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Synthesis of proteins in cells infected with herpesvirus
VIROLOGY
37,
56-61 (1969)
Synthesis
of Proteins
II. Flow of Structural TAMAR
BEN-PORAT,
Department
in Cells
Viral
Infected
with
Proteins from Cytoplasm
HIDEYO
SHIMONO,
of Microbiology, Research Laboratories, Philadelphia, Pennsylvania
AND
Herpesvirus to Nucleus’
ALBERT
Albert Einstein 19141
S. KAPLAN
Medical
Center,
Accepted September 12, 1968 In pseudorabies virus-infected cells, structural viral proteins are synthesized in the cytoplasm and move to the nucleus. When accumulation of progeny viral DNA in the nucleus is prevented by treatment of the infected cells with I-b-n-arabinofuranosylcytosine (Ara-C) the synthesis of structural viral proteins proceeds unaffected in the cytoplasm and movement of these proteins to the nucleus occurs. INTRODUCTION
unequivocally the site of synthesis of structural viral proteins.
It is well established that the capsids of the herpesviruses are assembled within the nucleus of infected cells (Morgan et al., 1954; Reissig and Melnick, 1955; Tournier et al., 1957; Felluga, 1963), but, until recently, little information has been available about the cellular site (nucleus or cytoplasm) of synthesis of structural viral proteins. In cells infected with pseudorabies (Pr) virus, there is a flow of proteins from the cytoplasm to the nucleus when a large proportion of the proteins being synthesized by the infected cells are structural viral proteins (Fujiwara and Kaplan, 1967). Similar results have been reported for cells infected with herpes simplex virus (Olshevsky et al., 1967). Although these results indicate that the cytoplasm is the site of synthesis of structural viral proteins, they do not constitute rigorous proof for this conclusion. The present paper is concerned with experiments designed to determine
MATERIALS
AND
METHODS
With the exceptions given below, the materials and methods were the same as in the preceding paper (Shimono et al., 1968). Solutions. Ca-t&on solution: 0.25 M sucrose, 0.03 M Tris-HCl (pH 7.4), 0.0018 M CaCl2, 0.01 M sodium azide, and 0.5% Triton X-100. Chemicals. Arginine-3H, arginine-14C, and thymidineJ4C were purchased from Schwarz BioResearch Inc. I-P-D-Arabinofuranosylcytosine (Ara-C) was a gift from the Upjohn Company. Cellfractirmation. The RN cells (8 X 106/ ml) were harvested by being scraped in Ca-Triton solution, then homogenized in a tight-fitting Dounce homogenizer, and centrifuged at 1000 g for 7 min. The pellet (the nuclear fraction) contained 15-17 % of the cellular protein and all the DNA. RESULTS
The Cytoplasmic Synthesis of Structural Viral Proteins and Their Flow to the Nucleus
1 This investigation was supported by grants from the National Institutes of Health (AI03362) and from the National Science Foundation (GB-4995), and by a U.S. Public Health Research Career Program Award (5-K3-AI-19,335) from the National Institute of Allergy and Infectious Diseases.
The major portion of the proteins found in a preparation of purified Pr virus migrates upon electrophoresis in polyacrylamide gels in one sharp peak (peak 2) with 56
PROTEIN
SYNTHESIS
IN HERPESVIRUS-INFECTED
a molecular weight of approximately 120,000 daltons (Shimono et al., 1968). Furthermore, most of the proteins synthesized by the infected cells that migrate in this position consist of structural viral proteins. The following experiments show that these proteins are synthesized in the cytoplasm and migrate thereafter to the nucleus of the infected cells, thereby demonstrating conclusively that the synthesis of structural viral proteins occurs in the cytoplasm. Infected RK cells mere pulsed with either arginine-3H or arginine-14C. The 3H-labeled cells were then further incubated with an excess of unlabeled arginine, as described in the legend to Table 1, the samples were mixed, and the nuclear and cytoplasmic fractions were separated, as described in Materials and Methods. Table 1 shows the amount of radioactivity associated with each fraction and confirms our previous results (Fujiwara and Kaplan, 1967): there is a flow of proteins from the cytoplasm to the nucleus in Pr virus-infected cells. That t’his flow is not an artifact due to differential TABLE 1 EVIDENCE FOR THE FLOW OF LABELED PROTEINS FROM THE CYTOPLASM TO THE NUCLEUS IN VIRUS-INFECTED Cmrsa
Sample
Pulse 3H Pulse-chase%
Cytoplasm
Nucleus
Cpm/ Percent sample of total
Cpm/ Percent sample of total
37,100 31,100
10,350 17,300
78.5 64.2
21.5 35.8
5 RK cultures were infected (adsorbed multiplicity, 10 PFU/cell) and incubated in EDS. At 5.5 hours after infection, the cultures were incubated in EDS/lO with either arginine-3H (7.5 &i/ml; specific activity, 7 Ci/mmole) or arginineW (0.75 &i/ml; specific activity, 250 mCi/ mmole) for 25 min. The cultures were washed once and EDS containing twice the regular amount of amino acids was added. The 3H-labeled cultures were harvested immediately; the 14C-labeled cultures were further incubated at 37” for 45 min and then harvested. The pulsed cultures were mixed with the pulse-chased cultures, the nuclei were separated from the cytoplasm, and radioactivity in each fraction was determined as described in Materials and Methods.
