VIROLOGY 67, 365-374 (1975)
Isolation Membrane
and Characterization Protein
of the Nonglycosylated
and a Nucleocapsid Paramyxovirus
JAMES J. McSHARRY,’
Complex
from the
SV5 l
RICHARD W. COMPANS, HENRY LACKLAND, PURNELL W. CHOPPIN
The Rockefeller
University, Accepted
AND
New York, New York 10021 May 12, 1975
The smallest polypeptide of the paramyxovirus SV5 (simian virus 5) the M protein, which is the major nonglycosylated protein of the viral envelope was isolated. The procedure used consisted of solubilization of the glycoproteins with Triton X-100, leaving an insoluble aggregate consisting of the protein subunit of the nucleocapsid (NP), the M protein, and a protein with a molecular weight of -50,000 (protein 5). Further treatment of this aggregate with 1% deoxycholate and 0.5 M urea followed by centrifugation on a discontinuous sucrose-on-cesium chloride gradient separated the aggregate into a fraction at the sucrose-cesium chloride interface consisting of the M polypeptide and a complex banding in 30% cesium chloride consisting of the nucleocapsid and protein 5. This technique yielded large quantities of highly purified M protein and a nucleocapsid complex that were amenable to biological and chemical analysis. Amino acid analysis of the purified membrane protein revealed a chemical composition of 64% neutral amino acids. The finding of protein 5 with the nucleocapsid after isolation on the cesium chloride gradient suggests that this protein may be associated with the nucleocapsid in the virion.
nucleocapsid; and a 50,000-dalton protein of undetermined function. The designaPrevious studies have shown that virions tions for the viral proteins are those sugof the paramyxovirus SV5 (simian virus 5) gested for paramyxoviruses by Scheid and contain five major proteins (Caliguiri et Choppin (1974). al., 1969; Klenk et al., 1970; Chen et al., Several of the biological activities associ1971; Mountcastle et al., 1970; Scheid et ated with paramyxovirus virions have been al., 1972; Choppin et al., 1972). These assigned to specific proteins. The neuramiinclude two glycoproteins (HN, 67,000 dal- nidase and hemagglutinating activities tons, and F, 56,000 daltons) that form the have been associated with the HN glycosurface projections of the virion; a nu- protein and the cell fusing and hemolyzing cleocapsid protein (NP, 60,000 daltons); a activities with the F glycoprotein (Scheid nonglycosylated envelope protein (M, et al., 1972; Scheid and Choppin, 1974; 43,000 daltons) that is the major protein of Homma and Ouchi, 1973). The proteins the viral envelope and is thought to be involved in the RNA transcriptase activity situated between the lipid bilayer and the of SV5 have not been established, although, in the case of Sendai virus, the ‘Presented at the 72nd Annual Meeting of the nucleocapsid protein and a nonAmerican Society for Microbiology, April 1972. Abglycosylated protein with a molecular stracts, Amer. Sot. Microbial. (1972), p. 215. weight of -75,000 have been found in a 2Present address: Department of Microbiology, complex with enzymatic activity (Stone et Albany Medical College, Albany, NY 12208. al., 1972; Marx et ul., 1974). In this report 3Present address: Worthington Biochemical Corporation, Freehold, NJ 07728. we describe the isolation of a nucleocapsid INTRODUCTION
365 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction
in any form reserved.
366
M&HARRY
complex and the M protein and give some of the physical and chemical characteristics of the purified SV5 M protein. MATERIALS
AND
METHODS
Cells. Cell cultures were prepared as previously described (Choppin, 1964; 1969). Monolayer cultures of the BHK21-F line of baby hamster kidney cells were grown in 32-0~ glass prescription bottles or 60-mm plastic petri dishes (Falcon Plastics, Los Angeles) in reinforced Eagle’s medium (REM) (Bablanian et al., 1965) with 10% calf serum and 10% tryptose phosphate broth. The MDBK line of bovine kidney cells was grown as a monolayer in plastic bottles or loo-mm plastic petri dishes in REM with 10% fetal calf serum. Virus. The W3 strain of the parainfluenza virus SV5 was propagated in MDBK cells and assayed on BHKBl-F cells as described previously (Choppin, 1964; Choppin and Compans, 1970). Propagation and purification of isotopitally labeld virus. The procedures for growth and purification of SV5 have been previously described (McSharry et al., 1971). Briefly, confluent monolayers of MDBK cells were infected with 2 ml of SV5 at a multiplicity of infection -10 plaque forming units per cell; then 8 ml of medium were added to each loo-mm plastic petri dish. The medium consisted of three parts REM without amino acids and one part REM, supplemented with 2% calf serum, and either 1 pCi/ml of 14C-reconstituted protein hydrolysate or 5 pCi/ml of 3H-reconstituted protein hydrolysate. Released virus was harvested 48 hr after infection and purified by sequential steps of clarification, precipitation with polyethylene glycol in the presence of NaCl, and isopycnic centrifugation in linear potassium tartrate gradients. The virus band was isolated, concentrated by centrifugation onto a cushion of potassium tartrate (40%, w/w) and dialyzed against 0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7.4. Chemicals and isotopes. Compnents for polyacrylamide gels were obtained from Canalco Industrial Corp., Rockville, Md; 3H- and’*C-reconstituted protein hydroly-
ET AL.
