Replication of semliki forest virus: An electron microscopic study

Acheson, Nicholas H.; Tamm, Igor

doi:10.1016/0042-6822(67)90261-9

Cited by 253 publications

(140 citation statements)

References 28 publications

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“…That such an extrapolation is justified is shown by a comparison between the data presented here for SF virus and data obtained for vesicular stomatitis virus (Table V), grown in the same cell line. Although the morphology of both viruses differs markedly (Acheson & Tamm, 1967;Nakai & Howatson, 1968), they have a similar phospholipid composition, and also the results of the phospholipid localization studies are similar. It can be concluded, therefore, that the phospholipid distribution in the plasma membrane of BHK-21 cells is as is summarized in Table V.…”

Section: Discussionmentioning

confidence: 89%

Phospholipid asymmetry in Semliki Forest virus grown on baby hamster kidney (BHK-21) cells

Meer¹,

Simons²,

Kamp³

et al. 1981

Biochemistry

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ABSTRACT:The distribution of the different phospholipid classes over the two leaflets of the membrane of Semliki Forest virus, grown on baby hamster kidney cells (BHK-21), was studied. To localize the phospholipids we have used a phosphatidylcholine-specific exchange protein, a nonspecific exchange protein, phospholipases A2 and C, sphingomyelinase, and the amino group labeling reagent trinitrobenzenesulfonate. When low concentrations of exchange proteins and phospholipases were used, only those phospholipids present in lysed or otherwise defective virions were detected. Up to 10% of the phospholipids are present in such particles, and this amount correlated well with the amount of ribonuclease-degradable RNA present in each virus preparation. In the actual localization experiments, carried out with higher concentrations of exchange proteins and phospholipases as well as with trinitrobenzenesulfonate, the various independent techniques An asymmetric distribution of the different phospholipid classes over the two membrane monolayers has been observed in a variety of biological membranes. The concept of lipid asymmetry has been best documented for plasma membranes from cells which do not contain additional intracellular membrane systems such as erythrocytes, bacteria, and viruses [for a review, see Op den Kamp (1979)l. Phospholipid localization studies on plasma membranes from nucleated eukaryotic cells are much more complicated and have been carried out mainly with derivatives of the plasma membrane. In general, two approaches have been followed to obtain plasma membrane derived structures.Through the uptake of latex beads by mouse LM' cells, phagolysosomes are formed, which were considered to represent inside-out plasma membrane derivatives (Sandra & Payielded identical results. The outer membrane layer of the virus contains 52% of the phosphatidylcholine, 22% of the phosphatidylethanolamine, and 33% of the sphingomyelin. Phosphatidylserine could not be localized. Altogether 30% of the total phospholipids could be assigned to the outer layer and 50% to the inner membrane leaflet whereas 20% could not be localized. Calculations, based on the size of the Semliki Forest virus and the number of phospholipid molecules per virion, indicate it to be unlikely that the phospholipids which could not be localized are present in the inner bilayer leaflet. The present data on phospholipid composition and distribution have been compared with previous results on the lipid distribution in plasma membrane derived structures. It is concluded that an asymmetric distribution of phospholipids in the plasma membrane of various mammalian cells is apparent.

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Section: Discussionmentioning

confidence: 89%

Phospholipid asymmetry in Semliki Forest virus grown on baby hamster kidney (BHK-21) cells

Meer¹,

Simons²,

Kamp³

et al. 1981

Biochemistry

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“…They are freely mobile in the lateral plane of the plasma membrane [18] but during virus budding they are collected to the virus budding sites where nucleocapsids are in close contact with plasma membrane. In these sites the nucleocapsids bud out of the cell enwrapped in a membrane which contains a full set of spike proteins (about 240) but devoid of host cell proteins [19]. Garoff and Simons suggested that the budding process depends on the formation of a noncovalent bonds between the capsid proteins and the cytoplasmic portion of a spike.…”