CELLS.
57
II.
CYTOPLASM
IO
20 Fraction
30
40
50
Number
FIG. 1. RK cultures were infected and incubated in EDS. At 5.5 hours after infection the cultures were incubated in EDS/lO with either leucine-3H (10 &i/ml; specific activity, 3.1 Ci/ mmole) or leucine-W (0.8 pCi/ml; specific activity, 135 mCi/mmole) for 25 min. The cultures were then washed and the medium was changed to EDS containing twice the normal amount of amino acids. The 3H-labeled cultures were harvested immediately; the W-labeled cultures were further incubated at 37” for 45 min and were then harvested. The 3H- and W-labeled cells were mixed, the cytoplaemic and nuclear fractions were separated and prepared for electrophoresis as described in Materials and Methods. An aliquot was used for electrophoresis on polyacrylamide gels. Filled circles, 3H; open circles, 14C.
leakage of proteins from the nuclei during their isolation has been demonstrated previously by radioautography (Fujiwara and Kaplan, 1967). Figure 1 shows a typical pattern of distribution in polyacrylamide gels of the labeled proteins in the cytoplasmic and in
58
BEN-PORAT,
SHIMONO,
the nuclear fractions obtained in an experiment similar to the one summarized in Table 1. During the chase period, the proportion of proteins migrating as peak 2 and peaks 6-8 increased in the nucleus. (A similar pulse-chase experiment was also performed with uninfected cells and no migration of proteins from the cytoplasm to the nucleus was observed.) Table 2 summarizes the quantitative aspects of the migration of the radioactive peak 2 proteins from the cytoplasm to the nucleus during the pulse-chase experiment illustrated in Fig. 1. In this experiment, the amount of peak 2 protein found in the nucleus after the chase was increased by a factor of two. This relatively low factor of increase was probably due to the length of the pulse (25 min). In other experiments, after a shorter pulse period (lo-min pulse followed by a 60-min chase), there was a 3-fold increase, after the chase, in the amount of peak 2 proteins associated with the nuclear fraction. However, since the radioactive proteins present in samples obtained after a short pulse migrated in the polyacrylamide gels in an atypical fashion (probably because of the presence of a relatively large proportion of labeled protein molecules which were in the process of being synthesized at the time of harvest), we considered it more advisable for illustrative purposes to use an experiment with a longer pulse period, even though it does not demonstrate as clearly the migration of peak 2 proteins from the cytoplasm to the nucleus. After the chase only 34% of the peak 2
AND
KAPLAN
proteins synthesized by the infected cells were found associated with the nuclear fraction in this experiment. Similar results were obtained in all experiments of this type that we have performed and we have never found more than 50% of the total peak 2 proteins synthesized during the pulse to be associated with the nucleus, probably because the procedure of cell fractionation caused leakage of some of these proteins or because of the exit of complet)ed capsids from the nucleus. Evidence that a large proportion of the peak 2 proteins synthesized by the cells during a similar pulse reached the nucleus comes from the fact, that they eventually became aggregated into particles (see Table 4). Since aggregation of viral particles probably occurs in the nucleus only, we conclude that these proteins reached the nucleus. Movement of Viral Proteins from the Cytoplasm to the Nucleus in Ara-C-Treatecl Cells
The experiments in this section are concerned with the reason for the flow of structural viral proteins from the cytoplasm to the nucleus. The simplest and therefore the most appealing model for this migration is that it is due to diffusion and that the formation of viral particles in the nucleus acts as a diffusion sink by removing free struct’ural viral proteins; these proteins would then be replaced in the nucleus by a continuous feed from the cytoplasm. Since viral DNA (which is known to accumulate in the nucleus) could play an active role in this process by serving as a center of condensation for the proteins, we TABLE 2 FLOWOFPEAK 2 PROTEINS FROMTHE CYTOPLASM tested whether viral proteins will migrate from the cytoplasm to the nucleus when TO THE NUCLEUS IN VIRUS-INFECTED CELLS~ viral DNA synthesis is inhibited. However, Radioas a first step it was necessary to determine Cytoplasm Nucleus activity in Sample whether, under these conditions, structural nucleus @pm) (v-4 (%I viral proteins are at all synthesized. This point is controversial and, in most systems, Pulse 3H 10,130 1,940 17.4 the synthesis of structural viral proteins Pulse-chase 14C 8,520 4,500 34.5 does not occur when viral DNA synhhesis is inhibited (see Kaplan and Ben-Porat, a The experiment was performed as described 1968). In the present experiments, we used in the legend to Fig. 1. The amount of 1% and 3H Ara-C (50 pg/ml) to inhibit the synthesis of in peak 2 obtained from the cytoplasmic and the viral DNA. Under these conditions, the nuclear fractions was determined.
