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Structural proteins of Semliki Forest virus and its nucleocapsid
VIROLOGT
2, 321-329 (1970)
Structural
Proteins
of Semliki
XICHOLAS The Eockefeller
Forest
H. ACHESON [Jniversity, Accepted
Virus AXD
and
IGOR
Its Nucleocapsid’ TAM31
New York, New York 10021 February
10, 19YO
The structural proteins of Semliki Forest virus and its nucleocapsid were investigated by SDS-polyacrylamide gel electrophoresis. Purified virus gives rise to three electrophoretically distinct bands of protein, and two of these bands are found in purified nucleocapsids. However, the protein in the minor nucleocapsid band probabl) represents a dimer of the major nucleocapsid protein. The minor band protein has twice the molecular weight of the protein in t,he major band, and contains the same relative proportions of lysine, valine, and leucine as the major band protein. The minor band occurs in varying amounts depending on the preparation analyzed. The nucleocapsid protein, with a molecular weight of -32,000, is relatively rich in lysine, a basic amino acid; the envelope protein, which has a molecular weight of -51,600, is relatively rich in leucine and valine, hydrophobic amino acids. There are approximately 2.5 molecules of envelope protein per molecule of nucleocapsid protein in Semliki Forest virus particles. INTRODUCTION
Semliki Forest virus, a group A arbovirus, consists of a spherical nucleocapsid tightly enclosed in a lipid-containing envelope (Osterrieth and Calberg-Bacq, 1966; Acheson and Tamm, 1967). Viral nucleocapsids can be isolated from extracts of infected chick embryo cells and purified by gentle methods (Acheson and Tamm, 1970). Complete virus particles can be purified from the extracellular fluids taken from the same cell cultures. The present paper reports the results of SDSpolyacrylamide gel electrophoresis of the proteins of both purified nucleocapsids and purified virus. Two published reports are in disagreement concerning the number of electrophoretically distinct structural proteins in Semliki Forest virus. Friedman (1968) found three distinct radioactive bands in gel electropherograms 1 This investiagtion was supported by research grant AI-03445 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service. 2 Dr. Acheson’s present address is: Swiss Institute for Experimental Cancer Research, 21 Rue du Bugnon, Lausanne, Switzerland.
of Semliki Forest virus proteins. Two bands were present in nucleocapsids and the third band presumably arose from the viral envelope. On the other hand, Hay et al. (1968) found only two bands of isotopically labeled protein in gels of Semliki Forest virus proteins. However, these authors found a third band in stained gels. We have found three bands of protein in purified virus and two bands in purified nucleocapsids, both in stained gels and in gels assayed for radioactivity. However, further analysis indicates that the polypeptides in the minor band of nucleocapsid protein are probably dimers of the polypeptides in the major band. This finding suggests that there are only two electrophoretically distinct polypeptide species in Semliki Forest virus particles, one in the nucleocapsid and one in the envelope. Data are also given on the molecular weights, relative proportions, and relative amino acid contents of the two viral structural proteins. MATERIALS
AND
METHODS
Virus and cells. Semliki Forest virus, Kumba strain, was grown in BHK21 cells as 321
322
ACHESON
previously described (Acheson and Tamm, 1967). Primary monolayer cultures of chick embryo cells were grown in loo-mm petri dishes in lactalbumin hydrolyzate medium containing 2 % calf serum. Chemicals awl bu$ers. Trypsin (Worthington Biochemical Corp., Freehold, New Jersey) and crystalline bovine serum albumin (BSA) (Armour Pharmaceutical Co., Chicago, Illinois) were used as st,andards of known molecular weights. Actinomycin D was a gift from Merck, Sharpe and Dohme Research Laboratories, Rahway, New Jersey. Potassium tartrate, crystal, was obtained from ,J. T. Baker Chemical Co., Phillipsburg, New ,Jersey. The following radioactively labeled compounds were used : L-leucine-4, 5-3H, 30-50 Ci/mmole and reconstituted protein hydrolyzate-3H, New England Nuclear Co., Boston, Massachusetts; L-14C-lysine, 300 mCi/mmole, L-14C-valine, 260 mCi/mmole, and reconstituted protein hydrolyzate-3H, 1 Ci/mmole, Schwarz BioResearch, Orangeburg, New York. Phosphate-sodium chloride (PN) buffer was 0.01 M sodium phosphate, pH 7.3,0.1 M NaCl. Labeling of nucleocapsids and virus. Monolayer cultures of chick embryo cells in lOOmm petri dishes were incubated for 1 hour at 37” with lo-50 PFU/cell of virus in 1.5 ml of reinforced Eagle’s medium (Bablanian et al., 1965) plus 1% BSA. In some cases cells were incubated with 1-2 pg/ml actinomycin D in 5 ml of medium per plate from 1 to 4 hours after infection to reduce cellular protein synthesis (Strauss et al., 1969; Friedman, 1968), and labeled amino acids were added at 4 hours. In other cases cells were not incubated with actinomycin D, and labeled amino acids were added 1 hour after infection. Labeled amino acids were always added in 5 ml per plate of fresh medium containing only Mo the normal concentration of those amino acids and 0.24.5 % BSA. Cells and medium were harvested at 9-12 hours after infection. Purification of nucleocapsids. The pelleted cells were Dounce homogenized and nucleocapsids were purified by sedimentation in three successive sucrose gradients by the method described in Acheson and Tamm
AND
TAMM
(1970). Purified nucleocapsids were dialyzed extensively against PN buffer prior to disruption and electrophoresis. Puri$cation of vii-us. The supernatant medium after pelleting the cells was clarified by centrifugation in the Spinco S30 rotor at 10,000 y for 15 min at 4”. Virus was pelleted from the supernatant by centrifugation in the S30 rotor at 74,000 g for 90 min at 4”. The virus pellets were resuspended in a small volume of 5 % (w/w) potassium tartrate in Pn’ buffer, and pipetted vigorously or Dounce homogenized to break up aggregates. The virus suspension was layered over a preformed linear 20-30 % (w/w) potassium tartrate gradient prepared in PN buffer and centrifuged in the SW 27 rotor at 90,000 g until the virus reached its equilibrium buoyant density (6-12 hours). The light-scattering band of virus near the middle of the tube was removed, diluted 1:2 with PN buffer, and rebanded in a second potassium tartrate gradient. The band was collected and dialyzed extensively against PN buffer before disruption and electrophoresis. SDS -polyacrylamide gel electrophoresis. Polyacrylamide gels, 12 cm in length, were polymerized in glass tubes 0.6 cm in diameter. The gels contained 7.5 % (w/v) acrylamide, 0.2 % N, N’-bismethylene acrylamide, 0.075 % ammonium persulfate, and 0.05% (v/v) N, N, N’ , N’-tetramethylethylenediamine (all obtained from Canal Industrial Corp., Rockville, Maryland), in 0.1 M sodium phosphate buffer, pH 7.2, with 0.1% SDS (Summers et al., 1965). A 0.5 cm spacer gel consisting of 2.5 % acrylamide was subsequently formed above each gel. Gels were prerun for 2 hours at 3 V/cm to remove unreacted persulfate. In some cases, 0.1 M reduced glutathione (Calbiochem, Los Angeles, California) was included in the upper (anodal) buffer chamber during prerunning and electrophoresis (Strauss et al., 1969). Nucleocapsid or virus samples in PN buffer were made 1% in SDS and 1% in /3mercaptoethanol or 0.01 M in dithiothreitol, and heated in a boiling water bath for 1 min. One-tenth volume of 60 % (w/w) sucrose and of bromophenol blue (tracking dye) were added, and 25-200 ~1 samples were layered onto gels. Electrophoresis was carried out at
