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Synthesis of proteins in cells infected with herpesvirus
VIROLOGY
41, 256-264 (1970)
Synthesis
of Proteins
IV. Analysis
in Cells
of the Proteins Cytoplasm
TAMAR
BEN-PORAT,
Department
HIDEYO
Infected
in Viral
with
Particles
Isolated
from the
and the Nucleus’ SHIMONO,
AND
ALBERT
of Microbiology, Research Laboratories, Albert Einstein Philadelphia,
Herpesvirus
Pennsylvania
S. KAPLAN
Medical
Center,
19141
Accepted February 9, 1970 Three types of pseudorabies virus particles can be isolated from infected rabbit kidney cells by centrifugation in sucrose density gradients. One type, isolated from the cytoplasm, contains DNA, behaves like extracellular virus, and probably represents a population of mature virus. A second type, isolated from the nucleus, also contains DNA but has a lower sedimentation value and 4-fold lower specific infectivity than cytoplasmic virus. A third type, also isolated from the nucleus, does not contain DNA and is not infectious. Electrophoretic analysis of the proteins present in the infectious nuclear and cytoplasmic viral particles revealed that they contain the same proteins but that the former possess about 30-357& less of at least two proteins normally present in mature virus. These two proteins probably represent proteins of the viral envelope. INTRODUCTION
The proteins synthesized by pseudorabies (Pr) virus-infected rabbit kidney (RK) cells differ from the proteins synthesized by uninfected cells with respect to their pattern of electrophoretic migration in polyacrylamide gels; they also contain more arginine but less methionine, phenylalanine, tyrosine, isoleucine, and lysine relative to leucine than the uninfected cell proteins. Furthermore, the proteins from Pr virusinfected RK cells, which migrate to different positions in the gels, differ from one another in their amino acid composition (Kaplan et al., 1970). We have been analyzing these proteins in order to sort out the structural viral proteins and to determine the specific structures they represent (membrane, capsid, or core). In a previous series of experiments, performed to characterize the structural viral we prepared partially purified proteins, 1This investigation was supported by a grant from the National Institutes of Health (AI-03362).
viral particles by a method which combines differential centrifugation with immunoprecipitation (Shimono et al., 1969). These virus preparations contained large amounts of proteins migrating as one major peak (designated as peak 2), as well as several other minor protein components. However, such virus preparations consisted not only of complete, but also of fragmented, viral particles, so that the relative amount of the various proteins assayed in these preparations was probably not a true measure of their relative amount in complete viral particles. Therefore, in the present experiments, which deal with the electrophoretic profiles of the proteins of viral particles isolated from the cytoplasm and from the nucleus, we have resorted to a different method of purification as a means of minimizing this difficulty. Since the herpesviruses derive their envelopes from the nuclear membrane as they enter the cytoplasm (Falke et al., 1959; Shipkey et al., 1967; Kii et al., 1968)) a 256
PROTEINS
IN
CYTOPLASMIC
AND
comparison of the proteins of the particles isolated from the nucleus and the cytoplasm should allow us to identify the proteins contained in the viral envelope. We have determined the quantitative differences between nuclear and cytoplasmic viral particles with respect to the various proteins migrating to different positions in polyacrylamide gels. The relative content of some amino acids in these proteins was also determined. The experiments described here indicate that the viral envelope consists of at least two proteins and that the DNA-containing viral particles, which can be isolated from the nucleus, are associated with some membrane material identical to that found in mature viral particles. MATERIALS
AND
METHODS
Virus and cell culture. The properties of Pr virus and the cultivation of RK monolayer cultures were described previously (Kaplan, 1957; Kaplan and Vatter, 1959; Ben-Porat and Kaplan, 1962). Primary R,K cells were grown in 90-mm petri dishes in EDS. In all experiments, the cultures were inoculated with approximately 20 plaque-forming units (PFU)/cell, unless otherwise noted. Media arzcl solutions. EDS: Eagle’s synthetic medium (Eagle, 1959) containing dialyzed bovine serum (5 %). EDSIlO and EDS/2: The same as EDS, except that the concentration of amino acids was reduced l/lOth or l/2 the normal amount. SDS: Sodium dodecyl sulfate was purified, and a stock solution (30 70) in water was prepared (Kaplan, 1969). TBSA: A buffer containing the same salts as PBS, except that the phosphate is replaced by Tris-HCl, 0.01 M, pH 7.5, plus 1% crystalline serum albumin. RSB: 0.01 :11 Tris-HCl, pH 7.4, 0.01 Ail KCl, and 0.0015 AI MgC12. Cell fractionation. The method described by Penman (1969) was used. RK cells (4 X 106) were harvested in 1 ml of RSB, homogenized, and the nuclear fraction separated from the cytoplasmic fraction by centrifugation. The nuclei were suspended in RSB, and both nuclear and cytoplasmic fractions were disrupted by sonic vibration in a Raytheon (10 kc/second; total power, 200 W) for 1 min at maximum power. In those experiments, in which virus was isolated by
NUCLEAR
HERPESVIRUS
PARTICLES
257
centrifugation in sucrose density gradients, the material was first clarified by centrifugation at 5000 g for 5 min. Pwi$cation of Pr virus by cerltrifugatiorh irb sucrose density gradients. Approximately 1 ml of the samples containing virus was layered on a linear sucrose gradient (15-30 % sucrose in TBSA) and centrifuged in the Spinco SW 25 rotor at 10,000 rpm for 140 min. Samples (approximately 1 ml) were collected through a hole pierced at the bottom of the tube. The virus band (approximately 4 ml) was mixed with unlabeled virus; TBSA was added slowly (tot’al volume, 30 ml), and the virus was collected by centrifugation in a Spinco No. 30 rotor at 25,000 rpm for 60 minutes. In some cases, the virus was centrifuged in sucrose gradients from which albumin had been omitted; the viral band was dialyzed and concentrated with Lyphogel (Gelman Instrument Co.). The electrophoretic profiles of the proteins of the virus prepared by both methods were identical. Preparatiou 0s samples for electrophoresis. The samples containing proteins to be electrophoresed were treated with DNase (20 Kg/ml) to reduce the viscosity. SDS (final concentration, 6 %) was added, and the samples were incubated at SO” for 15 min. Dithioerythritol (DTE) (final concentration, 0.025 M) was then added and the samples were incubated at 45” for 1 hour and dialyzed against 0.01 111 phosphate buffer (pH 7.2) containing 0.1% SDS. The proteins were separated by electrophoresis in polyacrylamide gels, essentially as described by Maize1 (1966). Electrophoresis was for 11.5 hours at 5 mA per 0.6 X 10 cm gel containing 7.5 70 polyacrylamide, 0.1 M sodium phosphate, and 0.1% SDS. The gels were sliced into fractions 2 mm wide, placed into vials, and dried at room temperature or in a 50” oven. One-tenth milliliter of HzOz (30 %) was added to the dried gels which were heated at 50” for 4 hours. This was followed by the addition of 0.5 ml KCS solubilizer (iZmersham/Searle) and tolueneethanol scintillation fluid (Tishler and Epstein, 1968). The samples were counted in a scintillation spectrometer set up for double counting.