PROTEIN
SYNTHESIS
IN
HERPESVIRUS-INFECTED
synthesis of DXA is inhibited in infected cells by about 95 70 to 98 70, as measured by the incorporation of t’hymidine-14C into Dn’A, and the synthesis of infectious virus is inhibited by a factor greater than 102; the synthesis of protein, as measured by the incorporation of arginine-3H is, however, not affected (see Table 3). [Ara-C inhibits the formation of infectious Pr virus by inhibiting the synthesis of viral DNA (Ben-Porat et al., 1968) .] The electrophoretic migration in polyacrylamide gels of the proteins synthesized by Ara-C-treated and untreated cells was determined. The pattern of migration of the proteins, as well as t’he amount of protein that migrated in peak 2, was similar in Ara-C-treated and untreated cells. Since the proteins that migrate as peak 2 are made up mostly of structural viral proteins (Shimono et al., 1968), we conclude that Ara-Ctreated infected cells synthesized structural viral proteins. Table 4 summarizes t’he results of a pulsechase experiment with virus-infected, AraC-treated and untreated cells carried out to determine whether peak 2 proteins become sedimentable, thereby indicating their agTABLE
3
EFFECT OFARA-C ONTHESYNTHESIS OFPROTEIN, DNA, AND INFECTIOUS YIRUS IN INFECTED RK CELLS" Sample
No Ara-C Ara-C (50 bdml)
Arginine Thymidine (cpm/ culture) 12,310 13,540
(cpm’ culture) 10,127 395
Infectivity (PFU/culture) 3.5 x 108 6.5 x 1C”
a Actively growing RK cells were infected (adsorbed multiplicity, 5 PFU/cell), washed, and incubated in EDS with or without Ara-C. Two hours after infection arginine-3H (0.1 &i/ml; specific activity, 0.3 Ci/mmole) and thymidine14C (0.04 &%/ml; specific activity, 0.1 Ci/mmole) were added to the cultures, which were further incubated until 10 hours post infection. The culby scraping the cells into tures were harvested the culture fluid, and the incorporation of label into DNA and protein, as well as t,he formation of infectious virus, was determined, as described in Materials and Methods.
CELLS. TABLE
59
II.
4
FORMATION OF PEAK 2 PROTEINS BND THEIR AGGREGATIONIN ARA-C-TREATED INFECTED CELLsa Pulse
Pulse-chase
Fraction Cpm/ sample No Ara-C Total sample Cell debris 20,0OOgsupernatant fluid 20,OOOgpellet (virus) Ara-C Total sample Cell debris 20,OOOgsupernatant fluid 20,OOOgpellet (virus)
Cpm in peak 2
Cpm/ sample
Cpm in peak 2
318,000 78,000 171,400
54,300 5,300 38,200
294,600 81,400 103,000
49,100 8,400 8,700
59,600
9,400
124,000
31,200
396,000 87,500 247,000
63,200 5,000 46,700
410,600 96,000 146,000
65,000 8,600 11,100
72,000
9,800
171,000
42,500
a RK cells were infected and incubated in EDS or in EDS plus Ara-C (50 pg/ml). Between 4.5 and 5.5 hours after infection, the cells were labeled by incubation in EDS/lO containing leutine-14C (2 &i/ml; specific activity, 135 mCi/ mmole) or EDS/lO containing Ara-C (50 pg/ml) and 1eucineJH (20 pCi/ml; specific activity, 3.1 Ci/mmole). The medium was changed to EDS containing twice the normal amount of amino acids with or without Ara-C. Part of the cultures was harvested immediately; another part was incubated further at 37” for 6 hours. The cells and culture fluids were collected, the ‘%-labeled, Bra-C-treated cultures were mixed with the 3Hlabeled, untreated cultures, the samples were sonicated, centrifuged, and the total radioactivity, as well as the amount present in the fractions of radioactive proteins migrating in peak 2 in polyacrylamide gels, was determined.
