed by a sucrose gradient in which it sedimented with a peak at 34 S. It contained double-stranded RNA which by mild ribonuclease treatment was converted to replicative forms I, II, and III, indicating that it was derived from replicative intermediates synthesizing 42 S and 26 S RNA. A similar structure was also isolated from infected cells labeled with [³H]uridine early in infection. The only virus-specific proteins associated with the complex were ns70, ns72, and ns86. The presence of ns70 and ns72 was confirmed by tryptic peptide mapping.     

The functions of oligosaccharide chains associated with influenza C viral glycoproteins

Summary The effect of a glycosylation inhibitor, tunicamycin (TM) on the replication of influenza C virus was investigated. Incorporation of [³H]-glucosamine into the gp88 glycoproteins of this virus was completely inhibited by TM at the concentrations higher than 0.25 µg/ml. Under these conditions, the synthesis of internal proteins NP and M was shown in TM-treated cells but the synthesis of gp88 was not. The disappearance of gp88 was however accompanied with the appearance of two new polypeptides with molecular weights of 80,000 (T80) and 76,000 (T76). While T80 was identified by peptide mapping as a host cell protein whose synthesis was enhanced by TM, T76 was shown to correspond to a nonglycosylated form of gp88. Pulse-chase experiments revealed that there was no significant difference in the intracellular stability of T76 and gp88. Although TM depressed the production of infectious progeny virus greater than 100-fold, only a five-fold decrease was observed in the release of noninfectious physical particles, suggesting that glycosylation is not essential for the formation of influenza C virus particles. However, the virions from TM-treated cells had a lower buoyant density in isopycnic sucrose gradients and lacked surface proteins in either glycosylated or nonglycosylated form.With 7 FiguresPresented in part at the international meeting of influenza virus hemagglutinin, Miki, Japan (September 7–9, 1984).     

Preliminary characterization of glycopeptides derived from glycoproteins specified by the Flavivirus Kunjin

The oligosaccharide components of the glycoproteins observed in Vero cells infected by the flavivirus Kunjin were separated by gel filtration following exhaustive Pronase digestion of radioactively labeled glycoproteins. The glycopeptides of gp59, gp56, gp53, and NV2 fell into four classes based on size: (i) 3600 to 4200 daltons, (ii) 2800 to 3100 daltons, (iii) 2300 to 2500 daltons, and (iv) 1500 to 1700 daltons. Glycopeptides of the first class were detected in gp59 and gp56, of the second in gp59, gp56, and gp53, and of the third and fourth classes in gp59, gp53, and NV2. Complex oligosaccharides containing fucose, galactose, mannose, and glucosamine were found in the first two classes and possibly in the third. Oligosaccharides in the smallest class were labeled with [³H]mannose and [³H]glucosamine only.     

Improved production of Epstein-Barr virus DNA for nucleic acid hybridization studies.

Large quantities of Epstein-Barr virus (EBV) DNA were prepared by superinfection of Raji cells with EBV. Virus DNA thus prepared showed a single symmetrical peak at 1.718 g/cm³ in CsCl analytical centrifugation and served as a good template for preparation of cRNA specific to EBV DNA. The virus DNA was also highly labeled in vivo with [³H]thymidine or ³²PO₄ for DNA-DNA reassociation studies. The yield of viral DNA from Raji cells, superinfected with EBV prepared from supernatant fluid of HRI cell culture, was 100- to 570-fold higher than that of viral DNA directly extracted from the same virus preparation.     

A new in vitro method (END) for detection and measurement of hog cholera virus and its antibody by means of effect of HC virus on newcastle disease virus in swine tissue culture

Summary A newin vitro method of neutralization test for hog cholera virus by means of the END method was established. The test is based on the finding that immune serum against hog cholera virus specifically inhibits the exalting effect of hog cholera virus on Newcastle disease virus in swine testicular cell culture. The serum-virus mixtures are incubated at 37° C for 60 minutes or at 4° C overnight and tested for infectivity by the END method described in the previous papers of this series. The test is not only simple enough for the routine use, but also highly reproducible and specific. The test may achieve wide application in studies of hog cholera virus.     

The localization of virus-specific double-stranded RNA of cowpea mosaic virus in subcellular fractions of infected Vigna leaves

The fraction of a homogenate of cowpea mosaic virus (CPMV) infected leaves sedimenting at 1000g for 15 min was further divided by centrifugation on a discontinuous gradient consisting of layers of 60, 45, and 20% sucrose. It was possible to separate the nuclei and nuclear fragments from most of the chloroplasts.Hybridization with [³H]uridine labeled CPMV RNA and electron microscopy were used to detect CPMV-specific double-stranded RNA in different fractions of the gradient. By hybridization 70–90% of the double-stranded RNA was found in the so called chloroplast-fraction. By electron microscopy it was confirmed that double-stranded RNAs with lengths characteristic for double-stranded viral RNAs occurred predominantly in this chloroplast-fraction. The chloroplast-fraction also contained vesiculated membrane structures and amorphous electron-dense material, which are characteristic structures from CPMV-infected cells, suggesting that CPMV-specific double-stranded RNA might be associated with either or both of these structures rather than with the chloroplasts.     