sate (Schwarz Mixture) from Schwarz BioResearch, Orangeburg, NY; polyethylene glycol 6000 from Amend Durg and Chemical Co., New York, NY; Triton x-100 from Beckman Instruments Inc., Fullerton, CA; sodium deoxycholate, urea, and sucrose, from Mann Research Laboratories, New York, NY; crystalline bovine serum albumin (BSA), from Armour Pharmaceutical Co., Chicago, IL. Nonidet P-40 (NP-40) was a gift of Dr. Daniel Rifkin. Removal of gl?coproteins. The solubilization of the spike glycoproteins of SV5 with Triton X-100 has been described in detail elsewhere (Scheid et al., 1972). Purified virus suspensions at a concentration of 0.5-1.0 mg of protein per ml in 0.01 M Tris buffer, pH 7.4, were treated with Triton X-100 at a final concentration of 10% (w/w) at 25” for 1 hr. The solubilized glycoproteins were separated from the insoluble viral proteins by centrifugation at 5000 g for 30 min in an SS-34 rotor in a Sorvall automatic superspeed refrigerated centrifuge (Ivan Sorvall, Inc., Norwalk, CT). The supernatant fluid was decanted, and the pellet was washed thoroughly with cold 0.01 M Tris buffer, pH 7.4, to remove excess Triton X-100 and resuspended in Tris buffer. Isolation of the SV5 membrane protein and nucleocapsid complex. The pellet was separated into the SV5 M protein and a nucleocapsid complex by a modification of the procedure described by Mountcastle et al. (1970). The pellet, at a protein concentration of 0.5-1.0 mg/ml, was treated with Nonidet P-40 and urea at a final concentration of 1% and 0.5 M, respectively. After thorough mixing, this was layered onto a discontinuous gradient consisting of 3.5 ml of 40% CsCl, 7.0 ml of 30% CsCl, 7.0 ml of 25% CsCl, and 10.5 ml of 5% sucrose prepared in Tris-EDTA-NaCl buffer. All cesium chloride and sucrose solutions were prepared on a w/w basis and contained 0.5 M urea to prevent viral proteins from sticking to the sides of the polyallomer centrifuge tube. The gradient was centrifuged at 90,000 g for 2.5 hr in a Beckman SW 27 rotor. Equal fractions were collected from the bottom of the gradient, and acid-precipitable radio-activity was deter-
ISOLATION
OF SV5 MEMBRANE
PROTEIN
367
X-100 and the glycoproteins and the M protein can be solubilized in this detergent plus 0.5-l A4 KC1 (Scheid et al., 1972). In order to separate the M protein from the glycoproteins, [3H]amino acidlabeled SV5 was treated with 10% Triton X-100 and the insoluble material was pelleted. Figure 1 shows the polypeptide composition of the insoluble pellet. The polypeptides of the resuspended pellet were examined by coelectrophroesis with 14Clabeled amino acid-labeled whole SV5 marker virions. This aggregate contains proteins NP, 5 and M in approximately the same relative amounts found in the marker virus. The two glycoproteins, HN and F, have been completely removed without affecting the nonglycosylated envelope protein, M. Whether the small peak of high molecular weight material around fraction 14 is a distinct viral protein has not been established. By treatment with NP-40 and urea followed by centrifugation in a discontinuous sucrose-cesium chloride gradient as described in Materials and Methods, the aggregate containing t’hese three proteins can be separated into two fractions. Figure 2 shows the profile of radioactivity in the gradient. Two distinct peaks of radioactivity are evident: peak I, which is a visible band, is in the region of the 30% cesium chloride layer and peak II, which is not visible, is at the interface between the sucrose-cesium chloride layers. The presence of radioactivity in peak II which does not correspond to a visible band suggests that some of the material in the aggregate becomes soluble as it enters the high salt at the sucrose-cesium chloride interface, and remains there because of its low density, whereas the remainder of the aggregate is not influenced by this exposure to high salt and sediments to its apparent density near the interface between the 25 and 30% RESULTS cesium chloride layers. Fractions from each Isolation of the SV5 Membrane Protein peak were isolated and dialyzed against and the Nucleocapsid Complex distilled water. The soluble material from Previous studies have shown that three peak II readily precipitated during dialysis of the five major proteins of SV5 are and more than 90% of the radioactivity was associated with the viral envelope and that easily collected by low speed centrifugation the two spike glycoproteins can be solubi- (5,000 g). The dialyzed material from both lized by treatment of the virion with Triton fractions was analyzed by polyacrylamide
mined after an aliquot of each fraction was precipitated in the presence of 0.1% BSA with 5% trichloroacetic acid (TCA) and filtered on cellulose nitrate filters (Millipore Filter Corp., Bedford, MA). The filters were washed with 5% TCA and 95% ethyl alcohol, dried and counted in toluene-liquiflour scintillation fluid in a Packared Tricarb liquid scintillation spectrometer. Polyacrylamide-gel electrophoresis. Viral proteins were dissociated in the presence of 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol by heating at 100” for 2 min (Maize1 et al., 1968). Procedures employed in the preparation of polyacrylamide gels, electrophoresis and the processing of gels for determination of radioactivity were previously described (Caliguiri et al., 1969; Scheid et al ,, 1972). Electron microscopy. The material to be examined was dialyzed against H,O and a drop was placed on a carbon-coated Formvar grid, stained with 2% phosphotungstic acid (pH 6.2) and examined in a Philips EM 300 microscope. ‘Amino acid analysis. Samples to be analyzed for their amino acid composition were dialyzed exhaustively against distilled water, extracted with choloroformmethanol and lyophilized. This material was hydrolyzed in 6 N HCL at 110” for 24 hr in U~CUUO(Moore and Stein, 1963), and the analysis was performed in a Beckman 120 C amino acid analyzer according to the procedure of Spackman et al. (1958). Halfcysteine and methionine residues were measured as cysteic acid and methionine sulfone after performic acid oxidation (Moore, 1963). Protein determination. Protein was estimated by the method of Lowry et al. (1951) with BSA as a standard.
368
ET AL.
McSHARRY 4
HJJ
20
NJ
;
5 6
40
60 number
Fraction
9 II3(
80
FIG. 1. Polypeptides found in the insoluble pellet of Triton X-loo-treated SV5 virions. [3H]amino acid-labeled SW was treated with 10% Triton X-100, and insoluble material was pelleted. An aliquot of the resuspended pellet was mixed with “C-labeled amino acid-labeled SV5 marker virus and the polypeptide composition was determined by SDS-polyacrylamide-gel electrophoresis. The solid line represents [3H]amino acid-labeled material, and the dashed line represents “C-labeled amino acid-labeled SV5 marker virus. In this and succeeding electropherograms, electrophoresis is from left to right on 7.5% gels at 3 V/cm for 16 hr. SV5 protein designations are: Spike glycoproteins, HN and F; nucleocapsid protein, NP nonglycosylated envelope protein, M; and protein 5.