gregation into viral particles. Immediately after the labeling period, about 66% of all the peak 2 proteins synthesized by the cells could not be sedimented at a force which sediments the virions; after the chase, however, only about 16% of these proteins were not sedimentable under these conditions. There was no significant difference in the behavior of these proteins in Ara-Ctreated and untreated cells. That the pellet obtained after centrifugation at 20,000 g
60
BEN-PORAT,
SHIlMONO,
indeed contained viral particles was further demonstrated by precipit,ation with specific antiserum. About 50-60% of the total radioactivity in these fractions obtained from both the Ara-C-t’reated and the untreated cells could thus be precipitated. The formation of viral particles in Ara-Ctreated cells would, in itself, indicate that in these cells peak 2 proteins do flow to the nucleus, since assembly of these proteins into particles normally occurs in the nucleus. However, if the absence of viral DNA affected this flow, assembly might possibly occur in the cytoplasm. We therefore determined whether t,he flow of peak 2 proteins from the cytoplasm to t,he nucleus occurred in Ara-C-treated cells in an experiment similar to that summarized in Fig. 1 and Table 2. The results of this experiment are summarized in Table 5. In the Ara-Ctreated cells, immediately after the pulse, about 14% of the total labeled protein, as well as of the labeled peak 2 proteins, were found in the nuclear fraction, and this proportion increased significantly after the chase. It is clear, therefore, that migration of peak 2 proteins to the nucleus occurred even when viral DNA synthesis was drastically reduced by Ara-C. However, in the untreated cells 37% of t’he peak 2 proteins were in the nuclear fraction after the chase as compared to 29% in Am-C treated cells. Thus, although migrat’ion occurred in the TABLE
5
MOVEMENT OF PROTEINS FROM THE CYTOPLASM TO THE NUCLEUS IN ARA-C-TREATED CELLSPercentage of radioactivity the nucleus Sample
Total
Peak 2
Pulse Pu1sechase Untreated Ara-C (50 /*g/ml)
14 13
in
32 28
Pulse Pulsechase 17 14
37 29
a The experiment was performed as described in the legend to Fig. 1 and the amount of 1% and aH in peak 2 obtained from the cytoplasmic and nuclear fractions was determined. The Ara-Ctreated cells were treated identically but Ara-C (50 rg/ml) was present in the culture fluid throughout the experiment.
AND
KAPLAN
Ara-C-treated cells, it was reduced. This reduction is significant and has been obtained repeatedly. DISCUSSION
The proteins synthesized by Pr virusinfected cells which migrate electrophoretitally in polyacrylamide gels in a position designated as peak 2 const’itute the major protein component of the viral particles (Shimono et al., 1968). In the present paper we show that these proteins are synthesized in the cytoplasm and move to the nucleus, where they are assembled into viral particles. The simplest and most appealing explanation for the mechanism for this migration is diffusion. Thus, if aggregation of viral proteins within the nucleus acts as a diffusion sink, the concentration gradient would be maintained and this would lead to an accumulation of these proteins in the nucleus. Since viral DNA within the nucleus seemed to be one possible agent responsible for the aggregation of viral proteins into particles, we tested whether accumulation of these proteins would occur in the nucleus in the absence of viral DNA synthesis. This was found to be the case. However, the migration of these proteins to the nucleus occurred less efficiently when viral DNA synthesis was inhibited. Whether t’his is due to the absence of viral DNA per se or to another factor that may be affected by these conditions is not clear. The fact that structural viral proteins migrate to the nucleus in Ara-C-treated cells does not eliminate the possibility that diffusion is the basis for this migration. However, our experiments do demonstrate that viral DNA does not play a major role in the migration of the proteins, and one would have to postulate the existence within the nucleus of another condensing factor responsible for the aggregation of the protein subunits into particles. Such a factor (presumably a protein) would, in contrast to the other virus-specific proteins, be synthesized in the nucleus, because aggregation would otherwise be expected to occur in the cytoplasm. Little is known about the internal proteins of the herpesviruses, and
PROTEIN
SYNTHESIS
IN HERPESVIRUS-INFECTED
the existence of such a condensing protein remains to be demonstrated. The inability of Ara-C to inhibit the synthesis of structural viral proteins in Pr virus-infected RK cells contradicts the results reported for other animal virus-cell systems, in which the synthesis of progeny viral DKA is required before structural viral proteins are synt,hesized (see Kaplan and Ben-Porat’, 1968). The difference may lie in the fact that in Pr virus-infected RK cells total inhibition of DNA synthesis is not achieved. Even at very high concentrations of Ara-C (50 ,ug/ml or more), DNA synthesis is inhibited by only 95-98 %. Alt’hough these conditions inhibit to a considerable extent the synthesis of viral DNA, some progeny viral DNA may, nevertheless, be formed and it seems that only a few of these molecules may suffice to lead to the formation of normal amounts of struct,ural viral proteins in this system. REFERENCES BEN-P• RAT, T., BROWN, M., and KAPLAN,
A. S. (1968). Effect of l-P-o-arabinofuranosylcytosine on DNA synthesis. II. In rabbit kidney cells infected with herpes viruses. Mol. Pharmacol. 4, 139-146.
CELLS. II.
61
FELLUGA, B. (1963). Electron microscopic observations on pseudorabies virus development in a line of pig kidney cells. Ann. Sclavo 5, 412-424. FUJI~ARA, S., and KAPLAN, A. S. (1967). Site of protein synthesis in cells infected with pseudorabies virus. V’Gology 32, 60-68. KAPLAN, A. S., and BEN-P• RAT, T. (1968). Metabolism of animal cells infected with nuclear 22, 427450. DNA viruses. Ann. Rev. Microbial. MORGAN, C., ELLISON, S. A., ROSE, H. M., and MOORE, D. H. (1954). Structure and development of viruses as observed in the electron microscope. I. Herpes simplex virus. J. Exptl. Med. 100, 195-202. OLSHEVSKY, U., LEVITT, J., and BECKER, Y. (1967). Studies on the synthesis of herpes simplex virions. V+oZogy 33, 323-334. REISSIG, M., ~~~MELNICE, J. L. (1955). The cellular changes produced in tissue cultures by herpes B virus correlated with the concurrent multiplication of the virus. J. Exptl. Med. 101, 341351. SHIMONO, H., BEN-P• RAT, T., and KAPLAN, A. S. (1968). Synthesis of proteins in cells infected with herpesvirus. I. Structural viral proteins. Virolow. 37, 49-55. TOURNIER, P., CATHALA, F., and BERNHARD, W. (1957). Ultrastructure et dbveloppement intracellulaire du virus de la varicelle observe au microscope Blectronique. Presse Med. 65, 12291234.