PROTEINS
OF SIGMLIKI
room temperature for 1 hour at 2 V/cm, to allow the proteins to enter the spacer gel, and then for 12-14 hours at 3 V/cm. Gels were extruded from the tubes and either stained or frozen on dry ice for slicing and counting. To stain, gels were fixed for 18 hours in 20 % (w/v) sulfosalicylic acid, stained for 18-24 hours in 0.25 % Coomassie blue, and washed in several changes of 7 % acetic acid for 24 hours. Frozen gels were sliced into l-mm segments (Fromageot and Zinder, 196s) which were placed into scintillation vials. Each slice was allowed to swell for 1-2 hours in 0.05 ml water. Then 0.2 ml NCS (Amersham-Searle, Inc.,) was added, and after 1-2 hours 0.3 ml more NCS was added. Vials were capped and stored overnight, and then 10 ml toluene containing 160 ml/gallon Liquifluor (New England Nuclear Corp.) were added. The samples were counted in a Packard Model 3375 liquid scintillation counter. RESULTS
Purification
of Virus awl Nucleocapsids
Virus was grown and purified as described in Materials and Methods. Fig. 1 shows the
__.J 0
38
35
30
FOREST
323
VIRUS
results of the second potassium tartrate gradient centrifugation of virus labeled with Iysine-14C and leucine-3H. There is a single peak of virus near the middle of the gradient at a density of approximately 1.1s g/ml. The profiles of absorbance and radioactivity overlap, and no other ultraviolet-absorbing or radioactive components are present. Sixty to SO% of the initial virus infectivity was recovered from this band before dialysis. Dialysis U.S.PN buffer prior to electrophoresis of viral proteins leads to disruption of most virus particles. Purified nucleocapsids sediment as a single peak in sucrose gradients, contain predominantly 45 S viral R?JA, and are free of particulate contamination as revealed by electron microscopy (Acheson and Tamm, 1970). Electyophoresis oj Nucleocapsid Proteix Purified nucleocapsids labeled with a 3Hamino acid mixture were disrupted and subjected to electrophoresis in SDS-polyacrylamide gels as described in Materials and Methods. Figure 2 shops the profile of radioactivity from such a gel, and in addition a stained gel of a similar sample. ,4 single
L I
25
20
15
IO
5
Fraction FIG. 1. Purification of Semliki Forest virus in a potassium tartrate density gradient. Cells were incubated with 1.3 pg/ml actinomycin D from 1 to 4.5 hours after infection. Virus was labeled with lysine-‘4C and leucine-3H from 4.5 to 9.5 hours after infection, then harvested and purified as described in Materials and Methods. Shown here are the results of the second successive equilibrium banding of virus in a 20-30yc potassium tartrate gradient. The gradient was collected by displacement, and absorbance at 254 nm was continuously monitored. Aliquots of l-ml fractions were assayed for acid-precipitable radioactivity on Whatman 3 MM titer paper disks (see Acheson and Tamm, 1970). Measurements of refractive index were made on selected fractions, and densities at 5” were calculated from an empirically determined relation between refractive index and density of potassium tartrate solutions.
324
ACHESON
AND
major band can be seen in both the stained and the counted gels. In addition, there is a minor band, very faint in the stained gel shown, which migrates more slowly than the major band. This minor band is consistently found both in stained gels and in gels assayed for radioactivity, whether nucleocapsids are disrupted in the presence of /3-mercaptoethanol or dithiothreitol, and whether or not glutathione is present in the gel during electrophoresis. Electrophoresis of Virus
Protein
Figure 3 shows the profile of radioactivity in an electropherogram of proteins from purified virus labeled with a 3H-amino acid mixture. A stained gel of a similar preparation is also shown. There are two major bands and a slower-migrating minor band in both the stained and the counted gels. The faster-migrating major band and the minor band correspond in position to the 2 bands of protein from purified nucleocapsids (Fig.
z
500
I-
400
/-
TAMM
2). The other major band is not found in nucleocapsids and thus probably represents the viral envelope protein. In some gels this band was very broad or had a shoulder as also reported by Strauss et al. (1969) for the analogous protein from Sindbis virus. Incorporation of 0.1 Ad glutathione into the gel and the anodal buffer (Strauss et al., 1969) reduced the half-width of this peak without affecting the behavior of the other two bands. In gels of nucleocapsid or virus protein which were first stained, then sliced and counted, the positions of the stained bands corresponded exactly with those of the radioactive peaks. No other bands were seen if freshly prepared samples were analyzed. Molecular
Weights of the Viral Proteins
Trypsin and BSA were included in some gels as standards, and the molecular weights of the three viral protein bands were estimated by the method of Shapiro et al. (1967).
300
u L ,‘:
200
100
00
0r1g1r-1
IO
20
30
40
50
60
70
Fraction FIG. 2. SDS-polyacrylamide gel electrophoresis of proteins from purified Semliki Forest virus nucleocapsids. Infected cells were incubated with a 3H-amino acid mixture from 1 to 12 hours after infection; nucleocapsids were harvested and purified. Radioactivity profile: nucleocapsids were disrupted in 170 SDS and 1% p-mercaptoethanol and subjected to electrophoresis in the presence of 0.1 M glutathione. One-millimeter slices of the frozen gel were counted as described in Materials and Met,hods. The direction of migrat,ion is from left to right, Stained gel: an identical sample was run in a similar gel which, however, did not contain glutathione. The gel was fixed and stained with Coomassie blue as described in Materials and Methods. The photograph of the stained gel was reproduced so that the major st,ained band is coincident with the major peak in the radioactivity profile.
PROTEIKS
OF SEMLIKI
FOREST
lYII~US
Fraction FIG. 3. Electrophoresis of protein from purified Semliki Forest virus. Infected cells were incubated with a 3H-amino acid mixture from 1 t,o 12 hours after infection; virus was subsequently harvested and purified. Radioactivity profile: virus was disrupted in lyO SDS and 17, P-mercaptoethanol and subjected to electrophoresis in a gel not, containing glutathione. Stained gel: a similar sample was I-L~ in a gel not containing glutathione. The gel was fixed and stained with Coomassie blue. The phot,ograph of t.he stained gel was reproduced so that the major stained bands are coincident with the major peaks in the radioactivity profile.
Figure 4 shows the results of one such experiment. In six experiments, the molecular weight of the protein in the major nucleocnpsid band was 31,00034,000, with a mean of 32,000. In four experiments, the molecular weight of the envelope protein was 50,000-53,000, with a mean of 51,500. in The minor band of nucleocapsid protein ran in the same position as BSA, at a molecular n-eight of 67,000. Is tlze Jlinor
Nucleocapsid Protein a Dimer?