6
12
18
Froctlon
24
30
36
number
FIG. 2. Fractionation of the Triton X-lOO-insoluble pellet of SV5 virions. Triton X-loo-treated [“HIamino acid-labeled SV5 pellet was resuspended in Tris buffer containing 1% NP-40 and 0.5 M urea and sedimented through a sucrose-CsCl gradient at 90,000 g for 2.5 hr. Fractions were collected from the bottom of the gradient, an aliquot was precipitated onto cellulose nitrate filters, and radioactivity was assayed.
gel electrophoresis and electron microscopy. Figure 3 shows an electropherogram of a portion of the material from peak I. Proteins NP and 5 are present in essentially the same ratio as they are in the marker virus; the M protein is present in a greatly reduced amount in relation to the
corresponding protein in the marker virus. The amount of the M protein in this complex is variable; however, attempts to rid the nucleocapsid complex completely of the M protein without removing protein 5 from the complex have been unsuccessful. Figure 4a is an electron micrograph of a
ISOLATION
OF SV5 MEMBRANE
Fractlan
PROTEIN
369
number
FIG. 3. Polypeptides
associated with the SV5 nucleocapsid complex. After dialysis against water, 13H]amino acid-labeled material from peak I of the gradient shown in Fig. 2 was mixed with “C-labeled amino acid-labeled SV5 marker virus and the polypeptide composition was determined by SDS-polyacrylamide-gel electrophoresis. The solid line represents [3H] amino acid-labeled material and the dashed line represents “C-labeled amino acid-labeled SV5 marker virus.
Fro. 4. Electron micrographs of isolated fractions of SV5 virions. (a) Nucleocapsid complex. Material from peak I of the sucrose-CsCl gradient after dialysis against water. Fragmented nucleocapsids are seen. (b) Membrane protein. Material from peak II of the sucrose-CsCl gradient after dialysis against water. Fibrous aggregates are seen. Preparations were negatively stained with phosphotungstic acid, pH 6.2. Magnification, x 80, 000.
370
negatively stained preparation of the material found in peak I. Typical SV5 nucleocapsid structures are evident along with some fragmented nucleocapsids. Thus, peak I of the gradient contained the morphologically identifiable SV5 nucleocapsid and consisted of the previously identified structural protein of the nucleocapsid, NP, and protein 5. These results suggest that the SV5 nucleocapsid complex consists of RNA and both polypeptides NP and 5. Figure 5 shows an electropherogram of an aliquot of the material found in peak II, i.e., the material that remained at the CsCl-sucrose interface. Only one 3Hlabeled amino acid-labeled polypeptide was found in this preparation and it corresponds to the M protein of the 14C-labeled amino acid-labeled SV5 marker virus. Figure 4b is an electron micrograph of a negatively stained preparation of the M protein found in peak II. Filamentous aggregates covered the field, but there is no evidence of an ordered structure. Observations at higher magnification did not reveal further details of the structure of the aggregate nor did electron micrographs of thin sections of the pelleted material. Quantitative
ET AL.
M&HARRY
Recovery of Components
The above results indicate that SV5 HN J
NP 4
virions can be fractionated into a glycoprotein fraction containing proteins HN and F, a nucleocapsid complex containing predominantly proteins NP and 5, and a membrane fraction containing only the M protein. These three fractions have been isolated using a gentle procedure involving the nonionic detergents Triton X-100 and NP-40 and centrifugation in the presence of sucrose and cesium chloride. Table 1 shows the recovery of radioactivity and protein in each fraction at the various steps throughout the procedure. Eight-six percent of the nucleocapsid complex and 69% of the membrane protein were recovered in the final steps. Yields of 1-2 mg of the M protein can be obtained from 10 mg of purified virions. In addition, a complex consisting essentially of the nucleocapsid protein and protein 5 was also obtained in large enough quantities for biochemical and biological studies. Amino Acid Composition Protein
of the Membrane
Table 2 shows the amino acid composition of the M protein presented as residues per 100 residues. These results are the averages of analyses of three different membrane preparations. Only the values for the number of cysteine residues varied by more than 5% over the three determinai
f
M c
0
Fractlan number [3H]amino acid-labeled material from peak II of the gradient amino acid-labeled SV5 marker virus and was dialyzed against water, an aliquot was mixed with “C-labeled polypeptides were separated by SDS-polyacrylamide-gel electrophoresis. The solid line represents the [3H]amino acid-labeled material, and the dashed line represents the ‘“C-labeled amino acid-labeled marker.
FIG. 5. Isolated SV5 membrane polypeptide.
ISOLATION TABLE
OF SV5 MEMBRANE
TABLE
1
RECOVERY OF SV5 PROTEINS AS DETERMINED BY BOTH RADIOACTIVITY AND PROTEIN DETERMINATIONS Radioactivity (cpm)
Fraction
Whole virions Triton X-100 supernatant Insoluble pellet Nucleocapsid fraction (Peak I) Membrane fraction (Peak II)
371
PROTEIN
Protein (mg)
Recovery (WY
7.0 x 107 2.5 x 10’
2.00 0.70
loo 95
4.5 x 107 2.4 x 10’
1.28 0.68
102 86
1.2 x 107
0.34
69
a Based on the percent of total viral protein represented by each polypeptide as determined by polyacrylamide-gel electrophoresis of SV5 grown in MDBK cells. The values expressed as percent of total protein are: HN = 22; NP = 29; F = 15; SV5 = 8; and M = 25.
tions. About 25% of the amino acid residues are hydrophobic and more than 64% of the amino acid residues are neutral amino acids. As was noted above, the membrane protein is soluble in the presence of detergent and high salt and precipitated when the salt was removed by dialysis against water, even in the continued presence of detergents. These physical properties and the amino acid composition indicate that the M protein is quite hydrophobic, as might be expected from its close association with the lipid bilayer of the viral envelope. DISCUSSION
This report describes a procedure for the isolation of the SV5 membrane protein M and a ribonucleoprotein complex consisting of proteins NP and 5. The membrane protein M was isolated in an electrophoretically pure form using a procedure that relies on the physicochemical properties of this protein. The solubility of the M protein in a nonionic detergent plus high salt and its insolubility in water, even in the presence of a nonionic detergent, suggested that it is a hydrophobic protein. The amino acid analyses which indicated that the protein consisted of 64% neutral amino acid residues confirmed the hydrophobic nature of the M protein. The chemical composition of only a few membrane proteins is known, so a comparison of the
2
AMINO ACID COMPOSITIONOF SV5 MEMBRANE PROTEIN Amino acid
Residues per 100 residues”
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine I r-Cystineb Valine Methionineb Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
6.8 2.0 4.8 7.8 7.1 7.6 11.0 6.5 9.9 5.0 2.0 7.3 2.1 5.7 8.1 2.6 3.8 N.D.
a Average of three determinations. b Not corrected for degradation. c N.D., not determined.
