Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA

Summary In subcellular extracts of Kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral RNA polymerase, RNA and proteins were associated mainly with cytoplasm. When the cytoplasmic extract (500g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or heavy membrane fractions, which contained three types of virus-induced morphologically distinct membrane structures. These dense fractions were treated with 0.5% NP 40 and the soluble material was again centrifuged through a sucrose gradient for analyses as before. Viral RNA polymerase activity was retained and was associated with replicative intermediate RNA and some replicative form RNA in the peak enzyme fractions sedimenting at 20 S to 40 S. Enrichment of NS 3 and of the small nonstructural proteins NS 2 A and NS 2 B/NS 4 A was apparent in these fractions which were well separated from the slow sedimenting structural proteins. No detergent-resistant structures in the heavy membrane fractions other than ribosome-like particles were visible. The data show that the RNA polymerase complex cosedimented with virus-induced membrane structures and remained associated with specific nonstructural proteins and replicative intermediate RNA after detergent treatment.     

Chemical and enzymatic characteristics of cytoplasmic membranes of poliovirus-infected HeLa cells

The density distribution of cytoplasmic membranes separated by centrifugation to equilibrium in discontinuous sucrose gradients is progressively altered after infection of HeLa cells with poliovirus. The most striking changes are a decrease in quantity and an increase in density of rough microsomes and a large increase in quantity of the smooth microsomal membranes with which viral RNA synthesis is associated.     

Replication of the flavivirus Kunjin: proteins, glycoproteins, and maturation associated with cell membranes.

Kunjin virus-infected Vero cells were disrupted and the membranes separated by equilibrium centrifugation in discontinuous sucrose density gradients. Relatively pure fractions of smooth, rough and plasma membranes were obtained. Radioisotopic labeling experiments showed that virus-specific RNA and protein syntheses occur predominantly on smooth and rough membranes, respectively. Electrophoretic profiles of viral proteins were similar in all membrane fractions. Glucosamine, mannose, galactose and fucose were incorporated into viral glycoproteins in decreasing proportions. In smooth membranes only, the envelope glycoprotein was deficient in glucosamine but not in mannose. Rapid transfer of newly synthesized proteins to plasma membranes occurred; some enrichment therein of the largest nonstructural protein was detected. In electron micrographs, mature virions were observed only in fractions containing rough membranes. In thin sections of whole cells at 24 hr postinfection, most mature virions were in vesicles, but in some cells virions and possibly precursor particles were in lamella-like arrays within cisternae bounded by modified membranes.     

Separation of bovine plasma lipoproteins by a rapid ultracentrifugation method

The recently described method of centrifugation with iodixanol for the rapid separation of human plasma lipoproteins was adapted to separate bovine plasma lipoproteins. Density gradients were generated by mixing plasma with iodixanol 12% (w/v), followed by centrifugation at 350,000 g and 16 degrees C for 3 h 10 min in a vertical rotor. Gradients were unloaded dense-end first into 10 fractions. Human very low density lipoprotein (VLDL; density < 1.011 g/ml), low density lipoprotein (LDL; density = 1.016-1.039 g/ml) and high density lipoprotein (HDL; density = 1.039-1.090 g/ml) were resolved well at densities considerably lower than those traditionally reported in salt gradients. In gradients generated from 12% iodixanol, bovine LDL and HDL exhibited even lower densities (1.016-1.028 and 1.016-1.048 g/ml, respectively) with all lipoproteins occurring at the lower density region of the gradient. In contrast, density gradients generated from layers of equal volumes of 6% and 12% iodixanol readily separated bovine HDL from VLDL, whilst LDL still overlapped with HDL. The latter accounts for >80% of all bovine lipoproteins and exists as two populations, namely light and heavy HDL. Gradients generated from two layers of iodixanol recovered bovine HDL in five fractions. The hypercholesterolaemia associated with lactation resulted in a modest shift in the profile of HDL cholesterol towards lipoprotein particles of lower density (light HDL). Significant between-farm differences were also detected in the density profiles of bovine plasma cholesterol. This new method is suitable for use in research and diagnosis in relation to lipoprotein metabolism disorders in cows.     