37,
56-61 (1969)
Synthesis
of Proteins
II. Flow of Structural TAMAR
BEN-PORAT,
Department
in Cells
Viral
Infected
with
Proteins from Cytoplasm
HIDEYO
SHIMONO,
of Microbiology, Research Laboratories, Philadelphia, Pennsylvania
AND
Herpesvirus to Nucleus’
ALBERT
Albert Einstein 19141
S. KAPLAN
Medical
Center,
Accepted September 12, 1968 In pseudorabies virus-infected cells, structural viral proteins are synthesized in the cytoplasm and move to the nucleus. When accumulation of progeny viral DNA in the nucleus is prevented by treatment of the infected cells with I-b-n-arabinofuranosylcytosine (Ara-C) the synthesis of structural viral proteins proceeds unaffected in the cytoplasm and movement of these proteins to the nucleus occurs. INTRODUCTION
unequivocally the site of synthesis of structural viral proteins.
It is well established that the capsids of the herpesviruses are assembled within the nucleus of infected cells (Morgan et al., 1954; Reissig and Melnick, 1955; Tournier et al., 1957; Felluga, 1963), but, until recently, little information has been available about the cellular site (nucleus or cytoplasm) of synthesis of structural viral proteins. In cells infected with pseudorabies (Pr) virus, there is a flow of proteins from the cytoplasm to the nucleus when a large proportion of the proteins being synthesized by the infected cells are structural viral proteins (Fujiwara and Kaplan, 1967). Similar results have been reported for cells infected with herpes simplex virus (Olshevsky et al., 1967). Although these results indicate that the cytoplasm is the site of synthesis of structural viral proteins, they do not constitute rigorous proof for this conclusion. The present paper is concerned with experiments designed to determine
MATERIALS
AND
METHODS
With the exceptions given below, the materials and methods were the same as in the preceding paper (Shimono et al., 1968). Solutions. Ca-t&on solution: 0.25 M sucrose, 0.03 M Tris-HCl (pH 7.4), 0.0018 M CaCl2, 0.01 M sodium azide, and 0.5% Triton X-100. Chemicals. Arginine-3H, arginine-14C, and thymidineJ4C were purchased from Schwarz BioResearch Inc. I-P-D-Arabinofuranosylcytosine (Ara-C) was a gift from the Upjohn Company. Cellfractirmation. The RN cells (8 X 106/ ml) were harvested by being scraped in Ca-Triton solution, then homogenized in a tight-fitting Dounce homogenizer, and centrifuged at 1000 g for 7 min. The pellet (the nuclear fraction) contained 15-17 % of the cellular protein and all the DNA. RESULTS
The Cytoplasmic Synthesis of Structural Viral Proteins and Their Flow to the Nucleus
1 This investigation was supported by grants from the National Institutes of Health (AI03362) and from the National Science Foundation (GB-4995), and by a U.S. Public Health Research Career Program Award (5-K3-AI-19,335) from the National Institute of Allergy and Infectious Diseases.
The major portion of the proteins found in a preparation of purified Pr virus migrates upon electrophoresis in polyacrylamide gels in one sharp peak (peak 2) with 56
PROTEIN
SYNTHESIS
IN HERPESVIRUS-INFECTED
a molecular weight of approximately 120,000 daltons (Shimono et al., 1968). Furthermore, most of the proteins synthesized by the infected cells that migrate in this position consist of structural viral proteins. The following experiments show that these proteins are synthesized in the cytoplasm and migrate thereafter to the nucleus of the infected cells, thereby demonstrating conclusively that the synthesis of structural viral proteins occurs in the cytoplasm. Infected RK cells mere pulsed with either arginine-3H or arginine-14C. The 3H-labeled cells were then further incubated with an excess of unlabeled arginine, as described in the legend to Table 1, the samples were mixed, and the nuclear and cytoplasmic fractions were separated, as described in Materials and Methods. Table 1 shows the amount of radioactivity associated with each fraction and confirms our previous results (Fujiwara and Kaplan, 1967): there is a flow of proteins from the cytoplasm to the nucleus in Pr virus-infected cells. That t’his flow is not an artifact due to differential TABLE 1 EVIDENCE FOR THE FLOW OF LABELED PROTEINS FROM THE CYTOPLASM TO THE NUCLEUS IN VIRUS-INFECTED Cmrsa
Sample
Pulse 3H Pulse-chase%
Cytoplasm
Nucleus
Cpm/ Percent sample of total
Cpm/ Percent sample of total
37,100 31,100
10,350 17,300
78.5 64.2
21.5 35.8
5 RK cultures were infected (adsorbed multiplicity, 10 PFU/cell) and incubated in EDS. At 5.5 hours after infection, the cultures were incubated in EDS/lO with either arginine-3H (7.5 &i/ml; specific activity, 7 Ci/mmole) or arginineW (0.75 &i/ml; specific activity, 250 mCi/ mmole) for 25 min. The cultures were washed once and EDS containing twice the regular amount of amino acids was added. The 3H-labeled cultures were harvested immediately; the 14C-labeled cultures were further incubated at 37” for 45 min and then harvested. The pulsed cultures were mixed with the pulse-chased cultures, the nuclei were separated from the cytoplasm, and radioactivity in each fraction was determined as described in Materials and Methods.