amino acid composition of the M protein with other membrane proteins is limited. However, recently Laver and Baker (1972) showed that the membrane proteins of two strains of influenza virus contain about 80% neutral amino acid residues. Thus, these membrane proteins have a hydrophobic character, a property compatible with their association with the lipid bilayer of the viral envelope. With another paramyxovirus, Newcastle disease virus, utilizing the solubility of the M protein in Triton X-100 and high salt described here, it has been possible to isolate the M protein by extracting the two glycoproteins and the M protein from virions by Triton X-100 on 1 M KC1 and then recovering the M protein by dialyzing away the KCl. The M protein precipitates under these conditions, leaving the glycoproteins in solution in the detergent (Scheid and Choppin, 1973). Thus, two methods are available for isolation of the M protein from paramyxoviruses, each taking advantage of the solubility of this protein in nonionic detergents in the presence of a high salt concentration. The association of a small amount of the
372
McSHARRY
M protein with the nucleocapsid complex after the separation of the aggregate on the sucrose-cesium chloride gradient suggests that there is some affinity between the nucleocapsid and the membrane protein. This finding supports previous studies (Compans et al., 1966; Compans and Choppin, 1971; Choppin et al., 1972) which suggest that, during the maturation of influenza and parainfluenza viruses, the nucleocapsid interacts only with portions of the plasma membrane which contain viral-specific proteins. With the use of vesicular stomatitis virus-SV5 phenotypitally mixed virions, evidence was also obtained that the nucleocapsid of these viruses recognizes the nonglycosylated membrane protein at the plasma membrane during virus assembly (McSharry et al., 1971). Evidence has also been obtained that a portion of the nucleocapsid protein subunit (NP) of paramyxoviruses which can be removed by proteolytic enzymes confers hydrophobicity on the nucleocapsid, and it has been suggested that this portion of NP is involved in the interaction with the M protein, perhaps a hydrophobic et al., 1970, interaction (Mountcastle 1974). Thus there are several lines of evidence suggesting an affinity between the M protein and the nucleocapsid and a role for this affinity in virus assembly. In addition to its providing a recognition site for the nucleocapsid at the plasma membrane, other functions have been suggested for the M protein in virions. It is thought to contribute to the organization of the lipid phase and the stability of the viral membrane, and it may play a role in maintaining a virus-specific domain within the plasma membrane of the infected cell which contains viral proteins but from which host cell proteins are excluded (Choppin et al., 1972; Landsberger et al., 1973; Choppin and Compans, 1975). Additional studies are required to establish the various functions of the M protein; however, the ability to isolate this protein on a preparative scale should permit further characterization of its physical, chemical, and biological properties. The finding of protein 5 associated with the nucleocapsid when it is isolated from
ET AL.
the CsCl-sucrose gradient suggests that this protein is associated with the nucleocapsid in a complex in the virion. However at the present time the possibility cannot be excluded that this association occurs when the virus is disrupted with detergent rather than preexisting in the virion. In any event, the affinity of these two proteins is sufficiently strong to keep them associated in the presence of the detergent and a high salt environment. It is noteworthy that with several other negative-strand viruses, virion-associated RNA transcriptase activity is associated with a complex that contains at least one other protein in addition to the nucleocapsid protein (Stone et al., 1971, 1972; Emerson and Wagner, 1972; Bishop and Roy, 1972; Caliguiri and Compans, 1974). In the case of Sendai virus, a major virion protein (P) with a molecular weight of approximately 75,000 is associated with the nucleocapsid in the transcriptase complex (Stone et al., 1971, 1972; Marx et al., 1974). A major SV5 protein of this size has not been isolated from SV5 virions, although a small amount of a 76,000-dalton protein has been seen in some preparations of SV5 (Caliguiri et al., 1969). The question therefore arises as to whether protein 5 in SV5 might be analogous to protein P of Sendai virus. There is no direct evidence on this point as yet, and this protein might serve other unknown functions. It is perhaps pertinent that no transcriptase activity was found in a preparation of nucleocapsid containing only the SV5 nucleocapsid protein subunit NP (Mountcastle, Compans, and Choppin, unpublished experiments). Additional studies are necessary to characterize further protein 5 and determine its bioligical role. Such studies should be facilitated by the ability to isolate it in association with the nucleocapsid by the procedure described here. ACKNOWLEDGMENTS This work was supported by Grants No. AI-05600 from the National Institutes of Allergy and Infectious Diseases, U.S. Public Health Service and BMS740983 from the National Science Foundation. J. J. M&harry was a postdoctoral fellow of NIAID. We are grateful for the assistance of Mrs. Ann
ISOLATION Erickson, Miss Cathleen lynn Santa Maria.
O’Connell
OF SV5 MEMBRANE
and Mrs. Caro-
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tion and reconstitutuon of the transcriptase and template activities of vesicular stomatitis B and T virions. J. Viral. 10, 297-309. HOMMA, M., and OUCHI, M (1973). Trypsin action on the growth of Sendai virus in tissue culture cells. III. Structural differentiation of Sendai viruses grown in eggs and tissue culture cells. J. Viral. 12, 1457-1465. KLENK, H. -D., CALIGUIRI. L. A., and CHOPPIN, P. W. (1970). The proteins of the parainfluenza virus SV5. II. The carbohydrate content and glycoproteins of the virion. Virology 42, 473-481. LANDSBERGER,F. R., COMI~ANS,R. W., CHOPPIN, P. W., and LENARD, J. (1973). Organization of the lipid phase in viral membranes. Effects of independent variation of the lipid and protein composition. Biochemistry 12,4498-4501. LAVER, W. G., and BAKER, N. (1972). Amino acid composition of polypeptides of influenza virus. J. Gen. Viral. 17, 61-68. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAIZEL, J. V., JR., WHITE, D. 0. and SCHARFF, M. D. (19681. The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of type 2. 7a and 23. Virology 36, 115-125. MARX, P. A., PORTNEH, .4., and KINGSBERRY, D. W. (19741. Sendai virion transcriptase complex: Polypeptide composition and inhibition by virion enveloped proteins. J. Viral. 13, 107-112. MCSHARRY, J. J., COMPANS,R. W., and CHOPPIN, P. W. (1971). Proteins of vesicular stomatitis virus and phenotypically mixed vesicular stomatitis virussimian virus 5 virions. J. Viral. 8, 722-729. MOORE, S. (1963). On the determination of cystine as cysteic acid. J. Biol. Chem. 238, 235-237. MOORE, S., and STEIN, W. H. (1963). Chromatographic determination of amino acids by the use of automatic recording equipment. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 6, pp. 819-831. Academic Press, New York. MOC’NTCASTLE,W. E., COMPANS, R. W., CALIGUIRI, L. A. and CHOPPIN, P. W. (1970). The nucleocapsid protein subunits of SV5, Newcastle disease virus and Sendai virus. J. Viral. 6, 677-684. MOUNTCASTLE, W. E., COMPANS,R. W., LACKLAND, H., and CHOPPIN, P. W. (19741. Proteolytic cleavage of subunits of the nucleocapsid of the paramyxovirus simian virus 5. J. Viral. 14, 1253-1261. SCHEID, A., CALIGUIRI, L. A., COMPANS, R. W., and CHOPPIN, P. W. (1972). Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger SV5 glycoprotein. Virology 50, 640-652.