25s
BEN-PORAT,
0 BOttOm
IO
SHIMONO,
IO
300
20
TOP
AND
Bottom
KAPLAN
20
30 TOP
ml
FIG. 1. Distribution
in sucrose gradients of viral particles obtained from the cytoplasmic and nuclear fractions of infected cells. RK cells (4 X 106) were infected and incubated in EDS/2. At 3 hours post infection, EDS/B containing leucine-W (1 &i/ml) and thymidineJH (2 ,.&i/ml) was added and the cultures were incubated to 11 hours post infection. The cells were harvested, and the cytoplasmic fraction was separated from the nuclear fraction; each fraction was sonicated and then centrifuged in sucrose gradients, as described in Materials and Methods. The distribution in the gradient of 3H and 1% in acid-precipitable molecules was determined.
Racliochemicals. LeucineJH (specific activity, 40 Ci/mmole) ; leucine-l*C (specific activity, 316 mCi/mmole) ; arginine-3H (specific activity, 45 Ci/mmole) ; argininel*C (specific activity, 220 mCi/mmole) ; tryptophan-3H (specific activity, 2.2 Ci/ mmole) were purchased from Schwarz BioResearch Labs. Thymidine-3H (specific activity, 5 Ci/mmole) and thymidine-2l*C (specific activity, 53 mCi/mmole) were purchased from New England Nuclear Corporation. Indirect immunoprecipitation. This procedure has been described in detail (Hamada and Kaplan, 1965). In brief, radioactively labeled proteins were treated with specific rooster y-globulin against Pr virus, and the complex formed was precipitated with antiserum against the r-globulin. The amount of radioactive material precipitated was a measure of radioactive antigen present in the sample. RESULTS
Distribution of Nuclear Viral Particles in Gradients
and Cytoplasmic Sucrose Density
Viral particles from the nuclear and cytoplasmic fractions of infected cells xvere
centrifuged in sucrose density gradients, as described in Materials and Methods. This method, which is similar to that used for the purification of herpes simplex virus (Olshevsky et al., 1967; Spear and Roizman, 1968), permits separation in good yield of particles of Pr virus from cellular material, o?zZyif the virus preparation is not pelleted by centrifugation prior to sedimentation in the sucrose density gradients. In our hands, centrifugation at high speed (70,000 g for 1 hour) causes aggregation of the viral particles which can be disaggregated only by methods that also disrupt a large proportion of the viral particles. Figure 1 shows the pattern of distribution in sucrose gradients of particles from the nuclear and cytoplasmic fractions of cells labeled with thymidine-3H and leucineJ4C. A similar pattern of distribution of herpes simplex virus has been reported (Olshevsky et al., 1967; Spear and Roizman 1968). Virus from the cytoplasm, as well as extracellular virus (the distribution of which in sucrose gradients was identical to that of cytoplasmic virus), formed a band, S1, which contained particles possessing DNA. Virus infectivity coincided with the Sr band. In addition to the Sr viral particles, the nuclear
PROTEINS
IN
CYTOPLASMIC
AND
fraction also yielded particles that lacked DNA and were not infectious and that formed a second band, designated SI1. (The macromolecules remaining at the top of the gradient were designated S,,,.) All the labeled macromolecules present in Sl and SI1 bands from the cytoplasm and the nucleus could be precipitated by the indirect immunoprecipitation procedure using antiserum against infectious Pr virus, as described in AIaterials and Methods. The DSA present in the Sr particles isolated from the extracellular fluid, from the cytoplasmic fraction, as well as from the nuclear fraction, was not sensitive to DNase. Tests of Purity
of Viral Particles
The next series of experiments was carried out to determine whether the Pr viral particles, which sediment as Sl or E&I, were contaminated with nonviral proteins. None of the proteins from uninfected cells labeled with leucine-3H for 16 hours, or from infected cells labeled similarly for 30 min (a period too short for the labeled proteins to aggregate into viral particles) banded in the same region of the sucrose gradient as the viral particles; all the radioactivity remained at the top of the gradient. There was also no unspecific adsorption of these proteins to viral particles, as demonstrated by the following experiment: Intact, unlabeled virus (5 X log PFU), or the same amount of virus which had been disrupted by sonication for (5min, was added to homogenates of infected cells that had been labeled with leucine-3H (100 $2i/ml) for 30 min or of uninfected cells labeled with leucine-3H (5 &$‘ml) for 16 hours. Virus labeled with thymidine-2-‘*C was also added as a marker, and the samples were centrifuged in sucrose gradients. In none of the samples did labeled protein band at the regions of the sucrose density gradient occupied by viral particles. We conclude therefore that viral particles do not adsorb unspecifically any of the cellular proteins and that the band of viral part.icles in the sucrose gradient probably is not contaminated with nonviral proteins. The particles present in the various bands were purified further, as described below; the protein composition of the virus prepara-
NUCLEAR
HERPESVIRUS
PARTICLES
2.39
tions was analyzed by polyacrylamidc gel electrophoresis before and after these purification procedures. Virus obtained after centrifugation in the sucrose gradients was dialyzed and concentrated by Lyphogel; this virus was then rebanded in sucrose gradients. The virus bands appeared in the expected positions and the distribution of the proteins in the polyacrylamide gels did not change after this procedure, and was essentially the same as indicated in Figs. 3-5. Thus, the particles obtained by centrifugation in sucrose gradients were not contaminat,ed with foreign proteins, at least not with proteins which can be removed by reccntrifugation. L4nalysis of Nuclear Viral Particles The S1 Pr viral particles from the cytoplasm have a slightly higher sedimentation
-
Cytoplasm
o---o
Nucleus
OL
h 5
IO
t-0 15
sottom
ml
20
25
30 TOP
2. Distribution in a sucrose gradient of DNA-containing viral particles obtained from the cytoplasmic and nuclear fractions of infected cells. RK cells (4 X 106) were infected and incubated in EDS containing either thymidine-1% (2 ,&i/ml) or thymidine-3H (20&i/ml). Ten hours post infection the cells were harvested and the cytoplasmic fraction was separated from the nuclear fract,ion, as described in Materials and Methods. The laClabeled cytoplasmic fraction was mixed with the 3H-labeled nuclear fraction, the mixture was sedimented through sucrose gradients, and the distribution in the gradient of 1°C and 3H in acidprecipitable molecules was determined. FIG.