Section: Discussionmentioning

confidence: 99%

The Effects of Octylglucoside on the Semliki Forest Virus Membrane

Helenius

Kartenbeck

1980

European Journal of Biochemistry

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Evidence is presented for a non-covalent interaction between the spike glycoprotein and the nucleocapsid of Semliki Forest virus. When isolated viruses were treated with the non-ionic detergent P-D-octylglucoside at neutral pH and low ionic strength the lipid bilayer membrane could be removed leaving most of the spike glycoproteins attached to the nucleocapsids. The interaction between capsids and membrane protein was sensitive to elevated pH and ionic strength, but at least partially reversible at neutral pH and low ionic strength. The possible role of this interaction in the viral structure and in the mechanism of budding of the virus from the host cell is discussed.Non-ionic detergents are widely used to solubilize, delipidate and isolate viral spike glycoproteins. They do not generally denature the proteins nor dissociate the non-covalent interactions between protein subunits [1,2]. This is illustrated by our studies on the Semliki Forest virus (SFV) membrane. The ionic detergents that we have tested dissociate the SFV spike proteins into their individual polypeptide chains [3,4], but the non-ionic Triton X-100 Cp-tert-octylphenylpolyoxyethylene9 ~ 10) leaves the spikes intact [5,6]. Triton X-100 has therefore been useful in characterising the structure and subunit composition of the spike protein. The SFV spike is composed of three non-covalently linked glycopolypeptides; El (apparent M , 49000), E2 (apparent M, 51 000) and E3 (apparent M , 10000) [6].In this paper we describe the effects of another non-ionic detergent, fl-D-octylglucoside. We have previously used this detergent to reconstitute the isolated viral spike proteins with phospholipid [7]. In studying its effects on the virus under different ionic conditions we observed features different from Triton X-100. This led to the demonstration of a reversible non-covalent interaction between the spike glycoprotein and the nucleocapsid surface. MATERIALS AND METHODS VirusA prototype strain of SFV was grown in baby hamster kidney BHK-21 cell monolayers and purified Sucrose Gradient Analysis oj Virus Dissociution 10 -50 % (w/w) sucrose gradients containing varying buffers and concentrations of octylglucoside were prepared in Spinco SW 50.1. centrifuge tubes. The samples (0.2 ml) containing a mixture of carrier-free 32P and [35S]methionine-labelled virus (about 2 x lo5 counts/min each) were applied on top usually without added detergent. The centrifugation was performed at 20°C for 30 min (35000 rev./min). Fractions (0.25 ml) were collected through the bottom of the tube, and aliquots (50 pl) were used for scintillation counting in a Triton X-lOO/toluene scintillation h i d . Aliquots were also subjected to chloroform/methanol extraction (3 : 4: 8 water :methanol: chloroform, by

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“…It is currently accepted that arboviruses consist of a ribonucleoprotein core surrounded by a membrane (envelope) which is derived partly from the host cell (1) (Fig. 4) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CSCL

Aaslestad¹,

Hoffman²,

Brown³

1968

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When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm3. Infectious Many laboratories have shown that considerable density heterogeneity exists in purified virus preparations. This is easily understood when the particles are deficient or devoid of nucleic acid or are mixed with soluble viral antigens. However, infectious virus of significantly different densities has also been observed, particularly among some of the enveloped viruses such as the arboviruses and the myxoviruses. Two representative arboviruses, Sindbis (4, 7) and Dengue-2 (10), have been characterized with respect to their buoyant densities in CsCl. In each case, considerable density heterogeneity was apparent, and, in some cases, virus specific hemagglutinins and complement-fixing antigens could be resolved from the major band of infectious virus. Sindbis and Semliki Forest virus hemagglutinins are considered to be identical with the virion, which is located in the envelope of the virus (6,8).In this report, we have determined that the buoyant density of the group A arbovirus, Eastern equine encephalitis (EEE), is 1.20 g/cm3 in CsCl. In addition, two other bands of viral material were observed; a light hemagglutinin which had a buoyant density of 1.18 g/cm3 and 1 Present address: Department of Microbiology, University of Georgia, Athens 30601.an infectious virus of a higher density (1.22 to 1.23 g/cm3). These two bands appear to be the salt-induced breakdown products of the EEE virion. MATERIALS AND METHODSVirus anid cell culture. The Louisiana strain of EEE virus, the origin and properties of which were reported by Brown (2), was used in all experiments. The virus was propagated on chick embryo (CE) monolayers and was assayed by the plaque technique described by Zebovitz and Brown (12). Virus was harvested 16 to 20 hr postinfection; titers averaged 4 X 108 to 8 X 108 plaque-forming units (PFU)/ml. Density gradient cenitrifugation. Before density gradient centrifugation, EEE virus was partially purified from 400 to 600 ml of tissue culture fluid that had been decanted from infected cells by adsorption onto AlPO4 gel (9). The virus was eluted from the gel in 0.3 M phosphate buffer (pH 8.0) and was concentrated by centrifugation at 65,000 X g for 60 min. The resulting pellet was allowed to resuspend overnight in borate-saline buffer (pH 9.0) containing 0.1% bovine serum albumin (3). Typically, only 15 to 20% of the total PFU were recovered; however, an overall purification of about 500-fold was obtained (2.0 X 1010 PFU/mg of protein). CsCl was added to 4.5 ml of the partially purified EEE virus to a mean density of 1.21 g/cm3. The virus particles were unstable in the salt when bovine serum 972

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Replication of semliki forest virus: An electron microscopic study

Cited by 253 publications

References 28 publications

Phospholipid asymmetry in Semliki Forest virus grown on baby hamster kidney (BHK-21) cells

Phospholipid asymmetry in Semliki Forest virus grown on baby hamster kidney (BHK-21) cells

The Effects of Octylglucoside on the Semliki Forest Virus Membrane

Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CSCL

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