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SCHEID, A., and CHOPPIN, P. W. (19’73). Isolation
and purification of the envelope proteins of Newcastle disease virus. J. Viral. 50, 640-652. SCHEID, A., and CHOPPIN, P. W. (1974). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57,
475-490. SPACKMAN,
D. H., STEIN, W. H. and MOORE, S. (1958).
ET AL. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. STONE, H. O., KINGSBERRY, D. W., and DARLINGTON, R. W. (1972). Sendai virus-induced transcriptase from infected cells: Polypeptides in the transcription complex. J. Viral. 10, 1037-1043. STONE, H. O., PORTNE~ A., and KINGSBERRY, D. W. (1971). Ribonucleic acid transcriptases in Sendai virions and infected cells. J. Viral. 8, 174-180.
Isolation Membrane
and Characterization Protein
of the Nonglycosylated
and a Nucleocapsid Paramyxovirus
JAMES J. McSHARRY,’
Complex
from the
SV5 l
RICHARD W. COMPANS, HENRY LACKLAND, PURNELL W. CHOPPIN
The Rockefeller
University, Accepted
AND
New York, New York 10021 May 12, 1975
The smallest polypeptide of the paramyxovirus SV5 (simian virus 5) the M protein, which is the major nonglycosylated protein of the viral envelope was isolated. The procedure used consisted of solubilization of the glycoproteins with Triton X-100, leaving an insoluble aggregate consisting of the protein subunit of the nucleocapsid (NP), the M protein, and a protein with a molecular weight of -50,000 (protein 5). Further treatment of this aggregate with 1% deoxycholate and 0.5 M urea followed by centrifugation on a discontinuous sucrose-on-cesium chloride gradient separated the aggregate into a fraction at the sucrose-cesium chloride interface consisting of the M polypeptide and a complex banding in 30% cesium chloride consisting of the nucleocapsid and protein 5. This technique yielded large quantities of highly purified M protein and a nucleocapsid complex that were amenable to biological and chemical analysis. Amino acid analysis of the purified membrane protein revealed a chemical composition of 64% neutral amino acids. The finding of protein 5 with the nucleocapsid after isolation on the cesium chloride gradient suggests that this protein may be associated with the nucleocapsid in the virion.
nucleocapsid; and a 50,000-dalton protein of undetermined function. The designaPrevious studies have shown that virions tions for the viral proteins are those sugof the paramyxovirus SV5 (simian virus 5) gested for paramyxoviruses by Scheid and contain five major proteins (Caliguiri et Choppin (1974). al., 1969; Klenk et al., 1970; Chen et al., Several of the biological activities associ1971; Mountcastle et al., 1970; Scheid et ated with paramyxovirus virions have been al., 1972; Choppin et al., 1972). These assigned to specific proteins. The neuramiinclude two glycoproteins (HN, 67,000 dal- nidase and hemagglutinating activities tons, and F, 56,000 daltons) that form the have been associated with the HN glycosurface projections of the virion; a nu- protein and the cell fusing and hemolyzing cleocapsid protein (NP, 60,000 daltons); a activities with the F glycoprotein (Scheid nonglycosylated envelope protein (M, et al., 1972; Scheid and Choppin, 1974; 43,000 daltons) that is the major protein of Homma and Ouchi, 1973). The proteins the viral envelope and is thought to be involved in the RNA transcriptase activity situated between the lipid bilayer and the of SV5 have not been established, although, in the case of Sendai virus, the ‘Presented at the 72nd Annual Meeting of the nucleocapsid protein and a nonAmerican Society for Microbiology, April 1972. Abglycosylated protein with a molecular stracts, Amer. Sot. Microbial. (1972), p. 215. weight of -75,000 have been found in a 2Present address: Department of Microbiology, complex with enzymatic activity (Stone et Albany Medical College, Albany, NY 12208. al., 1972; Marx et ul., 1974). In this report 3Present address: Worthington Biochemical Corporation, Freehold, NJ 07728. we describe the isolation of a nucleocapsid INTRODUCTION
365 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction
in any form reserved.
366
M&HARRY
complex and the M protein and give some of the physical and chemical characteristics of the purified SV5 M protein. MATERIALS
AND
METHODS
Cells. Cell cultures were prepared as previously described (Choppin, 1964; 1969). Monolayer cultures of the BHK21-F line of baby hamster kidney cells were grown in 32-0~ glass prescription bottles or 60-mm plastic petri dishes (Falcon Plastics, Los Angeles) in reinforced Eagle’s medium (REM) (Bablanian et al., 1965) with 10% calf serum and 10% tryptose phosphate broth. The MDBK line of bovine kidney cells was grown as a monolayer in plastic bottles or loo-mm plastic petri dishes in REM with 10% fetal calf serum. Virus. The W3 strain of the parainfluenza virus SV5 was propagated in MDBK cells and assayed on BHKBl-F cells as described previously (Choppin, 1964; Choppin and Compans, 1970). Propagation and purification of isotopitally labeld virus. The procedures for growth and purification of SV5 have been previously described (McSharry et al., 1971). Briefly, confluent monolayers of MDBK cells were infected with 2 ml of SV5 at a multiplicity of infection -10 plaque forming units per cell; then 8 ml of medium were added to each loo-mm plastic petri dish. The medium consisted of three parts REM without amino acids and one part REM, supplemented with 2% calf serum, and either 1 pCi/ml of 14C-reconstituted protein hydrolysate or 5 pCi/ml of 3H-reconstituted protein hydrolysate. Released virus was harvested 48 hr after infection and purified by sequential steps of clarification, precipitation with polyethylene glycol in the presence of NaCl, and isopycnic centrifugation in linear potassium tartrate gradients. The virus band was isolated, concentrated by centrifugation onto a cushion of potassium tartrate (40%, w/w) and dialyzed against 0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer, pH 7.4. Chemicals and isotopes. Compnents for polyacrylamide gels were obtained from Canalco Industrial Corp., Rockville, Md; 3H- and’*C-reconstituted protein hydroly-
ET AL.