Purification of bacterial endotoxins by zonal centrifugation

In this paper, we describe a cultivation procedure in large-capacity fermentors (200 liters) and the purification of the Salmonella crude extracts by means of zonal centrifugation in a sucrose gradient. Briefly, the endotoxin has been extracted from salmonellae with hypertonic solutions. The crude endotoxin was shown to contain several antigens, mainly r(1), r(2), r(3), or heterologous antigen. The heavy endotoxin was isolated and purified previously by enzymatic digestion, followed by a series of gel filtrations on Sephadex or Sepharose columns. We report the isolation and purification of heavy endotoxin in sucrose gradients, with the use of a B XIV titanium zonal rotor. From 150 to 300 mg of the crude material resuspended in a solution containing 1 m sodium chloride, 0.1 m sodium citrate, and 2.5% (w/w) sucrose, has been submitted to zonal centrifugation in gradients consisting of 5 to 25% (w/w) sucrose, also containing 1 m sodium chloride and 0.1 m sodium citrate. An overlay of 200 ml of 1 m sodium chloride-0.1 m sodium citrate was introduced after the sample. The separations were obtained after centrifugation for 3.5 hr at 35,000 rev/min (integralw(2)dt = 1.66 x 10(11)) at 4 C; the heavy endotoxin sedimented as heterogeneous material, from the middle toward the distal portions of the gradient. The heterologous antigens (r(1), r(2), r(3)), as well as bacterial proteins, remained in the sample zone. The heavy endotoxin recovered from the gradients was quite pure, as revealed by immunodiffusion tests against several antibacterial sera.     

Studies on the assembly of the envelope of Newcastle disease virus.

The association of the envelope proteins of Newcastle disease virus with membranes of infected BHK 21-F cells and their incorporation into mature envelopes has been investigated in a study employing cell fractionation. The principal fractions obtained by sucrose density gradient centrifugation of cytoplasmic extracts were rough endoplasmic reticulum and smooth membranes derived predominantly from smooth endoplasmic reticulum and Golgi apparatus. Furthermore, by adsorption to red blood cells it was possible to isolate virions and a hemadsorptive fraction of smooth membranes believed to be immediate precursors of mature envelopes. In addition to the cytoplasmic fractions, plasma membranes obtained as cell ghosts have been analyzed. Each fraction showed a distinct pattern of virus-specific proteins. Pulse-chase experiments indicated that glycoprotein HN and F_o were synthesized on the rough endoplasmic reticulum and transferred from there via smooth intracellular membranes to the plasma membrane and into virions. In the course of migration, F_o is converted to F. In contrast to the glycoproteins, protein M was found to be incorporated into the plasma membrane immediately after synthesis. Pulse-chase experiments also demonstrated that this protein appears in the hemagglutinating fraction of smooth membranes and in mature virions more rapidly than the glycoproteins. These results suggest that M is incorporated into membranes that contain already viral glycoproteins and that this process is one of the last steps in envelope assembly.     

Characterization of Kunjin virus RNA-dependent RNA polymerase: reinitiation of synthesis in vitro

RNA-dependent RNA polymerase (RDRP) activity was characterized in a cytoplasmic extract of Kunjin virus-infected Vero cells at 24 hr. The activity was influenced, possibly indirectly, by the length of prior treatment of infected cells with actinomycin D; however, 6 micrograms/ml actinomycin D and 10(-5) M alpha-amanitin in the RDRP assay had no effect. The replication complex was membrane-bound and Mg2+ was essential for RDRP activity. Incorporation was more dependent on exogenous UTP and GTP than ATP or CTP. The specific activity was low, and rate of incorporation of GMP decreased as the period of assay was increased; however, incorporation of label lasted for at least 60 min. RNA products were fractionated by LiCl precipitation, and kinetic studies showed that the sequence of accumulation of label was the same as that observed in vivo, viz., RI----RF----44 S RNA; limited reinitiation was also observed. This sequence of labeling also indicated that the in vitro RDRP activity was due to an enzyme capable of elongation, release, and reinitiation of Kunjin RNA synthesis and not merely end labeling or elongating preexisting RNA molecules. No labeled bands in urea-polyacrylamide gels were observed using extracts from mock-infected cells and hence the three RNA products of assays were readily identified in a single gel. The replication complex was still active after treatment with nonionic detergent, but no labeled 44 S RNA was detected in gels, even in the presence of RNasin in the assay which inhibited some nuclease activity. Antibodies to flavivirus-specific nonstructural proteins were preincubated with infected cell extracts in the presence and absence of detergent but no inhibition of RDRP activity was observed. However, anti-dsRNA plus detergent blocked activity by as much as 78% and label was found only in RF.     