260
BEN-PORAT, TABLE SPECIFIC
INFECTIVITY CYTOPLASMIC
SHIMONO,
AND
KAPLAN
1
7’-‘---
OF NUCLEAR PARTICLES~
Tube No.
cpm/tube
PFU/tube (W-5)
9 10 11
2170 3230 2300
7.2 12.0 5.4
8 9 10
1530 1860 1030
22.0 27.0 12.0
Nuclear
AND
6
h
---j-
-1
Cytoplasmic Fraction SI Virus
I ’ ’
x
Q x
PFU/cpm x 10-z SI
Cytoplasmic
3.3 3.7 2.4 Sr 14.6 14.5 11.5
a In this experiment RK cells (4 X 106) were infect,ed at a multiplicity of 5 PFU/cell, washed to remove unadsorbed virus, and incubated for 10 hours in EDS/B plus arginineJH (20 &i/ml). The cells were harvested and the cytoplasmic and nuclear fractions were centrifuged in sucrose gradients. The infectivity and the amount of radioact,ivity in the tubes containing the SI viral particles were assayed.
value than the SI viral particles from the nucleus, as demonstrated by mixing 14Clabeled cytoplasmic particles with 3H-labeled nuclear particles, followed by centrifugation in a sucrose gradient (Fig. 2). A similar finding has been reported for herpes simplex virus (Spring and Roizman, 1968). The specific infectivity of SI virus from the nucleus and cytoplasm differ (Table 1). Specific infectivity is defined here as the ratio of the number PFU to the number of particles (the assumption is made that the number of particles is directly proportional to the number of counts in the protein). Table 1 shows that the specific infectivity of nuclear virus was about 4 times lower, on the average, than that of cytoplasmic virus. [These results differ from those of Spring and Roizman (1968), who isolated herpes simplex viral particles from the nucleus that had a higher specific infectivity than the particles from the cytoplasm.] The specific infectivity was approximately the same throughout the band and it is likely therefore that each band is composed of a relatively homogeneous population of viral particles. Since the S, particles derived from the nucleus have a lower sedimentation value and lower specific infectivity than the SI
0
IO
20 Froctlon
30
40
50
Number
FIG. 3. Electropherogram SI cytoplasmic virus. RK
of the proteins in cells (8 X 106) were infected and incubated in EDS/B containing leucine1% (1.5&i/ml) and arginineJH (15pCi/ml) for 10 hours; the nuclear and cytoplasmic fractions were then separated. The viral bands obtained from these fractions after centrifugation in sucrose gradients were collected and prepared for electrophoresis, as described in Materials and Methods. Leucine-W, filled circles; arginineJH, open circles. 5
0
Nuclear Fraction SI Virus
IO
20 Fraction
FIG.
nuclear
30
40
50
-
Number
4. Electropherogram of the proteins band. See legend to Fig. 3.
in Sr
particles from the cytoplasm, it is evident that the two types of particles truly differ from each other. Comparison of the Proteins of Nuclear and Cytoplasmic Viral Particles Figures 3-5 show the electrophoretic patterns in polyacrylamide gels of the proteins of nuclear and cytoplasmic particles which band as Sr and &. The proteins
PROTEINS
IX
CYTOPLASMIC
AND
MJCLEAR
III
Nuclear Fraction Sp Virus
Fraction
Number
of t,he proteins FIG. 5. Electropherogram nluzlear band. See legend to Fig. 3.
in &I
from Sr particles derived from the cytoplasm (which are representative of mature viral particles) migrate in several distinct peaks of various heights. The relative amount of the various proteins migrating to different positions in the gels does not, however, necessarily reflect the protein constitution of infectious virus. Since the ratio of infectious to noninfectious virus is low in all preparations of viruses of the herpes group, the population of SI virus may contain particles that have lost some specific proteins but have nevertheless retained the same sedimentation characteristics as infectious virus. The SI nuclear particles possess the same proteins as the SI cytoplasmic particles, but the proteins migrating as peaks 3 and 6 seem to be present in smaller amounts in the preparations of the nuclear particles. To estimate the relative amount of the protein present in the different peaks in the Sr cytoplasmic and the SI nuclear particles, the following experiment was done. Cells were infected and labeled either with arginine-“H or with arginine-14C. Eleven hours post infection the cells were harvested and the nuclei were separated from the cytoplasm. The 14C-labeled SI cytoplasmic particles were mixed with the “H-labeled SI nuclear particles (or vice versa). The particles were treated and electrophoresed in polyacrylamide gels, as described in Materials and Methods. The differences in the protein makeup of the two types of viral particles were determined
HERPESVIRUS
261
PARTICLFX
from the ratio of 3H to 14C in the different protein peaks (Fig. 6). Table 2 summarizes the results of several experiments of the type illustrated in Fig. 6. The ratio of the number of counts in peak 2 derived from cytoplasmic and nuclear virus was taken as unity and the ratio in the other protein peaks was compared to that in peak 2. (The ratio 3H: 14C in peak 2 was used as a standard, because this peak consists of the major structural viral protein and is present in large amounts in viral particles obtained both from the nucleus and the cytoplasm.) There is about 30-3570 less of peaks 3 and 6 in the nuclear particles compared to the cytoplasmic particles. The amount of proteins migrating to peak 4a is also reduced in the nuclear particles but to a lesser extent than those migrating to peaks 3 and 6, and in some experiments there was no difference between the nuclear and the cytoplasmic particles in their content of these proteins. Since it is known that the viral particles acquire their envelopes as they leave the nucleus, these results suggest that peaks 3 and 6 consist, at least in part, of proteins composing the viral envelope. 3 ;--- -
IO
20 Fraction
30
40
50
Number
FIG. 6. Coelectrophoresis of SI nuclear and SI cytoplasmic particles. RK cells were infected and incubated between 3 and 11 hours post infection in EDS/P containing either arginine-1% (2 &i/ml) or arginine-3H (20 &i/ml). The nuclear and cytoplasmic fractions were separated, the cytoplasmic ‘Glabeled fraction was mixed with the W-labeled nuclear fraction and the mixture centrifuged in sucrose gradients. The band containing the SC particles was collected and prepared for electrophoresis, as described in Materials and Methods.
262
BEN-PORAT, TABLF:
SHIMONO,
2
REL~ITIVEAMOUNTOF
PROTEINS IN VIRAL P-IRFROM CYTOPLlShl AND KUTO ~IFFISRENT POSITIONS
TICLES ISOL.ITED CLEUS MIGRATING IN THE GELSa
Peak number Expt. No.
2
3
/
4a
1 6
Nuclear SI particles/cytoplasmic 1 2 3 4 5
! 1.00
0.70
0.84 1.00 1.00
0.76 0.65 0.63
1 Nuclear SII particles/cytoplasmic
~
7
,
8
SI particles 1.07 0.96 1.10 0.95
! 1.05 0.94 0.99 0.95
SI particles
1 2 0 See Text
and legend
to Fig. 6 for details.