sate (Schwarz Mixture) from Schwarz BioResearch, Orangeburg, NY; polyethylene glycol 6000 from Amend Durg and Chemical Co., New York, NY; Triton x-100 from Beckman Instruments Inc., Fullerton, CA; sodium deoxycholate, urea, and sucrose, from Mann Research Laboratories, New York, NY; crystalline bovine serum albumin (BSA), from Armour Pharmaceutical Co., Chicago, IL. Nonidet P-40 (NP-40) was a gift of Dr. Daniel Rifkin. Removal of gl?coproteins. The solubilization of the spike glycoproteins of SV5 with Triton X-100 has been described in detail elsewhere (Scheid et al., 1972). Purified virus suspensions at a concentration of 0.5-1.0 mg of protein per ml in 0.01 M Tris buffer, pH 7.4, were treated with Triton X-100 at a final concentration of 10% (w/w) at 25” for 1 hr. The solubilized glycoproteins were separated from the insoluble viral proteins by centrifugation at 5000 g for 30 min in an SS-34 rotor in a Sorvall automatic superspeed refrigerated centrifuge (Ivan Sorvall, Inc., Norwalk, CT). The supernatant fluid was decanted, and the pellet was washed thoroughly with cold 0.01 M Tris buffer, pH 7.4, to remove excess Triton X-100 and resuspended in Tris buffer. Isolation of the SV5 membrane protein and nucleocapsid complex. The pellet was separated into the SV5 M protein and a nucleocapsid complex by a modification of the procedure described by Mountcastle et al. (1970). The pellet, at a protein concentration of 0.5-1.0 mg/ml, was treated with Nonidet P-40 and urea at a final concentration of 1% and 0.5 M, respectively. After thorough mixing, this was layered onto a discontinuous gradient consisting of 3.5 ml of 40% CsCl, 7.0 ml of 30% CsCl, 7.0 ml of 25% CsCl, and 10.5 ml of 5% sucrose prepared in Tris-EDTA-NaCl buffer. All cesium chloride and sucrose solutions were prepared on a w/w basis and contained 0.5 M urea to prevent viral proteins from sticking to the sides of the polyallomer centrifuge tube. The gradient was centrifuged at 90,000 g for 2.5 hr in a Beckman SW 27 rotor. Equal fractions were collected from the bottom of the gradient, and acid-precipitable radio-activity was deter-
ISOLATION
OF SV5 MEMBRANE
PROTEIN
367
X-100 and the glycoproteins and the M protein can be solubilized in this detergent plus 0.5-l A4 KC1 (Scheid et al., 1972). In order to separate the M protein from the glycoproteins, [3H]amino acidlabeled SV5 was treated with 10% Triton X-100 and the insoluble material was pelleted. Figure 1 shows the polypeptide composition of the insoluble pellet. The polypeptides of the resuspended pellet were examined by coelectrophroesis with 14Clabeled amino acid-labeled whole SV5 marker virions. This aggregate contains proteins NP, 5 and M in approximately the same relative amounts found in the marker virus. The two glycoproteins, HN and F, have been completely removed without affecting the nonglycosylated envelope protein, M. Whether the small peak of high molecular weight material around fraction 14 is a distinct viral protein has not been established. By treatment with NP-40 and urea followed by centrifugation in a discontinuous sucrose-cesium chloride gradient as described in Materials and Methods, the aggregate containing t’hese three proteins can be separated into two fractions. Figure 2 shows the profile of radioactivity in the gradient. Two distinct peaks of radioactivity are evident: peak I, which is a visible band, is in the region of the 30% cesium chloride layer and peak II, which is not visible, is at the interface between the sucrose-cesium chloride layers. The presence of radioactivity in peak II which does not correspond to a visible band suggests that some of the material in the aggregate becomes soluble as it enters the high salt at the sucrose-cesium chloride interface, and remains there because of its low density, whereas the remainder of the aggregate is not influenced by this exposure to high salt and sediments to its apparent density near the interface between the 25 and 30% RESULTS cesium chloride layers. Fractions from each Isolation of the SV5 Membrane Protein peak were isolated and dialyzed against and the Nucleocapsid Complex distilled water. The soluble material from Previous studies have shown that three peak II readily precipitated during dialysis of the five major proteins of SV5 are and more than 90% of the radioactivity was associated with the viral envelope and that easily collected by low speed centrifugation the two spike glycoproteins can be solubi- (5,000 g). The dialyzed material from both lized by treatment of the virion with Triton fractions was analyzed by polyacrylamide
mined after an aliquot of each fraction was precipitated in the presence of 0.1% BSA with 5% trichloroacetic acid (TCA) and filtered on cellulose nitrate filters (Millipore Filter Corp., Bedford, MA). The filters were washed with 5% TCA and 95% ethyl alcohol, dried and counted in toluene-liquiflour scintillation fluid in a Packared Tricarb liquid scintillation spectrometer. Polyacrylamide-gel electrophoresis. Viral proteins were dissociated in the presence of 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol by heating at 100” for 2 min (Maize1 et al., 1968). Procedures employed in the preparation of polyacrylamide gels, electrophoresis and the processing of gels for determination of radioactivity were previously described (Caliguiri et al., 1969; Scheid et al ,, 1972). Electron microscopy. The material to be examined was dialyzed against H,O and a drop was placed on a carbon-coated Formvar grid, stained with 2% phosphotungstic acid (pH 6.2) and examined in a Philips EM 300 microscope. ‘Amino acid analysis. Samples to be analyzed for their amino acid composition were dialyzed exhaustively against distilled water, extracted with choloroformmethanol and lyophilized. This material was hydrolyzed in 6 N HCL at 110” for 24 hr in U~CUUO(Moore and Stein, 1963), and the analysis was performed in a Beckman 120 C amino acid analyzer according to the procedure of Spackman et al. (1958). Halfcysteine and methionine residues were measured as cysteic acid and methionine sulfone after performic acid oxidation (Moore, 1963). Protein determination. Protein was estimated by the method of Lowry et al. (1951) with BSA as a standard.
368
ET AL.