The polypeptide chains in the minor band of nucleocapsid protein have a molecular weight approximately twice that of the polypeptides in the major nucleocapsid band. This suggested that the polypeptides in the minor band may not represent a distinct species, but instead may be dimers of the polypeptide chains in the major band. Two further lines of evidence also favor this hypothesis. Amino acid ratios in the minor and major bands. Nucleocapsids were grown in the presence of two different amino acids, one
labeled with 3H and one with 14C. Purified nucleocapsids were disrupted and run on gels, and the ratios of counts in 14C and 3H were measured for each band. If the polypeptide chains within the two bands had different amino acid compositions, the ratios of 14C to 3H should be different. If, on the other hand, the polypeptide chains within the minor band were dimers of those within the major band, there should be no difference in their amino acid compositions and thus no significant difference between the 14C:3H ratios of the two bands. Two pairs of amino acids were used: lysine-14C with leucineJH, and valine-14C with leucine-“H. Gels were run both in the presence and the absence of 0.1 ild glutathione. Table 1 shows that for each pair of amino acids there was no significant difference between the 14C:3H ratios in the minor band and the major band. However, in gels of labeled virus the 14C:3H ratios in the envelope protein were significantly different from those in the nucleocapsid protein. Thus the polppeptide chains within the
326
ACHESON
SND
TAMM
80,000
i .-cm %
40,000
is 5 : z 2 20,000
IO,OOC
igin
I
I
I
I
I
I
t
I
I
2
3
4
5
6
7
8
Distance
of migration,
cm
FIG. 4. Determination of the molecular weight,s of Semliki Forest virus proteins. Ten micrograms each of trypsin and bovine serum albumin (filled circles) were denatured and mixed with disrupted nucleocapsid or virus protein (empty circles). The mixtures were run on gels and stained with Coomassie blue. The distances of migration of the standard proteins were plotted as a function of the logarithm of their molecular weights, and the molecular weights of the envelope and nucleocapsid prot,eins were estimated from their relative migration distances. The nucleocapsid protein dimer migrates in nearly the same position as bovine serum albumin.
major and minor bands of nucleocapsid protein contain the same relative proportion of three different amino acids: lysine, valine, and leucine. Relative proportiow of major ancl minor bands. If the minor band represented a distinct structural nucleocapsid protein it should be present in the same proportion to the major band regardless of the sample analyzed. If, on the other hand, the minor band contained a dimer of the protein in the major band, its relative amount might vary depending on the nucleocapsid preparation analyzed. Several different purified, labeled nucleocapsid preparations were sub-
jected to electrophoresis, and the amount of label in the minor band relative to that in the major band (set at 100) was calculated for each sample. The upper part of Table 2 shows that in samples labeled with a 3Hamino acid mixture there is a Z-fold spread among the values for the relative number of counts in the minor band. The lower part of the table shows the proportion of counts in the minor band from a sample labeled with lysineJ4C and one labeled with valineJ4C. These two values can be compared since the data from Table 1 show that the relative amount of lysine and valine in the two bands is the same. Again there is a
PROTEINS TBBLE lC:3II
OF SEMLIKI
xperimerit
acids
Valine-‘“C 1erlcine-3H
Lysine-14C 1eucine-3H
and
and
Band
Xi
VIRUS TABLE
1
RATIOS IN THE MUOR .IND MINOR BINDS K;UCLICOC.\PSID PROTEIN LABELED WITH Two I~IFFERENT A~XINO ACIDS’
Amino
FOREST
OF
‘WI3 3H: channels ratio i standard err&’
Major Minor Major Minor
1.40 1.39 1.38 1.36
f f f It
0.01 0.06 0.01 0.04
Major Minor Major Minor
2.13 2.oG 2.09 2.20
f 0.01 f. 0.07 f 0.01 f 0.08
2
REL.\TIVE INCORPOR.~TION OF R.~DIO.UXWE AMIXO ACIDS 1.~~0 THE MUOR UTD MINOR B.ZNDS OF NUCLEOC.~PSID PROTJCIX
Labeled amino acid
“H-Amino
acid mixture
100 ~ 13.8 ~ bO.7 100 15.4 f0.6 100 2.8, f0.4
Lvsine-14C
100 100
4.il +O.l 4.81 f0.2
Valine-14C
100 100
4.0 4.1
f0.2 zto.3
u In gels of purified virus doubly labeled in the same experiments, the 14C:3H channels ratios in the envelope protein band were 1.02 & 0.01 (valineJ4C and leucine-3H) and 0.65 =t 0.01 (lysinel*C and 1eucineJH). b Standard errors were computed from the following formula:
a The values for the minor band were calculated by setting the number of comits per minute in the major band at. 100. b Standard errors were computed from the following formula:
Standard error = d(l/W C + (C/W2 H where C = total counts in 1% channel, corrected for background; H = tot,al counts in 3H channel, correct,ed for background.
Standard error = N(dcpm/cpm) where cpm is the total cpm in the minor band, corrected for background, and N is the relative cpm in the minor band.
significant difference between the relative amounts of label in the minor bands from the two samples of nucleocapsids. Thus the proportion of nucleocapsid protein in the minor band is not constant from one sample to the next. In further support of this finding, storage of a sample of purified virus for 1 week at 4” brought about an increase in the amount of label in the minor nucleocapsid band and a corresponding decrease in the major nucleocapsid band. These results suggest that, depending on conditions of isolation and age of sample, a certain proportion of the nucleocapsid protein is present as a dimer. The dimerixation cannot be reversed by the standard methods used to disrupt viral proteins for SDSpolyacrylamide gel electrophoresis, nor by the incorporation of 0.1 AI glutathione in the buffers and gel (Strauss et al., 1969). It thus appears that there is only one electrophoretically distinct type of polypeptide chain, of molecular weight approximatelyL32,000, in nucleocapsids of Semliki Forest virus.