CELLS.
57
II.
CYTOPLASM
IO
20 Fraction
30
40
50
Number
FIG. 1. RK cultures were infected and incubated in EDS. At 5.5 hours after infection the cultures were incubated in EDS/lO with either leucine-3H (10 &i/ml; specific activity, 3.1 Ci/ mmole) or leucine-W (0.8 pCi/ml; specific activity, 135 mCi/mmole) for 25 min. The cultures were then washed and the medium was changed to EDS containing twice the normal amount of amino acids. The 3H-labeled cultures were harvested immediately; the W-labeled cultures were further incubated at 37” for 45 min and were then harvested. The 3H- and W-labeled cells were mixed, the cytoplaemic and nuclear fractions were separated and prepared for electrophoresis as described in Materials and Methods. An aliquot was used for electrophoresis on polyacrylamide gels. Filled circles, 3H; open circles, 14C.
leakage of proteins from the nuclei during their isolation has been demonstrated previously by radioautography (Fujiwara and Kaplan, 1967). Figure 1 shows a typical pattern of distribution in polyacrylamide gels of the labeled proteins in the cytoplasmic and in
58
BEN-PORAT,
SHIMONO,
the nuclear fractions obtained in an experiment similar to the one summarized in Table 1. During the chase period, the proportion of proteins migrating as peak 2 and peaks 6-8 increased in the nucleus. (A similar pulse-chase experiment was also performed with uninfected cells and no migration of proteins from the cytoplasm to the nucleus was observed.) Table 2 summarizes the quantitative aspects of the migration of the radioactive peak 2 proteins from the cytoplasm to the nucleus during the pulse-chase experiment illustrated in Fig. 1. In this experiment, the amount of peak 2 protein found in the nucleus after the chase was increased by a factor of two. This relatively low factor of increase was probably due to the length of the pulse (25 min). In other experiments, after a shorter pulse period (lo-min pulse followed by a 60-min chase), there was a 3-fold increase, after the chase, in the amount of peak 2 proteins associated with the nuclear fraction. However, since the radioactive proteins present in samples obtained after a short pulse migrated in the polyacrylamide gels in an atypical fashion (probably because of the presence of a relatively large proportion of labeled protein molecules which were in the process of being synthesized at the time of harvest), we considered it more advisable for illustrative purposes to use an experiment with a longer pulse period, even though it does not demonstrate as clearly the migration of peak 2 proteins from the cytoplasm to the nucleus. After the chase only 34% of the peak 2
AND
KAPLAN
proteins synthesized by the infected cells were found associated with the nuclear fraction in this experiment. Similar results were obtained in all experiments of this type that we have performed and we have never found more than 50% of the total peak 2 proteins synthesized during the pulse to be associated with the nucleus, probably because the procedure of cell fractionation caused leakage of some of these proteins or because of the exit of complet)ed capsids from the nucleus. Evidence that a large proportion of the peak 2 proteins synthesized by the cells during a similar pulse reached the nucleus comes from the fact, that they eventually became aggregated into particles (see Table 4). Since aggregation of viral particles probably occurs in the nucleus only, we conclude that these proteins reached the nucleus. Movement of Viral Proteins from the Cytoplasm to the Nucleus in Ara-C-Treatecl Cells
The experiments in this section are concerned with the reason for the flow of structural viral proteins from the cytoplasm to the nucleus. The simplest and therefore the most appealing model for this migration is that it is due to diffusion and that the formation of viral particles in the nucleus acts as a diffusion sink by removing free struct’ural viral proteins; these proteins would then be replaced in the nucleus by a continuous feed from the cytoplasm. Since viral DNA (which is known to accumulate in the nucleus) could play an active role in this process by serving as a center of condensation for the proteins, we TABLE 2 FLOWOFPEAK 2 PROTEINS FROMTHE CYTOPLASM tested whether viral proteins will migrate from the cytoplasm to the nucleus when TO THE NUCLEUS IN VIRUS-INFECTED CELLS~ viral DNA synthesis is inhibited. However, Radioas a first step it was necessary to determine Cytoplasm Nucleus activity in Sample whether, under these conditions, structural nucleus @pm) (v-4 (%I viral proteins are at all synthesized. This point is controversial and, in most systems, Pulse 3H 10,130 1,940 17.4 the synthesis of structural viral proteins Pulse-chase 14C 8,520 4,500 34.5 does not occur when viral DNA synhhesis is inhibited (see Kaplan and Ben-Porat, a The experiment was performed as described 1968). In the present experiments, we used in the legend to Fig. 1. The amount of 1% and 3H Ara-C (50 pg/ml) to inhibit the synthesis of in peak 2 obtained from the cytoplasmic and the viral DNA. Under these conditions, the nuclear fractions was determined.