Separation of bronchoalveolar cells from the guinea pig on continuous density gradients of Percoll: morphology and cytochemical properties of fractionated lung macrophages

Cells recovered by lavage from lungs of normal guinea pigs were centrifuged on continuous density gradients of colloidal silica (Percoll). The gradient was divided into six fractions based on the banding pattern of cells. This pattern was highly reproducible from animal to animal. Cell types in the fractions were identified by morphological and cytochemical criteria and the volume of the cells was determined by measuring their diameter and tritiated water space. More than 70% of the cells put on the gradient were recovered in the six fractions and there was no selective loss of cell types. Macrophages comprised more than 95% of the cells in fractions 3, 4, and 5. These fractions were of intermediate density (1.037-1.078 gm/ml) and together contained more than 85% of the recovered macrophages. Fraction 6 (density 1.078-1.130 gm/ml) was enriched for lymphocytes and granulocytes. Macrophages in fraction 5 were smaller, had more densely staining cytoplasm, and exhibited more nonspecific cytoplasmic esterase activity than macrophages in other fractions (5 greater than 4 greater than 3 greater than 2). These results indicate that density-gradient centrifugation on Percoll is an efficient method for purifying guinea pig alveolar macrophages and demonstrate that macrophages that differ in bouyant density also differ in morphologic and cytochemical properties. In a companion paper we report that macrophages in fractions 3, 4, and 5 differ functionally as well [9].     

Isolation and characterization of phagosomes containing Chlamydia psittaci from L cells.

The obligate intracellular procaryote Chlamydia psittaci enters host cells by a mechanism similar to, but distinct from, conventional phagocytosis. To better understand chlamydial uptake, L-cell phagosomes containing a single chlamydial cell were isolated and studied. Two rounds of dextran rate-zonal gradient centrifugation of L cells homogenized 1 h after infection with C. psittaci yielded phagosomes relatively free of other membranous structures. In double-label experiments, the phagosomes were enriched over 40-fold for radioactivity derived from chlamydiae as compared with the initial homogenate. Several lines of evidence showed that the structures isolated on dextran gradients were chlamydial phagosomes. These structures and free chlamydiae banded at different positions on discontinuous sucrose gradients. The difference was destroyed by the nonionic detergent Nonidet P-40, which disrupts plasma membranes but has no effect on C. psittaci. Material labeled on the surface of the L-cell plasma membrane cosedimented with the phagosome fractions. Electron microscopy of these fractions revealed structures having the appearance of a chlamydial elementary body surrounded by a unit membrane. Sodium dodecyl sulfate-polyacrylamide gels of the phagosome membranes displayed 10 major protein bands, less than the total number of surface-labeled proteins in the L-cell plasma membrane. Seven of the proteins of phagosome membranes had electrophoretic mobilities corresponding to those of proteins exposed on the surface of L cells. Two of them were cleaved by both trypsin and chymotrypsin, enzymes that decrease the susceptibility of L cells to infection with C. psittaci. These proteins may therefore be involved in the attachment and ingestion of C. psittaci by L cells.     

Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.     

Cycloheximide-Resistant Glycosylation in L Cells Infected with Chlamydia psittaci

L cells (mouse fibroblasts), uninfected and infected with the meningopneumonitis strain of Chlamydia psittaci, were labeled with [(14)C]glucosamine, and their membranous organelles were separated by isopycnic equilibrium centrifugation of whole cell homogenates on discontinuous sucrose gradients. Glycosylation of host membranes continued throughout the infection. Cycloheximide almost completely inhibited glycosylation in uninfected L cells, but it only partially inhibited the process in infected host cells. Cycloheximide-resistant glycosylation of membrane fractions with [(14)C]glucosamine increased as the infection proceeded and was probably due to the action of chlamydial enzymes. Modification of host membranes by glycosylation may play a role in the natural development of chlamydial infections.     

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514–516 nm with shoulders toward shorter wavelengths (470–490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.     

Buoyant density studies on equine arteritis virus

Summary The buoyant density of the Bucyrus strain of equine arteritis virus was studied by isodensity centrifugation on three types of gradients. The distribution patterns of infectivity were dependent on the type of gradient used. In sucrose gradients a single peak of infectivity at densities approximating 1.17 g/ml was obtained. From this gradient the total recovery of infectious virus varied between 80 and 90%. In cesium chloride gradients a rather broad band appeared in the form of a saddled peak with a range of densities from 1.180 to 1.215 g/ml, the total recovery being 90%. In potassium tartrate (KT) gradients two peaks were formed, one at 1.17 and another at 1.24 g/ml; the total recovery was 50%. Recentrifugation of fractions from KT gradient on sucrose gradients resulted in a changed distribution of infectivity for particles of higher density (1.24 g/ml).     

Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR

The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting.     

Chédiak-Higashi syndrome neutrophils are characterized by the absence of both normal azurophilic granules.