Further Data on Envelope arzd Nucleocapsid Proteins
I
In gels of proteins from purified virus labeled with a 3H-amino acid mixture, the ratio of label in the envelope protein to that in the nucleocapsid protein is approximately 4.0 (Table 3). This value should equal the ratio between the amounts of envelope protein and nucleocapsid protein in virus, because the 3H-amino acid mixture should label both proteins uniformly. Since the envelope protein has a molecular weight of 51,000, and the nucleocapsid protein a molecular weight of 32,000, there are approximately 32,000/51,000 X 4.0, or 2.5 molecules of envelope protein per molecule of nucleocapsid protein. Purified virus doubly labeled with lysine14Cand leucine-3H, or valine-14C and leucine3H, was also analyzed by electrophoresis. Two points of interest can be mentioned. First, the ratio of 14Cto 3H is constant in a number of fractions on both sides of the peak fractions of the envelope and nucleo-
328
ACHESON TABLE
3
RELATIVE INCORPOR.1TION OFRADIO.~CTI~E ACIDSINTOTHI:ESVELOPE.~NDNUCLEOC.IPSID PROTEINS IN VIRUS PARTICLES
Labeled amino acid
3H-Amino
acid mixture
Experiment
Ratio,
AMIKO
cpm in envelope protein n&~&&id protein
A B C
3.9 4.0 4.0
Lysine-W
1) E
1.6 1.5
Valine-14C
F G
5.2 5.2
D E F G
7.0 6.9 5.8 5.1
capsid protein bands. This result makes it unlikely that either band contains more than one distinct polypeptide species of slightly different electrophoretic mobilities. Second, the ratio of label in the envelope protein to that in the nucleocapsid protein is lower when lysine is used, and higher when valine or leucine is used, than with a mixture of amino acids (Table 3). This suggests that the nucleocapsid protein, which is associated with the viral RNA (Acheson and Tamm, 1970) is relatively rich in lysine, a basic amino acid. Also, the envelope protein, which is associated with lipids in a membranelike structure (Acheson and Tamm, 1967; Strauss et al., 1968; Friedman and Pastan, 1969), is richer in valine and leucine, hydrophobic amino acids. DISCUSSION
The data presented in this paper indicate that Semliki Forest virus contains only two structural proteins: an envelope protein, of molecular weight -51,000, and a nucleocapsid protein, of molecular weight -32,000. A third electrophoretically distinct band probably represents a dimer of the nucleocapsid protein which is not dissociated by the methods used. No other discrete protein species were detected in virus or nucleo-
AND
TAMM
capsids purified as described. Virus particles contain approximately 2.5 molecules of envelope protein per molecule of nucleocapsid protein. The nucleocapsid protein is relatively rich in lysine, a basic amino acid, and the envelope protein is relatively rich in leucine and valine, hydrophobic amino acids. On the basis of its structural proteins, Semliki Forest virus is thus very similar t’o Sindbis virus, which has an envelope protein of molecular weight -53,000 and a nucleocapsid protein of molecular weight -30,000 (Strauss et al., 1968, 1969). These two group -4 arboviruses are also alike in their antigenic determinants (Casals and Clarke, 1965), RNA size (Sonnabend et al., 1966; Pfefferkorn et al., 1967)) and morphology (Simpson and Hauser, 1968a, b), although Simpson and Hauser (196813) detected differences in nucleocapsid morphology in thin sections of the two viruses. The envelope proteins of both Sindbis and Semliki Forest viruses tend to form broad or double bands upon electrophoresis unless a reducing agent such as glutathione is present in the gel (Strauss et al., 1969). Sindbis virus nucleocapsid protein apparently does not have a tendency to dimerize as does Semliki Forest virus nucleocapsid protein. Our results are in essential agreement with those of Friedman (1968), who found three peaks of radioactivity in autoradiograms of gel electropherograms of Semliki Forest virus. However, in that report the possibility that the second nucleocapsid protein band might be a dimer of the major nucleocapsid protein was not discussed. The absence of the third band in electropherograms of isotopically labeled proteins of Semliki Forest virus, published by Hay et al. (1968), is complicated by the presence of a third band in their stained gels. It seems possible in this case (cf. their Fig. 3) that the third band (nucleocapsid dimer) appeared only as a small shoulder on the trailing edge of the labeled envelope protein band because the gels may not have been run long enough or at a high enough voltage to separate the two bands. If this is true, their results would agree with the present ones and with those of Friedman (1968).
PI:OTEINS
OF SUILTKI
ACKNOWLEDGRIENT We thank Lawrence A. Caliguiri for instruction in the techniclues of gel electrophoresis. REFERENCES ACHESON, N. II., and TAMM, 1. (1967). Replication of Semliki Forest virus: an elect,ron microscopic study. Virology 32, 128-143. ACHK~ON, N. II., and T,.IMM, I. (1970). Purification and propert,ies of Semliki Forest virus nucleocapsids. Virology 41, 306. BABLAXIAP~, R., EGGERS, H. J., and TAMM, I. (1965). St,udies on the mechanism of poliovirusinduced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations in cldtnred cells. Virology 26, 101% 113. CASALS, J., and CLARKE, D. H. (1965). Arboviruses; Group A. In “\‘iral and Rickettsial Infections of Man” (F. L. Horsfall and I. Tamm, eds.), pp. 583-605. Lippincott, Philadelphia. FRIEDMAN, R. M. (1968). Structural and nonstruct,ural proteins of an arbovirus. J. Viral. 2, 107G1080. FRIEDMAN, It. M., and P~STAN, I. (19G9). Nature and function of the structural phospholipids of an arbovirus. J. 3101. Viol. 40, 107-115. FKOM~GEOT, H. P. M., aud ZINDER, N. II. (1968). Growth of bacteriophage f2 in E. coli treated with rifampicin. Proc. Xall. Acad. Sci. CA’. 61, 184-191. Hay, A. J., SKFHIEL, J. J., and BUIZKE, D. C. (1968). Proteins synthesized in chick cells following infection with Semliki Forest virus. J. Gen. Viral. 3, 175-184. OSTERRIBTH, P. M., and C~LBERG-BACQ, C. M. (1966). Changes in morphology, infectivity, and
FOREST
\-IRCS
:12(3
haemagglutinating activity of Semliki ForcTst virus produced by the treatment with caseillasc C from Streplo7~yce.s alhrrs G. J. Germ. .llic,&iol. 43, 19-30. PFI:FFI:RBOIW, 15. II., BURW, 13. W., and CO.ZDY, H. M. (1967). lnt,racellrrlar conversion of the RNA of Sindbis virus to a double-strarltled form. Virology 33, 239-249. SHAPIRO, 8. L., \-I&-ELA, E., aud ~Iarz1:1,, J. I-., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis ill SDS-polyacrylamide gels. Biocheln. Biophys. Ites. (‘OWL~~~zcn.28, 815-820. SIMPSOX, It. W., alld I-Iaus~n, R. E. (1968:r). Basic structure of group A arbovirus strains hliddleburg, Sindbis, and Semliki Forest examined by negative staining. I-iro2ogy 34, 358-361. SIMPSON, 11. W., and I~AUSEI~, K. I<. (1968b). Structural differentiation of grolip A srboviruses based on nucleoid morphology in ultrathin sections. I’irologjj 34, 508-570. SOSNABWW, J. ,4., MAWIN, IS. AI., nd Mr:cs, E. (1967). Viral specific RNA’s in infected cells. L!Jatwx 213, 365-367. STRAUSS, J. II., JR., BURGE, B. W., and ~~.uc.um~,, J. E., JR. (1969). Sindbis virus jufection of chick and hamst,er cells: synthesis of virlls-specific proteins. J’irology 37, 367-376. STRAUSS, J.H.,Jll., B~TRGK, B.W., PFEFFERKORN, E. R., and DARNELL, J. E., JR. (1908). Identification of the membrane protein and “core” protein of Sindbis virus. Proc. h’all. 9 cad. &‘ci. C.S. 59, 533-537. SUMMERS, I). F., MAIZEL, J. I*., and II~RKELL, J. E., JR. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Xatl. Acad. Sci. lT.S. 54, 505-513.