If the viral particles from the nucleus that banded as S1 in the sucrose gradients were a mixture of fully enveloped and nonenveloped particles, one would expect an enrichment of enveloped particles at the faster moving part of the band (since the mature particles have a higher sedimentation value than the nuclear particles-see Pig. 2). To test this, S1 nuclear particles labeled with either leucine-l*C or leucine-3H were centrifuged in a sucrose gradient. The faster moving half of the 3H-labeled band was mixed with the slower moving part of the 14C-labeled band and the two were co-electrophoresed. No differences between the two were found; we conclude therefore that the band of Sr particles from the nucleus consists of a homogeneous population all of which contains less of the proteins migrating as peaks 3 and 6 than the cytoplasmic particles. Table 2 also summarizes experiments on the electrophoretic analysis of SI1 particles derived from the nucleus. There was a considerable reduction in many of the proteins that are normally present in mature virus, and these particles seem to be composed mainly of proteins migrating as peaks 2, 7, and 8. We have previously reported (Shimono et
AND
KAPLAN
al., 1969) on the electrophoretic analysis of the proteins of viral particles purified by differential centrifugation and indirect immunoprecipitation. These preparations probably contained a considerable amount of broken capsids as shown by experiments in which viral particles partially purified by differential centrifugation were centrifuged in sucrose gradients. About 20 %l of the labeled proteins that had sedimented at high speed migrated as bands Sr and Srr. Of the remainder which stayed at the top of the gradient, about 50% could be precipitated by the indirect immunoprecipitation procedure. Analysis of these proteins in acrylamide gels showed that their electrophoret’ic profile is similar to that of the Srr particles and consists, in large measure, of proteins that migrate as peaks 2, 7, and S. Thus, proteins migrating as peaks 2, 7, and 8 are present in structures that can be sedimented by high speed centrifugation and can be precipitated serologically. These are, however, not associated with viral particles that form distinct bands in sucrose gradients and probably represent broken capsids. Relative Amino Acid Corztent of the Difleererrt Peaks of Protein Derived from Cytoplasmic and Nuclear Viral Particles Sr nuclear virus possesses about one-third less of the proteins migrating as peaks 3 and 6 than Sr cytoplasmic virus. Thus each of these protein peaks from cytoplasmic virus could consist of at least two different polypeptides. One of these could be acquired by the virus as it leaves the nucleus and enters the cytoplasm; this protein would then be missing from the nuclear virus. On the other hand, each peak (3 and 6) might be composed only of one type of polypeptide, the Sr nuclear particles possessing fewer of these molecules than the Sr cytoplasmic particles. One way to determine whether the proteins migrating as peaks 3 and 6 are identical in the nuclear and cytoplasmic viruses is to determine their relative content of different amino acids. In these experiments, RB cells were labeled throughout the infective process with either leucine-l*C and arginine-3H or leucine-14C and tryptophan-3H’ The cells
TBBLE
DISCUSSIOX
3
I:.LTIO OF ~~HGININE .\ND TRPPTOPH.YN TO LCTCCINIC IN PROTI:INS ISOLITI:D FROM NUCLEAR AND CY~TOPL~YIIC VIRAL P.\RTICI,E+ TH.\T MIGK.YTI.: .\s I-)IFFEILlCNT PEAKS”
Peak number -___ SI Virus
2
1 3
( 4a
1
6
i
8
I Arginine Cytoplasmic Nuclear
3.70b 3.70
4.GO ' 3.70 3.60 i 4.50 4.50 ) 3.60 I 3.50 4.GO Tryptophan
Cytoplasmic iYurlear
“0::;
( ;:g
1 “0:;;
1 8:;;
/ 8:;;
(1RK cells (8 X 106) were infected and incubated bet-Teen 3 and 11 hours post, infection in EM/2 containing letlcine-‘“C (2 @.X/ml) and either arginine-3H (20 rCi/ml) or t,ryptophan-“H (20 ,uCi!ml). The nuclear and cytoplasmic fractions were separated, the SI viral particles were collected after centrifagation in a sucrnse gradient, and were electrophoresed, as described in Materials and Methods. * Itatio of 3H:1&C.
were harvested 11 hours post infection, the nuclei were separated from the cytoplasm, and the virus present in each was centrifuged in sucrose density gradients. The proteins associated with the S1 particles were electrophoresed and the 3H: W ratio in the various protein peaks was determined. The results are summarized in Table 3 (see also Figs. 3-5). It is clear that the ratio of arginine to leucine and tryptophan to leucine in peaks 3 and 6 was approximately the same in Sr nuclear virus and SI cytoplasmic virus. These results suggest that both particles consist of the same proteins, but that the SI nuclear particles contain less of the proteins migrating as peaks 3 and 6 than the cytoplasmic particles. That the Sr nuclear particles differ from the SI cytoplasmic particles in their lower content of some proteins is further substantiated by the experiments presented in the next paper of this series.
The experiments described in this paper show that three types of viral particles can be isolated from Pr virus-infected cultures. The first consists of mature viral particles present in the cytoplasm and the extracellular fluid. Two additional types of virus can be isolated from the nucleus: (1) particles which differ from the cytoplasmic and extracellular virus by their loner sedimentation constant and lolver specific infectivity; (2) particles devoid of DNA (and infectivity) which have a considerably lower sedimentstion value than the infectious particles. Electrophoretic analysis revealed that the SI nuclear particles and the SI cytoplasmic particles possess the same proteins; however, the nuclear particles contain about one-third less of the proteins migrating as peaks 3 and 6. Since l’r virus acquires its membrane upon exit from the nucleus, we attribute the difference between nuclear and cytoplnsmic particles to differences in their content of membrane material and conclude therefore that peaks 3 and 6 consist, at least partially, of membrane proteins. Since the relative content of arginine and of tryptophan n-ith respect to leucine in the proteins migrating as peaks 3 and 6 derived from SI nuclear and cytoplasmic particles are the same, it seems that nuclear virus has associated with it membrane material identical to that associated with cytoplasmic virus. This conclusion is reinforced by the experiments described in the next paper of this series which reports on the types of glycoproteins associated with nuclear and cytoplasmic virus. The implication of this finding will also be discussed in the next paper (part V of this series).