Purification and some physicochemical properties of sonchus yellow net virus

Sonchus yellow net virus (SYNV) was purified from a Nicotiana hybrid by Celite filtration and sucrose density gradient centrifugation. Infectious preparations sedimented at 1044 S in linear-log gradients and banded at 1.183 g/ml in sucrose equilibrium gradients. Electron microscopy of purified preparations revealed bacilliform particles (94 × 248 nm). The virions had internal cross striations with a periodicity of about 4.1 nm and surface projections about 6 nm long. The molecular weight of the virion, estimated from size and density, was about 9 × 10⁸. Nucleic acid from sodium dodecyl sulfate-disrupted virions was susceptible to RNase, sedimented in sucrose gradients at 44 S, and had a molecular weight of 4.42 × 10⁶ as estimated by polyacrylamide-gel electrophoresis. Four major polypeptides with average molecular weights of 76,800, 63,800, 45,500, and 39,500 were detected by gel electrophoresis. SYNV preparations reacted in gel diffusion tests with a homologous antiserum but not with antisera to broccoli necrotic yellows virus, lettuce nectrotic yellows virus, or sow thistle yellow vein virus.     

Chick embryo lethal orphan (CELO) virus: some physical and immunological properties

Chick embryo lethal orphan (CELO) virus was partially purified by equilibrium centrifugation in CsCl density gradients. The virus was found to band at 1.32 g/cm³, and this band was found to contain the peak infectivity titres of virus. Electron microscopy of partially purified CELO virus revealed icosahedral particles of 60–80 nm diameter, and with a capsid of 252 capsomeres. The thermal denaturation temperature (T_m) of CELO virus DNA indicated a base composition of 46% guanine-cytosine. CELO virus CF antigens, separated from virus particles during purification of the virus, had the same sedimentation properties in preformed linear sucrose gradients as did the CF antigen of adenovirus 7.     

Structural components of mouse mammary tumor virus. I. Polypeptides of the virion.

Mouse mammary tumor virus (mMTV) obtained from MJY-alpha cell cultures and analyzed by polyacrylamide gel electrophoresis possesses four major polypeptides and six to eight minor components. Three of the major proteins, with estimated molecular weights of 60,000, 52,000 and 37,000, are glycoproteins, as demonstrated by labeling with [³H]glucosamine. Labeling with [³⁵S]sulfate revealed significant amounts of sulfate associated with the 60,000 (gp60) and 52,000 (gp52) glycoproteins, whereas sulfate was minimally incorporated into the 37,000 (gp37) dalton glycoprotein. At least three glycopeptide species containing both [³H]glucosamine and [³⁵S]sulfate labels were obtained after extensive Pronase digestion of mMTV, indicating that [³⁵S]sulfate is covalently linked to the carbohydrate component of mMTV glycoproteins. Variations in the polypeptide pattern of mMTV were observed when virions were grown and labeled under different culture conditions. The amount of gp60 decreased substantially, with an apparent increase in a minor polypeptide at 33,000 daltons, if virions were harvested from stationary cultures or if culture medium was not changed daily during viral harvests. When virions containing gp60 were incubated with medium from stationary cultures or with stationary cell layers, the level of gp60 decreased with a corresponding increase in the amount of radioactivity at 33,000 daltons. Cleavage of gp60 and gp52 was demonstrated by treatment of purified mMTV virions with trypsin or alpha-chymotrypsin (1–150 μg/ml) for 1 min to 3 hr. Virions were morphologically unaltered after incubation with either protease; however, both gp60 and gp52 were absent from the polypeptide patterns. The data suggest that gp52 is cleaved to form 33,000, 22,000, and possibly 37,000 dalton glycoproteins which remain associated with virions. Other mMTV virion polypeptides were resistant to treatment with protease.     

Alteration of hepatic polyribosome structure and function in mice during hypothermia

Analysis of the distribution of RNA double labeled with [¹⁴C]- and [³H]orotic acid in ribosomal particles separated by sucrose density-gradient centrifugation and the selective dissociation of unprogrammed ribosomes showed that the increase in proportion of monomeric ribosomes caused by hypothermia is due entirely to the increase in the unprogrammed ribosome fraction at the expense of polyribosomes. Protein synthetic activities of polyribosomes in vivo in livers of hypothermic and control mice were compared by double labeling of nascent and soluble polypeptides with [¹⁴C]- and [³H]leucine. Incorporation of radioactivity was relatively higher in nascent polypeptides and lower in soluble polypeptides in hypothermic than in control mice. The results indicate that hypothermia affects protein synthesis in livers of mice by decreasing probably the rates of both initiation and elongation, but affecting the former more than the latter.     