2, 321-329 (1970)
Structural
Proteins
of Semliki
XICHOLAS The Eockefeller
Forest
H. ACHESON [Jniversity, Accepted
Virus AXD
and
IGOR
Its Nucleocapsid’ TAM31
New York, New York 10021 February
10, 19YO
The structural proteins of Semliki Forest virus and its nucleocapsid were investigated by SDS-polyacrylamide gel electrophoresis. Purified virus gives rise to three electrophoretically distinct bands of protein, and two of these bands are found in purified nucleocapsids. However, the protein in the minor nucleocapsid band probabl) represents a dimer of the major nucleocapsid protein. The minor band protein has twice the molecular weight of the protein in t,he major band, and contains the same relative proportions of lysine, valine, and leucine as the major band protein. The minor band occurs in varying amounts depending on the preparation analyzed. The nucleocapsid protein, with a molecular weight of -32,000, is relatively rich in lysine, a basic amino acid; the envelope protein, which has a molecular weight of -51,600, is relatively rich in leucine and valine, hydrophobic amino acids. There are approximately 2.5 molecules of envelope protein per molecule of nucleocapsid protein in Semliki Forest virus particles. INTRODUCTION
Semliki Forest virus, a group A arbovirus, consists of a spherical nucleocapsid tightly enclosed in a lipid-containing envelope (Osterrieth and Calberg-Bacq, 1966; Acheson and Tamm, 1967). Viral nucleocapsids can be isolated from extracts of infected chick embryo cells and purified by gentle methods (Acheson and Tamm, 1970). Complete virus particles can be purified from the extracellular fluids taken from the same cell cultures. The present paper reports the results of SDSpolyacrylamide gel electrophoresis of the proteins of both purified nucleocapsids and purified virus. Two published reports are in disagreement concerning the number of electrophoretically distinct structural proteins in Semliki Forest virus. Friedman (1968) found three distinct radioactive bands in gel electropherograms 1 This investiagtion was supported by research grant AI-03445 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service. 2 Dr. Acheson’s present address is: Swiss Institute for Experimental Cancer Research, 21 Rue du Bugnon, Lausanne, Switzerland.
of Semliki Forest virus proteins. Two bands were present in nucleocapsids and the third band presumably arose from the viral envelope. On the other hand, Hay et al. (1968) found only two bands of isotopically labeled protein in gels of Semliki Forest virus proteins. However, these authors found a third band in stained gels. We have found three bands of protein in purified virus and two bands in purified nucleocapsids, both in stained gels and in gels assayed for radioactivity. However, further analysis indicates that the polypeptides in the minor band of nucleocapsid protein are probably dimers of the polypeptides in the major band. This finding suggests that there are only two electrophoretically distinct polypeptide species in Semliki Forest virus particles, one in the nucleocapsid and one in the envelope. Data are also given on the molecular weights, relative proportions, and relative amino acid contents of the two viral structural proteins. MATERIALS
AND
METHODS
Virus and cells. Semliki Forest virus, Kumba strain, was grown in BHK21 cells as 321
322
ACHESON
previously described (Acheson and Tamm, 1967). Primary monolayer cultures of chick embryo cells were grown in loo-mm petri dishes in lactalbumin hydrolyzate medium containing 2 % calf serum. Chemicals awl bu$ers. Trypsin (Worthington Biochemical Corp., Freehold, New Jersey) and crystalline bovine serum albumin (BSA) (Armour Pharmaceutical Co., Chicago, Illinois) were used as st,andards of known molecular weights. Actinomycin D was a gift from Merck, Sharpe and Dohme Research Laboratories, Rahway, New Jersey. Potassium tartrate, crystal, was obtained from ,J. T. Baker Chemical Co., Phillipsburg, New ,Jersey. The following radioactively labeled compounds were used : L-leucine-4, 5-3H, 30-50 Ci/mmole and reconstituted protein hydrolyzate-3H, New England Nuclear Co., Boston, Massachusetts; L-14C-lysine, 300 mCi/mmole, L-14C-valine, 260 mCi/mmole, and reconstituted protein hydrolyzate-3H, 1 Ci/mmole, Schwarz BioResearch, Orangeburg, New York. Phosphate-sodium chloride (PN) buffer was 0.01 M sodium phosphate, pH 7.3,0.1 M NaCl. Labeling of nucleocapsids and virus. Monolayer cultures of chick embryo cells in lOOmm petri dishes were incubated for 1 hour at 37” with lo-50 PFU/cell of virus in 1.5 ml of reinforced Eagle’s medium (Bablanian et al., 1965) plus 1% BSA. In some cases cells were incubated with 1-2 pg/ml actinomycin D in 5 ml of medium per plate from 1 to 4 hours after infection to reduce cellular protein synthesis (Strauss et al., 1969; Friedman, 1968), and labeled amino acids were added at 4 hours. In other cases cells were not incubated with actinomycin D, and labeled amino acids were added 1 hour after infection. Labeled amino acids were always added in 5 ml per plate of fresh medium containing only Mo the normal concentration of those amino acids and 0.24.5 % BSA. Cells and medium were harvested at 9-12 hours after infection. Purification of nucleocapsids. The pelleted cells were Dounce homogenized and nucleocapsids were purified by sedimentation in three successive sucrose gradients by the method described in Acheson and Tamm
AND
TAMM
(1970). Purified nucleocapsids were dialyzed extensively against PN buffer prior to disruption and electrophoresis. Puri$cation of vii-us. The supernatant medium after pelleting the cells was clarified by centrifugation in the Spinco S30 rotor at 10,000 y for 15 min at 4”. Virus was pelleted from the supernatant by centrifugation in the S30 rotor at 74,000 g for 90 min at 4”. The virus pellets were resuspended in a small volume of 5 % (w/w) potassium tartrate in Pn’ buffer, and pipetted vigorously or Dounce homogenized to break up aggregates. The virus suspension was layered over a preformed linear 20-30 % (w/w) potassium tartrate gradient prepared in PN buffer and centrifuged in the SW 27 rotor at 90,000 g until the virus reached its equilibrium buoyant density (6-12 hours). The light-scattering band of virus near the middle of the tube was removed, diluted 1:2 with PN buffer, and rebanded in a second potassium tartrate gradient. The band was collected and dialyzed extensively against PN buffer before disruption and electrophoresis. SDS -polyacrylamide gel electrophoresis. Polyacrylamide gels, 12 cm in length, were polymerized in glass tubes 0.6 cm in diameter. The gels contained 7.5 % (w/v) acrylamide, 0.2 % N, N’-bismethylene acrylamide, 0.075 % ammonium persulfate, and 0.05% (v/v) N, N, N’ , N’-tetramethylethylenediamine (all obtained from Canal Industrial Corp., Rockville, Maryland), in 0.1 M sodium phosphate buffer, pH 7.2, with 0.1% SDS (Summers et al., 1965). A 0.5 cm spacer gel consisting of 2.5 % acrylamide was subsequently formed above each gel. Gels were prerun for 2 hours at 3 V/cm to remove unreacted persulfate. In some cases, 0.1 M reduced glutathione (Calbiochem, Los Angeles, California) was included in the upper (anodal) buffer chamber during prerunning and electrophoresis (Strauss et al., 1969). Nucleocapsid or virus samples in PN buffer were made 1% in SDS and 1% in /3mercaptoethanol or 0.01 M in dithiothreitol, and heated in a boiling water bath for 1 min. One-tenth volume of 60 % (w/w) sucrose and of bromophenol blue (tracking dye) were added, and 25-200 ~1 samples were layered onto gels. Electrophoresis was carried out at