BI
264
BEN-PORAT,
SHIMONO,
teins in cells infected with pseudorabies virus. J. Bacterial. 89, 1328-1334. KAPLAN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell technique. Virology 4, 435457. KAPLAN, A. S. (1969). Isopycnic banding of viral DNA in cesium chloride. In ‘
AND
KAPLAN
S. (1969). Preparation of purified nuclei and nucleoli from mammalian cells. In “Fundamental Techniques in T’irology” (K. Habel and N. P. Salzman, eds.), pp. 35-48, Academic Press New York. SNIMONO, H., BEN-P• RAT, T., and KAPLAN, A. S. (1969). Synthesis of proteins in cells infected with herpesvirus. I. Structural viral proteins. Virology 37, 49-55. SHIPKEY, F. H., ERLANDSON, 1~. A., BAILEY, R. B., BABCOCK, V. I., and SOUTHAM, C. M. (1967). Virus Biographies. II. Growth of herpes simplex virus in tissue culture. Ezptl. Mol. Palhol. 6, 39-67. SPEAR, P. G., and ROIZMAN, B. (1968). The proteins specified by herpes simplex virus. I. Time of synthesis, transfer into nuclei, and properties of proteins made in productively infected cells. Virology 36, 545-555. SPRING, S. B., and ROIZMAN, B. (1968). Herpes simplex virus products in productive and abortive infection. III. Differentiation of infectious virus derived from nucleus and cytoplasm with respect to stability and size. J. Viral. 2,979-985. TISHLER, P. V., and EPSTEIN, C. J. (1968). A convenient method of preparing polyacrylamide gels for liquid scintillation spectrometry. Anal. Biochem. 22, 89-98. PENMAN,
41, 256-264 (1970)
Synthesis
of Proteins
IV. Analysis
in Cells
of the Proteins Cytoplasm
TAMAR
BEN-PORAT,
Department
HIDEYO
Infected
in Viral
with
Particles
Isolated
from the
and the Nucleus’ SHIMONO,
AND
ALBERT
of Microbiology, Research Laboratories, Albert Einstein Philadelphia,
Herpesvirus
Pennsylvania
S. KAPLAN
Medical
Center,
19141
Accepted February 9, 1970 Three types of pseudorabies virus particles can be isolated from infected rabbit kidney cells by centrifugation in sucrose density gradients. One type, isolated from the cytoplasm, contains DNA, behaves like extracellular virus, and probably represents a population of mature virus. A second type, isolated from the nucleus, also contains DNA but has a lower sedimentation value and 4-fold lower specific infectivity than cytoplasmic virus. A third type, also isolated from the nucleus, does not contain DNA and is not infectious. Electrophoretic analysis of the proteins present in the infectious nuclear and cytoplasmic viral particles revealed that they contain the same proteins but that the former possess about 30-357& less of at least two proteins normally present in mature virus. These two proteins probably represent proteins of the viral envelope. INTRODUCTION
The proteins synthesized by pseudorabies (Pr) virus-infected rabbit kidney (RK) cells differ from the proteins synthesized by uninfected cells with respect to their pattern of electrophoretic migration in polyacrylamide gels; they also contain more arginine but less methionine, phenylalanine, tyrosine, isoleucine, and lysine relative to leucine than the uninfected cell proteins. Furthermore, the proteins from Pr virusinfected RK cells, which migrate to different positions in the gels, differ from one another in their amino acid composition (Kaplan et al., 1970). We have been analyzing these proteins in order to sort out the structural viral proteins and to determine the specific structures they represent (membrane, capsid, or core). In a previous series of experiments, performed to characterize the structural viral we prepared partially purified proteins, 1This investigation was supported by a grant from the National Institutes of Health (AI-03362).
viral particles by a method which combines differential centrifugation with immunoprecipitation (Shimono et al., 1969). These virus preparations contained large amounts of proteins migrating as one major peak (designated as peak 2), as well as several other minor protein components. However, such virus preparations consisted not only of complete, but also of fragmented, viral particles, so that the relative amount of the various proteins assayed in these preparations was probably not a true measure of their relative amount in complete viral particles. Therefore, in the present experiments, which deal with the electrophoretic profiles of the proteins of viral particles isolated from the cytoplasm and from the nucleus, we have resorted to a different method of purification as a means of minimizing this difficulty. Since the herpesviruses derive their envelopes from the nuclear membrane as they enter the cytoplasm (Falke et al., 1959; Shipkey et al., 1967; Kii et al., 1968)) a 256
PROTEINS
IN
CYTOPLASMIC
AND
comparison of the proteins of the particles isolated from the nucleus and the cytoplasm should allow us to identify the proteins contained in the viral envelope. We have determined the quantitative differences between nuclear and cytoplasmic viral particles with respect to the various proteins migrating to different positions in polyacrylamide gels. The relative content of some amino acids in these proteins was also determined. The experiments described here indicate that the viral envelope consists of at least two proteins and that the DNA-containing viral particles, which can be isolated from the nucleus, are associated with some membrane material identical to that found in mature viral particles. MATERIALS
AND
METHODS
Virus and cell culture. The properties of Pr virus and the cultivation of RK monolayer cultures were described previously (Kaplan, 1957; Kaplan and Vatter, 1959; Ben-Porat and Kaplan, 1962). Primary R,K cells were grown in 90-mm petri dishes in EDS. In all experiments, the cultures were inoculated with approximately 20 plaque-forming units (PFU)/cell, unless otherwise noted. Media arzcl solutions. EDS: Eagle’s synthetic medium (Eagle, 1959) containing dialyzed bovine serum (5 %). EDSIlO and EDS/2: The same as EDS, except that the concentration of amino acids was reduced l/lOth or l/2 the normal amount. SDS: Sodium dodecyl sulfate was purified, and a stock solution (30 70) in water was prepared (Kaplan, 1969). TBSA: A buffer containing the same salts as PBS, except that the phosphate is replaced by Tris-HCl, 0.01 M, pH 7.5, plus 1% crystalline serum albumin. RSB: 0.01 :11 Tris-HCl, pH 7.4, 0.01 Ail KCl, and 0.0015 AI MgC12. Cell fractionation. The method described by Penman (1969) was used. RK cells (4 X 106) were harvested in 1 ml of RSB, homogenized, and the nuclear fraction separated from the cytoplasmic fraction by centrifugation. The nuclei were suspended in RSB, and both nuclear and cytoplasmic fractions were disrupted by sonic vibration in a Raytheon (10 kc/second; total power, 200 W) for 1 min at maximum power. In those experiments, in which virus was isolated by
NUCLEAR
HERPESVIRUS
PARTICLES
257
centrifugation in sucrose density gradients, the material was first clarified by centrifugation at 5000 g for 5 min. Pwi$cation of Pr virus by cerltrifugatiorh irb sucrose density gradients. Approximately 1 ml of the samples containing virus was layered on a linear sucrose gradient (15-30 % sucrose in TBSA) and centrifuged in the Spinco SW 25 rotor at 10,000 rpm for 140 min. Samples (approximately 1 ml) were collected through a hole pierced at the bottom of the tube. The virus band (approximately 4 ml) was mixed with unlabeled virus; TBSA was added slowly (tot’al volume, 30 ml), and the virus was collected by centrifugation in a Spinco No. 30 rotor at 25,000 rpm for 60 minutes. In some cases, the virus was centrifuged in sucrose gradients from which albumin had been omitted; the viral band was dialyzed and concentrated with Lyphogel (Gelman Instrument Co.). The electrophoretic profiles of the proteins of the virus prepared by both methods were identical. Preparatiou 0s samples for electrophoresis. The samples containing proteins to be electrophoresed were treated with DNase (20 Kg/ml) to reduce the viscosity. SDS (final concentration, 6 %) was added, and the samples were incubated at SO” for 15 min. Dithioerythritol (DTE) (final concentration, 0.025 M) was then added and the samples were incubated at 45” for 1 hour and dialyzed against 0.01 111 phosphate buffer (pH 7.2) containing 0.1% SDS. The proteins were separated by electrophoresis in polyacrylamide gels, essentially as described by Maize1 (1966). Electrophoresis was for 11.5 hours at 5 mA per 0.6 X 10 cm gel containing 7.5 70 polyacrylamide, 0.1 M sodium phosphate, and 0.1% SDS. The gels were sliced into fractions 2 mm wide, placed into vials, and dried at room temperature or in a 50” oven. One-tenth milliliter of HzOz (30 %) was added to the dried gels which were heated at 50” for 4 hours. This was followed by the addition of 0.5 ml KCS solubilizer (iZmersham/Searle) and tolueneethanol scintillation fluid (Tishler and Epstein, 1968). The samples were counted in a scintillation spectrometer set up for double counting.