McSHARRY 4
HJJ
20
NJ
;
5 6
40
60 number
Fraction
9 II3(
80
FIG. 1. Polypeptides found in the insoluble pellet of Triton X-loo-treated SV5 virions. [3H]amino acid-labeled SW was treated with 10% Triton X-100, and insoluble material was pelleted. An aliquot of the resuspended pellet was mixed with “C-labeled amino acid-labeled SV5 marker virus and the polypeptide composition was determined by SDS-polyacrylamide-gel electrophoresis. The solid line represents [3H]amino acid-labeled material, and the dashed line represents “C-labeled amino acid-labeled SV5 marker virus. In this and succeeding electropherograms, electrophoresis is from left to right on 7.5% gels at 3 V/cm for 16 hr. SV5 protein designations are: Spike glycoproteins, HN and F; nucleocapsid protein, NP nonglycosylated envelope protein, M; and protein 5.
6
12
18
Froctlon
24
30
36
number
FIG. 2. Fractionation of the Triton X-lOO-insoluble pellet of SV5 virions. Triton X-loo-treated [“HIamino acid-labeled SV5 pellet was resuspended in Tris buffer containing 1% NP-40 and 0.5 M urea and sedimented through a sucrose-CsCl gradient at 90,000 g for 2.5 hr. Fractions were collected from the bottom of the gradient, an aliquot was precipitated onto cellulose nitrate filters, and radioactivity was assayed.
gel electrophoresis and electron microscopy. Figure 3 shows an electropherogram of a portion of the material from peak I. Proteins NP and 5 are present in essentially the same ratio as they are in the marker virus; the M protein is present in a greatly reduced amount in relation to the
corresponding protein in the marker virus. The amount of the M protein in this complex is variable; however, attempts to rid the nucleocapsid complex completely of the M protein without removing protein 5 from the complex have been unsuccessful. Figure 4a is an electron micrograph of a
ISOLATION
OF SV5 MEMBRANE
Fractlan
PROTEIN
369
number
FIG. 3. Polypeptides
associated with the SV5 nucleocapsid complex. After dialysis against water, 13H]amino acid-labeled material from peak I of the gradient shown in Fig. 2 was mixed with “C-labeled amino acid-labeled SV5 marker virus and the polypeptide composition was determined by SDS-polyacrylamide-gel electrophoresis. The solid line represents [3H] amino acid-labeled material and the dashed line represents “C-labeled amino acid-labeled SV5 marker virus.
Fro. 4. Electron micrographs of isolated fractions of SV5 virions. (a) Nucleocapsid complex. Material from peak I of the sucrose-CsCl gradient after dialysis against water. Fragmented nucleocapsids are seen. (b) Membrane protein. Material from peak II of the sucrose-CsCl gradient after dialysis against water. Fibrous aggregates are seen. Preparations were negatively stained with phosphotungstic acid, pH 6.2. Magnification, x 80, 000.
370
negatively stained preparation of the material found in peak I. Typical SV5 nucleocapsid structures are evident along with some fragmented nucleocapsids. Thus, peak I of the gradient contained the morphologically identifiable SV5 nucleocapsid and consisted of the previously identified structural protein of the nucleocapsid, NP, and protein 5. These results suggest that the SV5 nucleocapsid complex consists of RNA and both polypeptides NP and 5. Figure 5 shows an electropherogram of an aliquot of the material found in peak II, i.e., the material that remained at the CsCl-sucrose interface. Only one 3Hlabeled amino acid-labeled polypeptide was found in this preparation and it corresponds to the M protein of the 14C-labeled amino acid-labeled SV5 marker virus. Figure 4b is an electron micrograph of a negatively stained preparation of the M protein found in peak II. Filamentous aggregates covered the field, but there is no evidence of an ordered structure. Observations at higher magnification did not reveal further details of the structure of the aggregate nor did electron micrographs of thin sections of the pelleted material. Quantitative
ET AL.
M&HARRY
Recovery of Components
The above results indicate that SV5 HN J
NP 4
virions can be fractionated into a glycoprotein fraction containing proteins HN and F, a nucleocapsid complex containing predominantly proteins NP and 5, and a membrane fraction containing only the M protein. These three fractions have been isolated using a gentle procedure involving the nonionic detergents Triton X-100 and NP-40 and centrifugation in the presence of sucrose and cesium chloride. Table 1 shows the recovery of radioactivity and protein in each fraction at the various steps throughout the procedure. Eight-six percent of the nucleocapsid complex and 69% of the membrane protein were recovered in the final steps. Yields of 1-2 mg of the M protein can be obtained from 10 mg of purified virions. In addition, a complex consisting essentially of the nucleocapsid protein and protein 5 was also obtained in large enough quantities for biochemical and biological studies. Amino Acid Composition Protein
of the Membrane
Table 2 shows the amino acid composition of the M protein presented as residues per 100 residues. These results are the averages of analyses of three different membrane preparations. Only the values for the number of cysteine residues varied by more than 5% over the three determinai
f
M c
0
Fractlan number [3H]amino acid-labeled material from peak II of the gradient amino acid-labeled SV5 marker virus and was dialyzed against water, an aliquot was mixed with “C-labeled polypeptides were separated by SDS-polyacrylamide-gel electrophoresis. The solid line represents the [3H]amino acid-labeled material, and the dashed line represents the ‘“C-labeled amino acid-labeled marker.
FIG. 5. Isolated SV5 membrane polypeptide.
ISOLATION TABLE
OF SV5 MEMBRANE
TABLE
1
RECOVERY OF SV5 PROTEINS AS DETERMINED BY BOTH RADIOACTIVITY AND PROTEIN DETERMINATIONS Radioactivity (cpm)
Fraction
Whole virions Triton X-100 supernatant Insoluble pellet Nucleocapsid fraction (Peak I) Membrane fraction (Peak II)
371
PROTEIN
Protein (mg)
Recovery (WY
7.0 x 107 2.5 x 10’
2.00 0.70
loo 95
4.5 x 107 2.4 x 10’
1.28 0.68
102 86
1.2 x 107
0.34
69
a Based on the percent of total viral protein represented by each polypeptide as determined by polyacrylamide-gel electrophoresis of SV5 grown in MDBK cells. The values expressed as percent of total protein are: HN = 22; NP = 29; F = 15; SV5 = 8; and M = 25.