PROTEINS
OF SIGMLIKI
room temperature for 1 hour at 2 V/cm, to allow the proteins to enter the spacer gel, and then for 12-14 hours at 3 V/cm. Gels were extruded from the tubes and either stained or frozen on dry ice for slicing and counting. To stain, gels were fixed for 18 hours in 20 % (w/v) sulfosalicylic acid, stained for 18-24 hours in 0.25 % Coomassie blue, and washed in several changes of 7 % acetic acid for 24 hours. Frozen gels were sliced into l-mm segments (Fromageot and Zinder, 196s) which were placed into scintillation vials. Each slice was allowed to swell for 1-2 hours in 0.05 ml water. Then 0.2 ml NCS (Amersham-Searle, Inc.,) was added, and after 1-2 hours 0.3 ml more NCS was added. Vials were capped and stored overnight, and then 10 ml toluene containing 160 ml/gallon Liquifluor (New England Nuclear Corp.) were added. The samples were counted in a Packard Model 3375 liquid scintillation counter. RESULTS
Purification
of Virus awl Nucleocapsids
Virus was grown and purified as described in Materials and Methods. Fig. 1 shows the
__.J 0
38
35
30
FOREST
323
VIRUS
results of the second potassium tartrate gradient centrifugation of virus labeled with Iysine-14C and leucine-3H. There is a single peak of virus near the middle of the gradient at a density of approximately 1.1s g/ml. The profiles of absorbance and radioactivity overlap, and no other ultraviolet-absorbing or radioactive components are present. Sixty to SO% of the initial virus infectivity was recovered from this band before dialysis. Dialysis U.S.PN buffer prior to electrophoresis of viral proteins leads to disruption of most virus particles. Purified nucleocapsids sediment as a single peak in sucrose gradients, contain predominantly 45 S viral R?JA, and are free of particulate contamination as revealed by electron microscopy (Acheson and Tamm, 1970). Electyophoresis oj Nucleocapsid Proteix Purified nucleocapsids labeled with a 3Hamino acid mixture were disrupted and subjected to electrophoresis in SDS-polyacrylamide gels as described in Materials and Methods. Figure 2 shops the profile of radioactivity from such a gel, and in addition a stained gel of a similar sample. ,4 single
L I
25
20
15
IO
5
Fraction FIG. 1. Purification of Semliki Forest virus in a potassium tartrate density gradient. Cells were incubated with 1.3 pg/ml actinomycin D from 1 to 4.5 hours after infection. Virus was labeled with lysine-‘4C and leucine-3H from 4.5 to 9.5 hours after infection, then harvested and purified as described in Materials and Methods. Shown here are the results of the second successive equilibrium banding of virus in a 20-30yc potassium tartrate gradient. The gradient was collected by displacement, and absorbance at 254 nm was continuously monitored. Aliquots of l-ml fractions were assayed for acid-precipitable radioactivity on Whatman 3 MM titer paper disks (see Acheson and Tamm, 1970). Measurements of refractive index were made on selected fractions, and densities at 5” were calculated from an empirically determined relation between refractive index and density of potassium tartrate solutions.
324
ACHESON
AND
major band can be seen in both the stained and the counted gels. In addition, there is a minor band, very faint in the stained gel shown, which migrates more slowly than the major band. This minor band is consistently found both in stained gels and in gels assayed for radioactivity, whether nucleocapsids are disrupted in the presence of /3-mercaptoethanol or dithiothreitol, and whether or not glutathione is present in the gel during electrophoresis. Electrophoresis of Virus
Protein
Figure 3 shows the profile of radioactivity in an electropherogram of proteins from purified virus labeled with a 3H-amino acid mixture. A stained gel of a similar preparation is also shown. There are two major bands and a slower-migrating minor band in both the stained and the counted gels. The faster-migrating major band and the minor band correspond in position to the 2 bands of protein from purified nucleocapsids (Fig.
z
500
I-
400
/-
TAMM
2). The other major band is not found in nucleocapsids and thus probably represents the viral envelope protein. In some gels this band was very broad or had a shoulder as also reported by Strauss et al. (1969) for the analogous protein from Sindbis virus. Incorporation of 0.1 Ad glutathione into the gel and the anodal buffer (Strauss et al., 1969) reduced the half-width of this peak without affecting the behavior of the other two bands. In gels of nucleocapsid or virus protein which were first stained, then sliced and counted, the positions of the stained bands corresponded exactly with those of the radioactive peaks. No other bands were seen if freshly prepared samples were analyzed. Molecular
Weights of the Viral Proteins
Trypsin and BSA were included in some gels as standards, and the molecular weights of the three viral protein bands were estimated by the method of Shapiro et al. (1967).
300
u L ,‘:
200
100
00
0r1g1r-1
IO
20
30
40
50
60
70
Fraction FIG. 2. SDS-polyacrylamide gel electrophoresis of proteins from purified Semliki Forest virus nucleocapsids. Infected cells were incubated with a 3H-amino acid mixture from 1 to 12 hours after infection; nucleocapsids were harvested and purified. Radioactivity profile: nucleocapsids were disrupted in 170 SDS and 1% p-mercaptoethanol and subjected to electrophoresis in the presence of 0.1 M glutathione. One-millimeter slices of the frozen gel were counted as described in Materials and Met,hods. The direction of migrat,ion is from left to right, Stained gel: an identical sample was run in a similar gel which, however, did not contain glutathione. The gel was fixed and stained with Coomassie blue as described in Materials and Methods. The photograph of the stained gel was reproduced so that the major st,ained band is coincident with the major peak in the radioactivity profile.
PROTEIKS
OF SEMLIKI
FOREST
lYII~US
Fraction FIG. 3. Electrophoresis of protein from purified Semliki Forest virus. Infected cells were incubated with a 3H-amino acid mixture from 1 t,o 12 hours after infection; virus was subsequently harvested and purified. Radioactivity profile: virus was disrupted in lyO SDS and 17, P-mercaptoethanol and subjected to electrophoresis in a gel not, containing glutathione. Stained gel: a similar sample was I-L~ in a gel not containing glutathione. The gel was fixed and stained with Coomassie blue. The phot,ograph of t.he stained gel was reproduced so that the major stained bands are coincident with the major peaks in the radioactivity profile.
Figure 4 shows the results of one such experiment. In six experiments, the molecular weight of the protein in the major nucleocnpsid band was 31,00034,000, with a mean of 32,000. In four experiments, the molecular weight of the envelope protein was 50,000-53,000, with a mean of 51,500. in The minor band of nucleocapsid protein ran in the same position as BSA, at a molecular n-eight of 67,000. Is tlze Jlinor
Nucleocapsid Protein a Dimer?