25s
BEN-PORAT,
0 BOttOm
IO
SHIMONO,
IO
300
20
TOP
AND
Bottom
KAPLAN
20
30 TOP
ml
FIG. 1. Distribution
in sucrose gradients of viral particles obtained from the cytoplasmic and nuclear fractions of infected cells. RK cells (4 X 106) were infected and incubated in EDS/2. At 3 hours post infection, EDS/B containing leucine-W (1 &i/ml) and thymidineJH (2 ,.&i/ml) was added and the cultures were incubated to 11 hours post infection. The cells were harvested, and the cytoplasmic fraction was separated from the nuclear fraction; each fraction was sonicated and then centrifuged in sucrose gradients, as described in Materials and Methods. The distribution in the gradient of 3H and 1% in acid-precipitable molecules was determined.
Racliochemicals. LeucineJH (specific activity, 40 Ci/mmole) ; leucine-l*C (specific activity, 316 mCi/mmole) ; arginine-3H (specific activity, 45 Ci/mmole) ; argininel*C (specific activity, 220 mCi/mmole) ; tryptophan-3H (specific activity, 2.2 Ci/ mmole) were purchased from Schwarz BioResearch Labs. Thymidine-3H (specific activity, 5 Ci/mmole) and thymidine-2l*C (specific activity, 53 mCi/mmole) were purchased from New England Nuclear Corporation. Indirect immunoprecipitation. This procedure has been described in detail (Hamada and Kaplan, 1965). In brief, radioactively labeled proteins were treated with specific rooster y-globulin against Pr virus, and the complex formed was precipitated with antiserum against the r-globulin. The amount of radioactive material precipitated was a measure of radioactive antigen present in the sample. RESULTS
Distribution of Nuclear Viral Particles in Gradients
and Cytoplasmic Sucrose Density
Viral particles from the nuclear and cytoplasmic fractions of infected cells xvere
centrifuged in sucrose density gradients, as described in Materials and Methods. This method, which is similar to that used for the purification of herpes simplex virus (Olshevsky et al., 1967; Spear and Roizman, 1968), permits separation in good yield of particles of Pr virus from cellular material, o?zZyif the virus preparation is not pelleted by centrifugation prior to sedimentation in the sucrose density gradients. In our hands, centrifugation at high speed (70,000 g for 1 hour) causes aggregation of the viral particles which can be disaggregated only by methods that also disrupt a large proportion of the viral particles. Figure 1 shows the pattern of distribution in sucrose gradients of particles from the nuclear and cytoplasmic fractions of cells labeled with thymidine-3H and leucineJ4C. A similar pattern of distribution of herpes simplex virus has been reported (Olshevsky et al., 1967; Spear and Roizman 1968). Virus from the cytoplasm, as well as extracellular virus (the distribution of which in sucrose gradients was identical to that of cytoplasmic virus), formed a band, S1, which contained particles possessing DNA. Virus infectivity coincided with the Sr band. In addition to the Sr viral particles, the nuclear
PROTEINS
IN
CYTOPLASMIC
AND
fraction also yielded particles that lacked DNA and were not infectious and that formed a second band, designated SI1. (The macromolecules remaining at the top of the gradient were designated S,,,.) All the labeled macromolecules present in Sl and SI1 bands from the cytoplasm and the nucleus could be precipitated by the indirect immunoprecipitation procedure using antiserum against infectious Pr virus, as described in AIaterials and Methods. The DSA present in the Sr particles isolated from the extracellular fluid, from the cytoplasmic fraction, as well as from the nuclear fraction, was not sensitive to DNase. Tests of Purity
of Viral Particles
The next series of experiments was carried out to determine whether the Pr viral particles, which sediment as Sl or E&I, were contaminated with nonviral proteins. None of the proteins from uninfected cells labeled with leucine-3H for 16 hours, or from infected cells labeled similarly for 30 min (a period too short for the labeled proteins to aggregate into viral particles) banded in the same region of the sucrose gradient as the viral particles; all the radioactivity remained at the top of the gradient. There was also no unspecific adsorption of these proteins to viral particles, as demonstrated by the following experiment: Intact, unlabeled virus (5 X log PFU), or the same amount of virus which had been disrupted by sonication for (5min, was added to homogenates of infected cells that had been labeled with leucine-3H (100 $2i/ml) for 30 min or of uninfected cells labeled with leucine-3H (5 &$‘ml) for 16 hours. Virus labeled with thymidine-2-‘*C was also added as a marker, and the samples were centrifuged in sucrose gradients. In none of the samples did labeled protein band at the regions of the sucrose density gradient occupied by viral particles. We conclude therefore that viral particles do not adsorb unspecifically any of the cellular proteins and that the band of viral part.icles in the sucrose gradient probably is not contaminated with nonviral proteins. The particles present in the various bands were purified further, as described below; the protein composition of the virus prepara-
NUCLEAR
HERPESVIRUS
PARTICLES
2.39
tions was analyzed by polyacrylamidc gel electrophoresis before and after these purification procedures. Virus obtained after centrifugation in the sucrose gradients was dialyzed and concentrated by Lyphogel; this virus was then rebanded in sucrose gradients. The virus bands appeared in the expected positions and the distribution of the proteins in the polyacrylamide gels did not change after this procedure, and was essentially the same as indicated in Figs. 3-5. Thus, the particles obtained by centrifugation in sucrose gradients were not contaminat,ed with foreign proteins, at least not with proteins which can be removed by reccntrifugation. L4nalysis of Nuclear Viral Particles The S1 Pr viral particles from the cytoplasm have a slightly higher sedimentation
-
Cytoplasm
o---o
Nucleus
OL
h 5
IO
t-0 15
sottom
ml
20
25
30 TOP
2. Distribution in a sucrose gradient of DNA-containing viral particles obtained from the cytoplasmic and nuclear fractions of infected cells. RK cells (4 X 106) were infected and incubated in EDS containing either thymidine-1% (2 ,&i/ml) or thymidine-3H (20&i/ml). Ten hours post infection the cells were harvested and the cytoplasmic fraction was separated from the nuclear fract,ion, as described in Materials and Methods. The laClabeled cytoplasmic fraction was mixed with the 3H-labeled nuclear fraction, the mixture was sedimented through sucrose gradients, and the distribution in the gradient of 1°C and 3H in acidprecipitable molecules was determined. FIG.