tions. About 25% of the amino acid residues are hydrophobic and more than 64% of the amino acid residues are neutral amino acids. As was noted above, the membrane protein is soluble in the presence of detergent and high salt and precipitated when the salt was removed by dialysis against water, even in the continued presence of detergents. These physical properties and the amino acid composition indicate that the M protein is quite hydrophobic, as might be expected from its close association with the lipid bilayer of the viral envelope. DISCUSSION
This report describes a procedure for the isolation of the SV5 membrane protein M and a ribonucleoprotein complex consisting of proteins NP and 5. The membrane protein M was isolated in an electrophoretically pure form using a procedure that relies on the physicochemical properties of this protein. The solubility of the M protein in a nonionic detergent plus high salt and its insolubility in water, even in the presence of a nonionic detergent, suggested that it is a hydrophobic protein. The amino acid analyses which indicated that the protein consisted of 64% neutral amino acid residues confirmed the hydrophobic nature of the M protein. The chemical composition of only a few membrane proteins is known, so a comparison of the
2
AMINO ACID COMPOSITIONOF SV5 MEMBRANE PROTEIN Amino acid
Residues per 100 residues”
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine I r-Cystineb Valine Methionineb Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
6.8 2.0 4.8 7.8 7.1 7.6 11.0 6.5 9.9 5.0 2.0 7.3 2.1 5.7 8.1 2.6 3.8 N.D.
a Average of three determinations. b Not corrected for degradation. c N.D., not determined.
amino acid composition of the M protein with other membrane proteins is limited. However, recently Laver and Baker (1972) showed that the membrane proteins of two strains of influenza virus contain about 80% neutral amino acid residues. Thus, these membrane proteins have a hydrophobic character, a property compatible with their association with the lipid bilayer of the viral envelope. With another paramyxovirus, Newcastle disease virus, utilizing the solubility of the M protein in Triton X-100 and high salt described here, it has been possible to isolate the M protein by extracting the two glycoproteins and the M protein from virions by Triton X-100 on 1 M KC1 and then recovering the M protein by dialyzing away the KCl. The M protein precipitates under these conditions, leaving the glycoproteins in solution in the detergent (Scheid and Choppin, 1973). Thus, two methods are available for isolation of the M protein from paramyxoviruses, each taking advantage of the solubility of this protein in nonionic detergents in the presence of a high salt concentration. The association of a small amount of the
372
McSHARRY
M protein with the nucleocapsid complex after the separation of the aggregate on the sucrose-cesium chloride gradient suggests that there is some affinity between the nucleocapsid and the membrane protein. This finding supports previous studies (Compans et al., 1966; Compans and Choppin, 1971; Choppin et al., 1972) which suggest that, during the maturation of influenza and parainfluenza viruses, the nucleocapsid interacts only with portions of the plasma membrane which contain viral-specific proteins. With the use of vesicular stomatitis virus-SV5 phenotypitally mixed virions, evidence was also obtained that the nucleocapsid of these viruses recognizes the nonglycosylated membrane protein at the plasma membrane during virus assembly (McSharry et al., 1971). Evidence has also been obtained that a portion of the nucleocapsid protein subunit (NP) of paramyxoviruses which can be removed by proteolytic enzymes confers hydrophobicity on the nucleocapsid, and it has been suggested that this portion of NP is involved in the interaction with the M protein, perhaps a hydrophobic et al., 1970, interaction (Mountcastle 1974). Thus there are several lines of evidence suggesting an affinity between the M protein and the nucleocapsid and a role for this affinity in virus assembly. In addition to its providing a recognition site for the nucleocapsid at the plasma membrane, other functions have been suggested for the M protein in virions. It is thought to contribute to the organization of the lipid phase and the stability of the viral membrane, and it may play a role in maintaining a virus-specific domain within the plasma membrane of the infected cell which contains viral proteins but from which host cell proteins are excluded (Choppin et al., 1972; Landsberger et al., 1973; Choppin and Compans, 1975). Additional studies are required to establish the various functions of the M protein; however, the ability to isolate this protein on a preparative scale should permit further characterization of its physical, chemical, and biological properties. The finding of protein 5 associated with the nucleocapsid when it is isolated from
ET AL.
the CsCl-sucrose gradient suggests that this protein is associated with the nucleocapsid in a complex in the virion. However at the present time the possibility cannot be excluded that this association occurs when the virus is disrupted with detergent rather than preexisting in the virion. In any event, the affinity of these two proteins is sufficiently strong to keep them associated in the presence of the detergent and a high salt environment. It is noteworthy that with several other negative-strand viruses, virion-associated RNA transcriptase activity is associated with a complex that contains at least one other protein in addition to the nucleocapsid protein (Stone et al., 1971, 1972; Emerson and Wagner, 1972; Bishop and Roy, 1972; Caliguiri and Compans, 1974). In the case of Sendai virus, a major virion protein (P) with a molecular weight of approximately 75,000 is associated with the nucleocapsid in the transcriptase complex (Stone et al., 1971, 1972; Marx et al., 1974). A major SV5 protein of this size has not been isolated from SV5 virions, although a small amount of a 76,000-dalton protein has been seen in some preparations of SV5 (Caliguiri et al., 1969). The question therefore arises as to whether protein 5 in SV5 might be analogous to protein P of Sendai virus. There is no direct evidence on this point as yet, and this protein might serve other unknown functions. It is perhaps pertinent that no transcriptase activity was found in a preparation of nucleocapsid containing only the SV5 nucleocapsid protein subunit NP (Mountcastle, Compans, and Choppin, unpublished experiments). Additional studies are necessary to characterize further protein 5 and determine its bioligical role. Such studies should be facilitated by the ability to isolate it in association with the nucleocapsid by the procedure described here. ACKNOWLEDGMENTS This work was supported by Grants No. AI-05600 from the National Institutes of Allergy and Infectious Diseases, U.S. Public Health Service and BMS740983 from the National Science Foundation. J. J. M&harry was a postdoctoral fellow of NIAID. We are grateful for the assistance of Mrs. Ann
ISOLATION Erickson, Miss Cathleen lynn Santa Maria.
O’Connell
OF SV5 MEMBRANE
and Mrs. Caro-
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and purification of the envelope proteins of Newcastle disease virus. J. Viral. 50, 640-652. SCHEID, A., and CHOPPIN, P. W. (1974). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57,
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ET AL. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. STONE, H. O., KINGSBERRY, D. W., and DARLINGTON, R. W. (1972). Sendai virus-induced transcriptase from infected cells: Polypeptides in the transcription complex. J. Viral. 10, 1037-1043. STONE, H. O., PORTNE~ A., and KINGSBERRY, D. W. (1971). Ribonucleic acid transcriptases in Sendai virions and infected cells. J. Viral. 8, 174-180.