Translation of 26 S virus-specific RNA from Semliki Forest virus-infected cells in vitro

Two types of virus-specific RNA are associated with polyribosomes of BHK-21 cells 3 hr after infection with Semliki Forest virus. These RNA species sediment at about 42 S and 26 S, respectively, in sucrose density gradients. Addition of the 26 S RNA after isolation from polyribosomes to an in vitro protein-synthesizing system derived from Krebs II ascites cells stimulated the incorporation of [³⁵S]methionine into one protein. This protein has the same electrophoretic mobility on SDS polyacrylamide gels and an identical fingerprint pattern after tryptic digestion as the core protein of purified Semliki Forest virus. 26 S RNA prepared by extraction of total RNA with phenol from infected cells and repeated sucrose density-gradient centrifugation also stimulated protein synthesis in vitro. A possible role of the 26 S RNA as mRNA for all viral structural proteins is discussed.     

Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55–65 nm in diameter with delicate spike projections were detected in the 1.14–1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33–40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10⁷ virions/ml.     

Antibody-complement interactions with purified lymphocytic choriomeningitis virus

Immune complex formation and immune virolysis of the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) were examined. LCMV, harvested from acutely infected BHK cells propagated in the absence of serum, was purified by methanol precipitation and sucrose density gradient centrifugation. The 31- and 23-S RNA species of LCMV were labeled under conditions of actinomycin D treatment of cells that prevented the labeling of cell ribosomes in the virion. The addition of guinea pig antibody (Ab) to LCMV lowered viral infectivity and enhanced the sedimentation rate but not the density of [U-³H]LCMV and ¹²⁵iodine-surface protein-labeled LCMV. This suggested that Ab aggregated the virions into faster sedimenting complexes. The addition of complement (C) to the LCMV-Ab complex further reduced the infectivity and increased both the sedimentation rate and density of [¹²⁵I]LCMV. When [U-³H]LCMV was reacted with Ab and C and subjected to rate zonal sedimentation, the RNA label remained at the top of the gradient rather than sedimenting with the ¹²⁵I-surface protein-membrane label. This observation, which indicated that Ab and C lysed the virus, was confirmed by electron microscopy showing first coating of the viral membrane with electron dense material, then swelling and rupture of the membrane and release of viral core material. Using sera deficient in specified C components, it was found that the C-mediated inactivation proceeded via the classical C pathway. Sera deficient in latter C components (beyond C3) augmented the inactivation of LCMV-Ab complexes, but this inactivation was far more extensive when a complete C source was used. Evidence suggesting that immune and persistently infected mice produce C-fixing Ab to the surface of the LCM virion was documented.     

Cystein 402 of HIV gp120 is essential for CD4-binding and resistance of gp120 to intracellular degradation

Summary A DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp120 was excised from an SV40-based expression vector containing gp160, and subcloned into phage M13 for site-directed mutagenesis. Mutant vectors were constructed and CV-1 cells were transfected with constructs, where Cys₄₀₂ was substituted for a serine, and metabolically labelled with [³H]-N-acetylglucosamine (GlcN). Radioimmunoprecipitation with an hyperimmunserum, specific for gp120/gp160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp160, whereas gp120 was replaced by [³H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80–105k, suggesting increased sensitivity of mutantenv gene products to proteolysis after cleavage to gp120. Wild type gp120 and gp160 bound to CD4, whereas neither gp160 nor gp120 from mutant-transfected cell lysates did bind to CD4. Altogether the results indicated that Cys₄₀₂, probably by participating in a disulfide bridge, is essential for (i) the CD4-binding ability ofenv gene products and for (ii) the physical stability of gp120.     

Properties of Aleutian disease virus assayed with feline kidney cells

Summary Properties of Aleutian disease virus (ADV) were studied using feline kidney cells, line CRFK, to assay virus by the induction of nuclear antigen. ADV nuclear antigen was detected by immunofluorescent staining. Titers of virus obtained from mink spleens at 10–8 days after infection were usually between 10³ and 10⁵ infectious units per gram of spleen. ADV was purified by fluorocarbon extraction, differential centrifugation, biogel A-15 chromatography and CsCl equilibrium centrifugation. The molecular weight of the virus was estimated to be 3–5 × 10⁶ daltons. The density of antigen-inducing virus in equilibrium CsCl gradients was 1.32–1.34 g/cm³. On velocity sucrose gradients, antigen-inducing virus had a sedimentation coefficient of approximately 110S. The virus was not neutralized by sera from chronically infected mink and ferrets and by sera from experimentally infected mink. ADV was resistant to ionic and nonionic detergents and lipid solvents. The titer of partially purified virus was reduced as much as 700-fold by proteolytic enzymes but not by DNase or RNase. The virus was inactivated slowly at 56° C; the initial half-life was 90 minutes. It is concluded that the properties of ADV can be determined by assay in CRFK cells, thus facilitating virological study of the disease.With 4 Figures     

Replication of vaccinia DNA in mouse L cells. II. In vitro DNA synthesis in cytoplasmic extracts.