260
BEN-PORAT, TABLE SPECIFIC
INFECTIVITY CYTOPLASMIC
SHIMONO,
AND
KAPLAN
1
7’-‘---
OF NUCLEAR PARTICLES~
Tube No.
cpm/tube
PFU/tube (W-5)
9 10 11
2170 3230 2300
7.2 12.0 5.4
8 9 10
1530 1860 1030
22.0 27.0 12.0
Nuclear
AND
6
h
---j-
-1
Cytoplasmic Fraction SI Virus
I ’ ’
x
Q x
PFU/cpm x 10-z SI
Cytoplasmic
3.3 3.7 2.4 Sr 14.6 14.5 11.5
a In this experiment RK cells (4 X 106) were infect,ed at a multiplicity of 5 PFU/cell, washed to remove unadsorbed virus, and incubated for 10 hours in EDS/B plus arginineJH (20 &i/ml). The cells were harvested and the cytoplasmic and nuclear fractions were centrifuged in sucrose gradients. The infectivity and the amount of radioact,ivity in the tubes containing the SI viral particles were assayed.
value than the SI viral particles from the nucleus, as demonstrated by mixing 14Clabeled cytoplasmic particles with 3H-labeled nuclear particles, followed by centrifugation in a sucrose gradient (Fig. 2). A similar finding has been reported for herpes simplex virus (Spring and Roizman, 1968). The specific infectivity of SI virus from the nucleus and cytoplasm differ (Table 1). Specific infectivity is defined here as the ratio of the number PFU to the number of particles (the assumption is made that the number of particles is directly proportional to the number of counts in the protein). Table 1 shows that the specific infectivity of nuclear virus was about 4 times lower, on the average, than that of cytoplasmic virus. [These results differ from those of Spring and Roizman (1968), who isolated herpes simplex viral particles from the nucleus that had a higher specific infectivity than the particles from the cytoplasm.] The specific infectivity was approximately the same throughout the band and it is likely therefore that each band is composed of a relatively homogeneous population of viral particles. Since the S, particles derived from the nucleus have a lower sedimentation value and lower specific infectivity than the SI
0
IO
20 Froctlon
30
40
50
Number
FIG. 3. Electropherogram SI cytoplasmic virus. RK
of the proteins in cells (8 X 106) were infected and incubated in EDS/B containing leucine1% (1.5&i/ml) and arginineJH (15pCi/ml) for 10 hours; the nuclear and cytoplasmic fractions were then separated. The viral bands obtained from these fractions after centrifugation in sucrose gradients were collected and prepared for electrophoresis, as described in Materials and Methods. Leucine-W, filled circles; arginineJH, open circles. 5
0
Nuclear Fraction SI Virus
IO
20 Fraction
FIG.
nuclear
30
40
50
-
Number
4. Electropherogram of the proteins band. See legend to Fig. 3.
in Sr
particles from the cytoplasm, it is evident that the two types of particles truly differ from each other. Comparison of the Proteins of Nuclear and Cytoplasmic Viral Particles Figures 3-5 show the electrophoretic patterns in polyacrylamide gels of the proteins of nuclear and cytoplasmic particles which band as Sr and &. The proteins
PROTEINS
IX
CYTOPLASMIC
AND
MJCLEAR
III
Nuclear Fraction Sp Virus
Fraction
Number
of t,he proteins FIG. 5. Electropherogram nluzlear band. See legend to Fig. 3.
in &I
from Sr particles derived from the cytoplasm (which are representative of mature viral particles) migrate in several distinct peaks of various heights. The relative amount of the various proteins migrating to different positions in the gels does not, however, necessarily reflect the protein constitution of infectious virus. Since the ratio of infectious to noninfectious virus is low in all preparations of viruses of the herpes group, the population of SI virus may contain particles that have lost some specific proteins but have nevertheless retained the same sedimentation characteristics as infectious virus. The SI nuclear particles possess the same proteins as the SI cytoplasmic particles, but the proteins migrating as peaks 3 and 6 seem to be present in smaller amounts in the preparations of the nuclear particles. To estimate the relative amount of the protein present in the different peaks in the Sr cytoplasmic and the SI nuclear particles, the following experiment was done. Cells were infected and labeled either with arginine-“H or with arginine-14C. Eleven hours post infection the cells were harvested and the nuclei were separated from the cytoplasm. The 14C-labeled SI cytoplasmic particles were mixed with the “H-labeled SI nuclear particles (or vice versa). The particles were treated and electrophoresed in polyacrylamide gels, as described in Materials and Methods. The differences in the protein makeup of the two types of viral particles were determined
HERPESVIRUS
261
PARTICLFX
from the ratio of 3H to 14C in the different protein peaks (Fig. 6). Table 2 summarizes the results of several experiments of the type illustrated in Fig. 6. The ratio of the number of counts in peak 2 derived from cytoplasmic and nuclear virus was taken as unity and the ratio in the other protein peaks was compared to that in peak 2. (The ratio 3H: 14C in peak 2 was used as a standard, because this peak consists of the major structural viral protein and is present in large amounts in viral particles obtained both from the nucleus and the cytoplasm.) There is about 30-3570 less of peaks 3 and 6 in the nuclear particles compared to the cytoplasmic particles. The amount of proteins migrating to peak 4a is also reduced in the nuclear particles but to a lesser extent than those migrating to peaks 3 and 6, and in some experiments there was no difference between the nuclear and the cytoplasmic particles in their content of these proteins. Since it is known that the viral particles acquire their envelopes as they leave the nucleus, these results suggest that peaks 3 and 6 consist, at least in part, of proteins composing the viral envelope. 3 ;--- -
IO
20 Fraction
30
40
50
Number
FIG. 6. Coelectrophoresis of SI nuclear and SI cytoplasmic particles. RK cells were infected and incubated between 3 and 11 hours post infection in EDS/P containing either arginine-1% (2 &i/ml) or arginine-3H (20 &i/ml). The nuclear and cytoplasmic fractions were separated, the cytoplasmic ‘Glabeled fraction was mixed with the W-labeled nuclear fraction and the mixture centrifuged in sucrose gradients. The band containing the SC particles was collected and prepared for electrophoresis, as described in Materials and Methods.
262
BEN-PORAT, TABLF:
SHIMONO,
2
REL~ITIVEAMOUNTOF
PROTEINS IN VIRAL P-IRFROM CYTOPLlShl AND KUTO ~IFFISRENT POSITIONS
TICLES ISOL.ITED CLEUS MIGRATING IN THE GELSa
Peak number Expt. No.
2
3
/
4a
1 6
Nuclear SI particles/cytoplasmic 1 2 3 4 5
! 1.00
0.70
0.84 1.00 1.00
0.76 0.65 0.63
1 Nuclear SII particles/cytoplasmic
~
7
,
8
SI particles 1.07 0.96 1.10 0.95
! 1.05 0.94 0.99 0.95
SI particles
1 2 0 See Text
and legend
to Fig. 6 for details.