Neutrophils from two Chédiak-Higashi syndrome brothers were isolated, suspended in heparinized sucrose, lysed, and filtered. The granule-rich filtrate was centrifuged on a sucrose gradient (rho = 1.287-1.10 g/ml) at a mean force of 95,000g for 4 hours. The gradients contained one band at rho = 1.18 g/ml (band C) which was broader than normal and lacked normal bands A, rho = 1.22 g/ml, and B, rho = 1.20 g/ml. Gradient fractions were assayed for enzyme activities and protein. No marker enzymes identified densities normally occupied by bands A and B, and of the enzymes measured, only lysozyme showed peak activity with band C. Thus, only normal specific granules were present. Two azurophil granules, normally present and separable, were absent. Also identified was eosinophil granule peroxidase at rho = 1.24 g/ml (band E). Alkaline phosphatase, not a granule marker, was twice normal at the normal density, rho = 1.14-1.15 g/ml, consistent with an increase in unidentified membranes. A lysate gradient suggested that the giant azurophilic granules were rho = 1.25-1.27 g/ml. These neutrophils contain blue-grey or slate-grey giant granules, which are not truly azurophilic or basophilic, but should continue to be identified as azurophilic to conform to the convention making "azurophilic" and "peroxidase-positive" synonymous. The eosinophils contain normal eosinophil granules as well as giant inclusion granules. In contrast, neutrophils are deficient in both normal azurophilic granules.     

Changes in buoyant density relationships of two cell types of Coxiella burneti phase I.

Coxiella burneti phase I, purified from a formalin-inactivated yolk-sac vaccine, was separated into two bands of morphologically distinct cell types when subjected to sucrose gradient centrifugation. Recycling of the less dense, rod-shaped cells in unbuffered sucrose gradients (pH 5.5 to 6.0) resulted in the formation of bands having the location and appearance of the original two bands. Recycling of the denser band of larger ovoid-shaped cells yielded a single band, suggesting that the larger cell type arose from the smaller cell. In contrast to vaccine-derived rickettsiae, live, cell culture-propagated phase I organisms formed a single band in unbuffered sucrose gradients, at the same density as the upper band of the vaccine preparation. Centrifugation of cell culture-derived rickettsiae for 26 to 48 h in sucrose gradients of pH 5.5 resulted in the formation of a second band, at the same density as the lower band of the vaccine preparation. This did not occur in gradients of pH 7.0. Treatment of cell culture-propagated rickettsiae with formalin or germicidal ultraviolet radiation induced a total shift of the less dense cell population to a zone of higher density when centrifuged isopycnically in CsC1 gradients. This density change did not occur in sucrose gradients, suggesting a difference in the effect of these treatments on the permeability of the cell membrane to sucrose and CsC1.     

Proteins associated with the poliovirus RNA replication complex

The poliovirus RNA replication complex is a heterogeneous structure associated with smooth-surfaced cytoplasmic membranes. The membrane-bound complex has an average buoyant density of 1.08 g/cm³ in potassium tartrate. After lysis of membranes the replication complex sediments heterogeneously in the 75 to 265 S region of linear sucrose gradients. The peaks of pulse-labeled virus-specific RNA and viral RNA polymerase activity coincide and sediment at 155 S. Virus-specific protein sediments as a peak at 110 S with a faster sedimenting shoulder.     

Proteases in different membrane fractions of Acinetobacter calcoaceticus

Distinct protease activities were found in membrane fractions from Acinetobacter calcoaceticus grown on acetate-NH4+ medium until early stationary phase. Mechanical or enzymatic cell disintegration followed by membrane fractionation through sucrose gradient revealed higher activities in the outer membrane than in the cytoplasmic membrane. Using azocasein and synthetic p-nitroanilides as substrates we found very low proteinase activities in intracytoplasmic membrane fractions. However, these fractions contained a significant aminopeptidase activity which was absent from cell envelope membranes. Peptidolytic activities in intracytoplasmic membranes of gram-negative bacteria have not been described before.     

Preparation and crossed immunoelectrophoretic analysis of cytoplasmic and outer membrane fractions from Neisseria gonorrhoeae.

Cell envelopes were obtained from lysates of Neisseria gonorrhoeae, colony type T1, prepared with lysozyme, ethylenediaminetetraacetate, and Brij 58. This preparation was separated into cytoplasmic (inner) and outer membrane fractions by equilibrium sucrose density gradient centrifugation. The former fraction was 10-fold enriched in L-lactate dehydrogenase activity with respect to the latter. On the basis of buoyant density in sucrose, polypeptide patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enzymatic activity, these preparations appear similar to cytoplasmic and outer membrane preparations from other gram-negative bacteria. The membrane preparations were analyzed by high-resolution crossed immunoelectrophoretic procedures. This technique permitted the identification of antigens originating from the structural components of the gonococcal cell. Among those found to be cytoplasmic membrane components was the fast-moving antigen which occurs widely in gram-negative bacteria.