The polypeptide chains in the minor band of nucleocapsid protein have a molecular weight approximately twice that of the polypeptides in the major nucleocapsid band. This suggested that the polypeptides in the minor band may not represent a distinct species, but instead may be dimers of the polypeptide chains in the major band. Two further lines of evidence also favor this hypothesis. Amino acid ratios in the minor and major bands. Nucleocapsids were grown in the presence of two different amino acids, one
labeled with 3H and one with 14C. Purified nucleocapsids were disrupted and run on gels, and the ratios of counts in 14C and 3H were measured for each band. If the polypeptide chains within the two bands had different amino acid compositions, the ratios of 14C to 3H should be different. If, on the other hand, the polypeptide chains within the minor band were dimers of those within the major band, there should be no difference in their amino acid compositions and thus no significant difference between the 14C:3H ratios of the two bands. Two pairs of amino acids were used: lysine-14C with leucineJH, and valine-14C with leucine-“H. Gels were run both in the presence and the absence of 0.1 ild glutathione. Table 1 shows that for each pair of amino acids there was no significant difference between the 14C:3H ratios in the minor band and the major band. However, in gels of labeled virus the 14C:3H ratios in the envelope protein were significantly different from those in the nucleocapsid protein. Thus the polppeptide chains within the
326
ACHESON
SND
TAMM
80,000
i .-cm %
40,000
is 5 : z 2 20,000
IO,OOC
igin
I
I
I
I
I
I
t
I
I
2
3
4
5
6
7
8
Distance
of migration,
cm
FIG. 4. Determination of the molecular weight,s of Semliki Forest virus proteins. Ten micrograms each of trypsin and bovine serum albumin (filled circles) were denatured and mixed with disrupted nucleocapsid or virus protein (empty circles). The mixtures were run on gels and stained with Coomassie blue. The distances of migration of the standard proteins were plotted as a function of the logarithm of their molecular weights, and the molecular weights of the envelope and nucleocapsid prot,eins were estimated from their relative migration distances. The nucleocapsid protein dimer migrates in nearly the same position as bovine serum albumin.
major and minor bands of nucleocapsid protein contain the same relative proportion of three different amino acids: lysine, valine, and leucine. Relative proportiow of major ancl minor bands. If the minor band represented a distinct structural nucleocapsid protein it should be present in the same proportion to the major band regardless of the sample analyzed. If, on the other hand, the minor band contained a dimer of the protein in the major band, its relative amount might vary depending on the nucleocapsid preparation analyzed. Several different purified, labeled nucleocapsid preparations were sub-
jected to electrophoresis, and the amount of label in the minor band relative to that in the major band (set at 100) was calculated for each sample. The upper part of Table 2 shows that in samples labeled with a 3Hamino acid mixture there is a Z-fold spread among the values for the relative number of counts in the minor band. The lower part of the table shows the proportion of counts in the minor band from a sample labeled with lysineJ4C and one labeled with valineJ4C. These two values can be compared since the data from Table 1 show that the relative amount of lysine and valine in the two bands is the same. Again there is a
PROTEINS TBBLE lC:3II
OF SEMLIKI
xperimerit
acids
Valine-‘“C 1erlcine-3H
Lysine-14C 1eucine-3H
and
and
Band
Xi
VIRUS TABLE
1
RATIOS IN THE MUOR .IND MINOR BINDS K;UCLICOC.\PSID PROTEIN LABELED WITH Two I~IFFERENT A~XINO ACIDS’
Amino
FOREST
OF
‘WI3 3H: channels ratio i standard err&’
Major Minor Major Minor
1.40 1.39 1.38 1.36
f f f It
0.01 0.06 0.01 0.04
Major Minor Major Minor
2.13 2.oG 2.09 2.20
f 0.01 f. 0.07 f 0.01 f 0.08
2
REL.\TIVE INCORPOR.~TION OF R.~DIO.UXWE AMIXO ACIDS 1.~~0 THE MUOR UTD MINOR B.ZNDS OF NUCLEOC.~PSID PROTJCIX
Labeled amino acid
“H-Amino
acid mixture
100 ~ 13.8 ~ bO.7 100 15.4 f0.6 100 2.8, f0.4
Lvsine-14C
100 100
4.il +O.l 4.81 f0.2
Valine-14C
100 100
4.0 4.1
f0.2 zto.3
u In gels of purified virus doubly labeled in the same experiments, the 14C:3H channels ratios in the envelope protein band were 1.02 & 0.01 (valineJ4C and leucine-3H) and 0.65 =t 0.01 (lysinel*C and 1eucineJH). b Standard errors were computed from the following formula:
a The values for the minor band were calculated by setting the number of comits per minute in the major band at. 100. b Standard errors were computed from the following formula:
Standard error = d(l/W C + (C/W2 H where C = total counts in 1% channel, corrected for background; H = tot,al counts in 3H channel, correct,ed for background.
Standard error = N(dcpm/cpm) where cpm is the total cpm in the minor band, corrected for background, and N is the relative cpm in the minor band.
significant difference between the relative amounts of label in the minor bands from the two samples of nucleocapsids. Thus the proportion of nucleocapsid protein in the minor band is not constant from one sample to the next. In further support of this finding, storage of a sample of purified virus for 1 week at 4” brought about an increase in the amount of label in the minor nucleocapsid band and a corresponding decrease in the major nucleocapsid band. These results suggest that, depending on conditions of isolation and age of sample, a certain proportion of the nucleocapsid protein is present as a dimer. The dimerixation cannot be reversed by the standard methods used to disrupt viral proteins for SDSpolyacrylamide gel electrophoresis, nor by the incorporation of 0.1 AI glutathione in the buffers and gel (Strauss et al., 1969). It thus appears that there is only one electrophoretically distinct type of polypeptide chain, of molecular weight approximatelyL32,000, in nucleocapsids of Semliki Forest virus.