If the viral particles from the nucleus that banded as S1 in the sucrose gradients were a mixture of fully enveloped and nonenveloped particles, one would expect an enrichment of enveloped particles at the faster moving part of the band (since the mature particles have a higher sedimentation value than the nuclear particles-see Pig. 2). To test this, S1 nuclear particles labeled with either leucine-l*C or leucine-3H were centrifuged in a sucrose gradient. The faster moving half of the 3H-labeled band was mixed with the slower moving part of the 14C-labeled band and the two were co-electrophoresed. No differences between the two were found; we conclude therefore that the band of Sr particles from the nucleus consists of a homogeneous population all of which contains less of the proteins migrating as peaks 3 and 6 than the cytoplasmic particles. Table 2 also summarizes experiments on the electrophoretic analysis of SI1 particles derived from the nucleus. There was a considerable reduction in many of the proteins that are normally present in mature virus, and these particles seem to be composed mainly of proteins migrating as peaks 2, 7, and 8. We have previously reported (Shimono et
AND
KAPLAN
al., 1969) on the electrophoretic analysis of the proteins of viral particles purified by differential centrifugation and indirect immunoprecipitation. These preparations probably contained a considerable amount of broken capsids as shown by experiments in which viral particles partially purified by differential centrifugation were centrifuged in sucrose gradients. About 20 %l of the labeled proteins that had sedimented at high speed migrated as bands Sr and Srr. Of the remainder which stayed at the top of the gradient, about 50% could be precipitated by the indirect immunoprecipitation procedure. Analysis of these proteins in acrylamide gels showed that their electrophoret’ic profile is similar to that of the Srr particles and consists, in large measure, of proteins that migrate as peaks 2, 7, and S. Thus, proteins migrating as peaks 2, 7, and 8 are present in structures that can be sedimented by high speed centrifugation and can be precipitated serologically. These are, however, not associated with viral particles that form distinct bands in sucrose gradients and probably represent broken capsids. Relative Amino Acid Corztent of the Difleererrt Peaks of Protein Derived from Cytoplasmic and Nuclear Viral Particles Sr nuclear virus possesses about one-third less of the proteins migrating as peaks 3 and 6 than Sr cytoplasmic virus. Thus each of these protein peaks from cytoplasmic virus could consist of at least two different polypeptides. One of these could be acquired by the virus as it leaves the nucleus and enters the cytoplasm; this protein would then be missing from the nuclear virus. On the other hand, each peak (3 and 6) might be composed only of one type of polypeptide, the Sr nuclear particles possessing fewer of these molecules than the Sr cytoplasmic particles. One way to determine whether the proteins migrating as peaks 3 and 6 are identical in the nuclear and cytoplasmic viruses is to determine their relative content of different amino acids. In these experiments, RB cells were labeled throughout the infective process with either leucine-l*C and arginine-3H or leucine-14C and tryptophan-3H’ The cells
TBBLE
DISCUSSIOX
3
I:.LTIO OF ~~HGININE .\ND TRPPTOPH.YN TO LCTCCINIC IN PROTI:INS ISOLITI:D FROM NUCLEAR AND CY~TOPL~YIIC VIRAL P.\RTICI,E+ TH.\T MIGK.YTI.: .\s I-)IFFEILlCNT PEAKS”
Peak number -___ SI Virus
2
1 3
( 4a
1
6
i
8
I Arginine Cytoplasmic Nuclear
3.70b 3.70
4.GO ' 3.70 3.60 i 4.50 4.50 ) 3.60 I 3.50 4.GO Tryptophan
Cytoplasmic iYurlear
“0::;
( ;:g
1 “0:;;
1 8:;;
/ 8:;;
(1RK cells (8 X 106) were infected and incubated bet-Teen 3 and 11 hours post, infection in EM/2 containing letlcine-‘“C (2 @.X/ml) and either arginine-3H (20 rCi/ml) or t,ryptophan-“H (20 ,uCi!ml). The nuclear and cytoplasmic fractions were separated, the SI viral particles were collected after centrifagation in a sucrnse gradient, and were electrophoresed, as described in Materials and Methods. * Itatio of 3H:1&C.
were harvested 11 hours post infection, the nuclei were separated from the cytoplasm, and the virus present in each was centrifuged in sucrose density gradients. The proteins associated with the S1 particles were electrophoresed and the 3H: W ratio in the various protein peaks was determined. The results are summarized in Table 3 (see also Figs. 3-5). It is clear that the ratio of arginine to leucine and tryptophan to leucine in peaks 3 and 6 was approximately the same in Sr nuclear virus and SI cytoplasmic virus. These results suggest that both particles consist of the same proteins, but that the SI nuclear particles contain less of the proteins migrating as peaks 3 and 6 than the cytoplasmic particles. That the Sr nuclear particles differ from the SI cytoplasmic particles in their lower content of some proteins is further substantiated by the experiments presented in the next paper of this series.
The experiments described in this paper show that three types of viral particles can be isolated from Pr virus-infected cultures. The first consists of mature viral particles present in the cytoplasm and the extracellular fluid. Two additional types of virus can be isolated from the nucleus: (1) particles which differ from the cytoplasmic and extracellular virus by their loner sedimentation constant and lolver specific infectivity; (2) particles devoid of DNA (and infectivity) which have a considerably lower sedimentstion value than the infectious particles. Electrophoretic analysis revealed that the SI nuclear particles and the SI cytoplasmic particles possess the same proteins; however, the nuclear particles contain about one-third less of the proteins migrating as peaks 3 and 6. Since l’r virus acquires its membrane upon exit from the nucleus, we attribute the difference between nuclear and cytoplnsmic particles to differences in their content of membrane material and conclude therefore that peaks 3 and 6 consist, at least partially, of membrane proteins. Since the relative content of arginine and of tryptophan n-ith respect to leucine in the proteins migrating as peaks 3 and 6 derived from SI nuclear and cytoplasmic particles are the same, it seems that nuclear virus has associated with it membrane material identical to that associated with cytoplasmic virus. This conclusion is reinforced by the experiments described in the next paper of this series which reports on the types of glycoproteins associated with nuclear and cytoplasmic virus. The implication of this finding will also be discussed in the next paper (part V of this series).
BI
264
BEN-PORAT,
SHIMONO,
teins in cells infected with pseudorabies virus. J. Bacterial. 89, 1328-1334. KAPLAN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell technique. Virology 4, 435457. KAPLAN, A. S. (1969). Isopycnic banding of viral DNA in cesium chloride. In ‘
AND
KAPLAN
S. (1969). Preparation of purified nuclei and nucleoli from mammalian cells. In “Fundamental Techniques in T’irology” (K. Habel and N. P. Salzman, eds.), pp. 35-48, Academic Press New York. SNIMONO, H., BEN-P• RAT, T., and KAPLAN, A. S. (1969). Synthesis of proteins in cells infected with herpesvirus. I. Structural viral proteins. Virology 37, 49-55. SHIPKEY, F. H., ERLANDSON, 1~. A., BAILEY, R. B., BABCOCK, V. I., and SOUTHAM, C. M. (1967). Virus Biographies. II. Growth of herpes simplex virus in tissue culture. Ezptl. Mol. Palhol. 6, 39-67. SPEAR, P. G., and ROIZMAN, B. (1968). The proteins specified by herpes simplex virus. I. Time of synthesis, transfer into nuclei, and properties of proteins made in productively infected cells. Virology 36, 545-555. SPRING, S. B., and ROIZMAN, B. (1968). Herpes simplex virus products in productive and abortive infection. III. Differentiation of infectious virus derived from nucleus and cytoplasm with respect to stability and size. J. Viral. 2,979-985. TISHLER, P. V., and EPSTEIN, C. J. (1968). A convenient method of preparing polyacrylamide gels for liquid scintillation spectrometry. Anal. Biochem. 22, 89-98. PENMAN,