PROTEIN
SYNTHESIS
IN
HERPESVIRUS-INFECTED
synthesis of DXA is inhibited in infected cells by about 95 70 to 98 70, as measured by the incorporation of t’hymidine-14C into Dn’A, and the synthesis of infectious virus is inhibited by a factor greater than 102; the synthesis of protein, as measured by the incorporation of arginine-3H is, however, not affected (see Table 3). [Ara-C inhibits the formation of infectious Pr virus by inhibiting the synthesis of viral DNA (Ben-Porat et al., 1968) .] The electrophoretic migration in polyacrylamide gels of the proteins synthesized by Ara-C-treated and untreated cells was determined. The pattern of migration of the proteins, as well as t’he amount of protein that migrated in peak 2, was similar in Ara-C-treated and untreated cells. Since the proteins that migrate as peak 2 are made up mostly of structural viral proteins (Shimono et al., 1968), we conclude that Ara-Ctreated infected cells synthesized structural viral proteins. Table 4 summarizes t’he results of a pulsechase experiment with virus-infected, AraC-treated and untreated cells carried out to determine whether peak 2 proteins become sedimentable, thereby indicating their agTABLE
3
EFFECT OFARA-C ONTHESYNTHESIS OFPROTEIN, DNA, AND INFECTIOUS YIRUS IN INFECTED RK CELLS" Sample
No Ara-C Ara-C (50 bdml)
Arginine Thymidine (cpm/ culture) 12,310 13,540
(cpm’ culture) 10,127 395
Infectivity (PFU/culture) 3.5 x 108 6.5 x 1C”
a Actively growing RK cells were infected (adsorbed multiplicity, 5 PFU/cell), washed, and incubated in EDS with or without Ara-C. Two hours after infection arginine-3H (0.1 &i/ml; specific activity, 0.3 Ci/mmole) and thymidine14C (0.04 &%/ml; specific activity, 0.1 Ci/mmole) were added to the cultures, which were further incubated until 10 hours post infection. The culby scraping the cells into tures were harvested the culture fluid, and the incorporation of label into DNA and protein, as well as t,he formation of infectious virus, was determined, as described in Materials and Methods.
CELLS. TABLE
59
II.
4
FORMATION OF PEAK 2 PROTEINS BND THEIR AGGREGATIONIN ARA-C-TREATED INFECTED CELLsa Pulse
Pulse-chase
Fraction Cpm/ sample No Ara-C Total sample Cell debris 20,0OOgsupernatant fluid 20,OOOgpellet (virus) Ara-C Total sample Cell debris 20,OOOgsupernatant fluid 20,OOOgpellet (virus)
Cpm in peak 2
Cpm/ sample
Cpm in peak 2
318,000 78,000 171,400
54,300 5,300 38,200
294,600 81,400 103,000
49,100 8,400 8,700
59,600
9,400
124,000
31,200
396,000 87,500 247,000
63,200 5,000 46,700
410,600 96,000 146,000
65,000 8,600 11,100
72,000
9,800
171,000
42,500
a RK cells were infected and incubated in EDS or in EDS plus Ara-C (50 pg/ml). Between 4.5 and 5.5 hours after infection, the cells were labeled by incubation in EDS/lO containing leutine-14C (2 &i/ml; specific activity, 135 mCi/ mmole) or EDS/lO containing Ara-C (50 pg/ml) and 1eucineJH (20 pCi/ml; specific activity, 3.1 Ci/mmole). The medium was changed to EDS containing twice the normal amount of amino acids with or without Ara-C. Part of the cultures was harvested immediately; another part was incubated further at 37” for 6 hours. The cells and culture fluids were collected, the ‘%-labeled, Bra-C-treated cultures were mixed with the 3Hlabeled, untreated cultures, the samples were sonicated, centrifuged, and the total radioactivity, as well as the amount present in the fractions of radioactive proteins migrating in peak 2 in polyacrylamide gels, was determined.