Cytoplasmic extracts prepared from vaccinia virus-infected L cells catalyzed the incorporation of labeled deoxynucleotide triphosphates into DNA which hybridized with vaccinia virus DNA. The incorporation of [³H]thymidine 5′ triphosphate ([³H]TTP) into DNA was shown to be dependent on the presence of all four deoxynucleoside triphosphates and incorporation was stimulated twofold by the addition of ATP, NAD, and ribonucleoside triphosphates. The incorporation of [³H]TTP in vitro was linear for 10 min and continued at a reduced rate for 30 min at 30°. The viral DNA synthesized in vitro was analyzed by sedimentation in alkaline-sucrose gradients and by isopycnic centrifugation in CsCl gradients. Alkaline-sucrose sedimentation analysis showed that replication of in vitro labeled DNA was discontinuous. Small fragments (～10 S) were synthesized in vitro in 10–30 sec which appeared to elongate so that after 30 min of synthesis the in vitro synthesized molecules cosedimented with in vivo labeled viral DNA species of 10–70 S. No molecules of greater length than mature viral single-stranded DNA (Type 1, 70–72 S) were observed when cell extracts prepared 3 hr postinfection were employed. Replication of viral DNA in vitro was symmetrical. No evidence for circular or superhelical DNA duplex molecules was obtained when in vitro synthesized DNA was analyzed by equilibrium density centrifugation in CsCl containing ethidium bromide.     

The site of cleavage in infected cells and polypeptides of representative paramyxoviruses grown in cultured cells of the chorioallantoic membrane

Summary Cultured cells of the chorioallantoic membrane (CAM) fulfilled the need of using the same cell system that was permissive for representative paramyxoviruses to carry out studies on the biosynthesis of their glycoproteins in infected cells.The polypeptide composition of the respective paramyxoviruses [Newcastle disease virus (NDV), paramyxovirus Yucapipa (PMY), and Sendai virus], grown in eggs and CAM-cells, was essentially identical. In egg-grown PMY a large glycoprotein (LGP) was present but only in some CAM-grown preparations of virus labeled with [³H]-glucosamine and rarely in [³⁵S]-methionine or [³H]-amino acids (valine, leucine, and tyrosine) labeled viruses.The site of cleavage of precursor F₀ to F_1,2 was not the same. In contrast to the cleavage of Sendai virus glycoprotein, cleavage was intracellular in NDV and PMY infected cells. Homologous antisera against the glycoproteins failed to inhibit cleavage of HN₀ or F₀ in cells infected with the representative paramyxoviruses.With 8 FiguresOn leave from: Department of Microbiology, California State University—Los Angeles, Los Angeles, CA 90032, U.S.A.     

Tissue-Binding Properties of the Cholera Toxin

[¹²⁵I]choleragen was employed to study further the tissue-binding properties of highly purified choleragen. It was observed that [¹²⁵I]choleragen was bound when combined with mucosal homogenates from all regions of the gastrointestinal tract of adult guinea pigs. Gastric, duodenal, jejunal, and ileal mucosa appeared equally effective in toxin-binding capacity. Preparations of large intestinal mucosa could bind an exceptionally larger amount of toxin. The binding property of small intestinal homogenates could not be attributed to any particular fraction after differential centrifugation; rather, the toxin receptor appeared to be associated with several sizes of particles containing cell membrane components. Although binding to mammalian cells was easily demonstrable, no binding to several types of bacterial cells was observed. The toxin receptor was found to be a “universal component” of many mammalian cell membranes, since specific binding of the toxin to a variety of guinea pig tissues was clearly demonstrated. [¹²⁵I]choleragen binding to all tissues, with the exception of those prepared from brain and large intestinal mucosa, could be inhibited by preincubation of the tissue homogenates with unlabeled choleragen but not with comparable concentrations of normal rabbit serum proteins. The determination of the specificity of [¹²⁵I]choleragen binding to brain and large intestinal mucosal homogenates was hampered by the continual release of soluble receptor from the homogenates, both of which contained the highest concentration of cholera toxin receptor. The data support and extend observations that cholera toxin binding to tissue receptor(s) is a very specific reaction, and further indicate that binding may occur with a variety of tissues to different degrees.