Further Data on Envelope arzd Nucleocapsid Proteins
I
In gels of proteins from purified virus labeled with a 3H-amino acid mixture, the ratio of label in the envelope protein to that in the nucleocapsid protein is approximately 4.0 (Table 3). This value should equal the ratio between the amounts of envelope protein and nucleocapsid protein in virus, because the 3H-amino acid mixture should label both proteins uniformly. Since the envelope protein has a molecular weight of 51,000, and the nucleocapsid protein a molecular weight of 32,000, there are approximately 32,000/51,000 X 4.0, or 2.5 molecules of envelope protein per molecule of nucleocapsid protein. Purified virus doubly labeled with lysine14Cand leucine-3H, or valine-14C and leucine3H, was also analyzed by electrophoresis. Two points of interest can be mentioned. First, the ratio of 14Cto 3H is constant in a number of fractions on both sides of the peak fractions of the envelope and nucleo-
328
ACHESON TABLE
3
RELATIVE INCORPOR.1TION OFRADIO.~CTI~E ACIDSINTOTHI:ESVELOPE.~NDNUCLEOC.IPSID PROTEINS IN VIRUS PARTICLES
Labeled amino acid
3H-Amino
acid mixture
Experiment
Ratio,
AMIKO
cpm in envelope protein n&~&&id protein
A B C
3.9 4.0 4.0
Lysine-W
1) E
1.6 1.5
Valine-14C
F G
5.2 5.2
D E F G
7.0 6.9 5.8 5.1
capsid protein bands. This result makes it unlikely that either band contains more than one distinct polypeptide species of slightly different electrophoretic mobilities. Second, the ratio of label in the envelope protein to that in the nucleocapsid protein is lower when lysine is used, and higher when valine or leucine is used, than with a mixture of amino acids (Table 3). This suggests that the nucleocapsid protein, which is associated with the viral RNA (Acheson and Tamm, 1970) is relatively rich in lysine, a basic amino acid. Also, the envelope protein, which is associated with lipids in a membranelike structure (Acheson and Tamm, 1967; Strauss et al., 1968; Friedman and Pastan, 1969), is richer in valine and leucine, hydrophobic amino acids. DISCUSSION
The data presented in this paper indicate that Semliki Forest virus contains only two structural proteins: an envelope protein, of molecular weight -51,000, and a nucleocapsid protein, of molecular weight -32,000. A third electrophoretically distinct band probably represents a dimer of the nucleocapsid protein which is not dissociated by the methods used. No other discrete protein species were detected in virus or nucleo-
AND
TAMM
capsids purified as described. Virus particles contain approximately 2.5 molecules of envelope protein per molecule of nucleocapsid protein. The nucleocapsid protein is relatively rich in lysine, a basic amino acid, and the envelope protein is relatively rich in leucine and valine, hydrophobic amino acids. On the basis of its structural proteins, Semliki Forest virus is thus very similar t’o Sindbis virus, which has an envelope protein of molecular weight -53,000 and a nucleocapsid protein of molecular weight -30,000 (Strauss et al., 1968, 1969). These two group -4 arboviruses are also alike in their antigenic determinants (Casals and Clarke, 1965), RNA size (Sonnabend et al., 1966; Pfefferkorn et al., 1967)) and morphology (Simpson and Hauser, 1968a, b), although Simpson and Hauser (196813) detected differences in nucleocapsid morphology in thin sections of the two viruses. The envelope proteins of both Sindbis and Semliki Forest viruses tend to form broad or double bands upon electrophoresis unless a reducing agent such as glutathione is present in the gel (Strauss et al., 1969). Sindbis virus nucleocapsid protein apparently does not have a tendency to dimerize as does Semliki Forest virus nucleocapsid protein. Our results are in essential agreement with those of Friedman (1968), who found three peaks of radioactivity in autoradiograms of gel electropherograms of Semliki Forest virus. However, in that report the possibility that the second nucleocapsid protein band might be a dimer of the major nucleocapsid protein was not discussed. The absence of the third band in electropherograms of isotopically labeled proteins of Semliki Forest virus, published by Hay et al. (1968), is complicated by the presence of a third band in their stained gels. It seems possible in this case (cf. their Fig. 3) that the third band (nucleocapsid dimer) appeared only as a small shoulder on the trailing edge of the labeled envelope protein band because the gels may not have been run long enough or at a high enough voltage to separate the two bands. If this is true, their results would agree with the present ones and with those of Friedman (1968).
PI:OTEINS
OF SUILTKI
ACKNOWLEDGRIENT We thank Lawrence A. Caliguiri for instruction in the techniclues of gel electrophoresis. REFERENCES ACHESON, N. II., and TAMM, 1. (1967). Replication of Semliki Forest virus: an elect,ron microscopic study. Virology 32, 128-143. ACHK~ON, N. II., and T,.IMM, I. (1970). Purification and propert,ies of Semliki Forest virus nucleocapsids. Virology 41, 306. BABLAXIAP~, R., EGGERS, H. J., and TAMM, I. (1965). St,udies on the mechanism of poliovirusinduced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations in cldtnred cells. Virology 26, 101% 113. CASALS, J., and CLARKE, D. H. (1965). Arboviruses; Group A. In “\‘iral and Rickettsial Infections of Man” (F. L. Horsfall and I. Tamm, eds.), pp. 583-605. Lippincott, Philadelphia. FRIEDMAN, R. M. (1968). Structural and nonstruct,ural proteins of an arbovirus. J. Viral. 2, 107G1080. FRIEDMAN, It. M., and P~STAN, I. (19G9). Nature and function of the structural phospholipids of an arbovirus. J. 3101. Viol. 40, 107-115. FKOM~GEOT, H. P. M., aud ZINDER, N. II. (1968). Growth of bacteriophage f2 in E. coli treated with rifampicin. Proc. Xall. Acad. Sci. CA’. 61, 184-191. Hay, A. J., SKFHIEL, J. J., and BUIZKE, D. C. (1968). Proteins synthesized in chick cells following infection with Semliki Forest virus. J. Gen. Viral. 3, 175-184. OSTERRIBTH, P. M., and C~LBERG-BACQ, C. M. (1966). Changes in morphology, infectivity, and
FOREST
\-IRCS
:12(3
haemagglutinating activity of Semliki ForcTst virus produced by the treatment with caseillasc C from Streplo7~yce.s alhrrs G. J. Germ. .llic,&iol. 43, 19-30. PFI:FFI:RBOIW, 15. II., BURW, 13. W., and CO.ZDY, H. M. (1967). lnt,racellrrlar conversion of the RNA of Sindbis virus to a double-strarltled form. Virology 33, 239-249. SHAPIRO, 8. L., \-I&-ELA, E., aud ~Iarz1:1,, J. I-., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis ill SDS-polyacrylamide gels. Biocheln. Biophys. Ites. (‘OWL~~~zcn.28, 815-820. SIMPSOX, It. W., alld I-Iaus~n, R. E. (1968:r). Basic structure of group A arbovirus strains hliddleburg, Sindbis, and Semliki Forest examined by negative staining. I-iro2ogy 34, 358-361. SIMPSON, 11. W., and I~AUSEI~, K. I<. (1968b). Structural differentiation of grolip A srboviruses based on nucleoid morphology in ultrathin sections. I’irologjj 34, 508-570. SOSNABWW, J. ,4., MAWIN, IS. AI., nd Mr:cs, E. (1967). Viral specific RNA’s in infected cells. L!Jatwx 213, 365-367. STRAUSS, J. II., JR., BURGE, B. W., and ~~.uc.um~,, J. E., JR. (1969). Sindbis virus jufection of chick and hamst,er cells: synthesis of virlls-specific proteins. J’irology 37, 367-376. STRAUSS, J.H.,Jll., B~TRGK, B.W., PFEFFERKORN, E. R., and DARNELL, J. E., JR. (1908). Identification of the membrane protein and “core” protein of Sindbis virus. Proc. h’all. 9 cad. &‘ci. C.S. 59, 533-537. SUMMERS, I). F., MAIZEL, J. I*., and II~RKELL, J. E., JR. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Xatl. Acad. Sci. lT.S. 54, 505-513.