gregation into viral particles. Immediately after the labeling period, about 66% of all the peak 2 proteins synthesized by the cells could not be sedimented at a force which sediments the virions; after the chase, however, only about 16% of these proteins were not sedimentable under these conditions. There was no significant difference in the behavior of these proteins in Ara-Ctreated and untreated cells. That the pellet obtained after centrifugation at 20,000 g
60
BEN-PORAT,
SHIlMONO,
indeed contained viral particles was further demonstrated by precipit,ation with specific antiserum. About 50-60% of the total radioactivity in these fractions obtained from both the Ara-C-t’reated and the untreated cells could thus be precipitated. The formation of viral particles in Ara-Ctreated cells would, in itself, indicate that in these cells peak 2 proteins do flow to the nucleus, since assembly of these proteins into particles normally occurs in the nucleus. However, if the absence of viral DNA affected this flow, assembly might possibly occur in the cytoplasm. We therefore determined whether t,he flow of peak 2 proteins from the cytoplasm to t,he nucleus occurred in Ara-C-treated cells in an experiment similar to that summarized in Fig. 1 and Table 2. The results of this experiment are summarized in Table 5. In the Ara-Ctreated cells, immediately after the pulse, about 14% of the total labeled protein, as well as of the labeled peak 2 proteins, were found in the nuclear fraction, and this proportion increased significantly after the chase. It is clear, therefore, that migration of peak 2 proteins to the nucleus occurred even when viral DNA synthesis was drastically reduced by Ara-C. However, in the untreated cells 37% of t’he peak 2 proteins were in the nuclear fraction after the chase as compared to 29% in Am-C treated cells. Thus, although migrat’ion occurred in the TABLE
5
MOVEMENT OF PROTEINS FROM THE CYTOPLASM TO THE NUCLEUS IN ARA-C-TREATED CELLSPercentage of radioactivity the nucleus Sample
Total
Peak 2
Pulse Pu1sechase Untreated Ara-C (50 /*g/ml)
14 13
in
32 28
Pulse Pulsechase 17 14
37 29
a The experiment was performed as described in the legend to Fig. 1 and the amount of 1% and aH in peak 2 obtained from the cytoplasmic and nuclear fractions was determined. The Ara-Ctreated cells were treated identically but Ara-C (50 rg/ml) was present in the culture fluid throughout the experiment.
AND
KAPLAN
Ara-C-treated cells, it was reduced. This reduction is significant and has been obtained repeatedly. DISCUSSION
The proteins synthesized by Pr virusinfected cells which migrate electrophoretitally in polyacrylamide gels in a position designated as peak 2 const’itute the major protein component of the viral particles (Shimono et al., 1968). In the present paper we show that these proteins are synthesized in the cytoplasm and move to the nucleus, where they are assembled into viral particles. The simplest and most appealing explanation for the mechanism for this migration is diffusion. Thus, if aggregation of viral proteins within the nucleus acts as a diffusion sink, the concentration gradient would be maintained and this would lead to an accumulation of these proteins in the nucleus. Since viral DNA within the nucleus seemed to be one possible agent responsible for the aggregation of viral proteins into particles, we tested whether accumulation of these proteins would occur in the nucleus in the absence of viral DNA synthesis. This was found to be the case. However, the migration of these proteins to the nucleus occurred less efficiently when viral DNA synthesis was inhibited. Whether t’his is due to the absence of viral DNA per se or to another factor that may be affected by these conditions is not clear. The fact that structural viral proteins migrate to the nucleus in Ara-C-treated cells does not eliminate the possibility that diffusion is the basis for this migration. However, our experiments do demonstrate that viral DNA does not play a major role in the migration of the proteins, and one would have to postulate the existence within the nucleus of another condensing factor responsible for the aggregation of the protein subunits into particles. Such a factor (presumably a protein) would, in contrast to the other virus-specific proteins, be synthesized in the nucleus, because aggregation would otherwise be expected to occur in the cytoplasm. Little is known about the internal proteins of the herpesviruses, and
PROTEIN
SYNTHESIS
IN HERPESVIRUS-INFECTED
the existence of such a condensing protein remains to be demonstrated. The inability of Ara-C to inhibit the synthesis of structural viral proteins in Pr virus-infected RK cells contradicts the results reported for other animal virus-cell systems, in which the synthesis of progeny viral DKA is required before structural viral proteins are synt,hesized (see Kaplan and Ben-Porat’, 1968). The difference may lie in the fact that in Pr virus-infected RK cells total inhibition of DNA synthesis is not achieved. Even at very high concentrations of Ara-C (50 ,ug/ml or more), DNA synthesis is inhibited by only 95-98 %. Alt’hough these conditions inhibit to a considerable extent the synthesis of viral DNA, some progeny viral DNA may, nevertheless, be formed and it seems that only a few of these molecules may suffice to lead to the formation of normal amounts of struct,ural viral proteins in this system. REFERENCES BEN-P• RAT, T., BROWN, M., and KAPLAN,
A. S. (1968). Effect of l-P-o-arabinofuranosylcytosine on DNA synthesis. II. In rabbit kidney cells infected with herpes viruses. Mol. Pharmacol. 4, 139-146.
CELLS. II.
61
FELLUGA, B. (1963). Electron microscopic observations on pseudorabies virus development in a line of pig kidney cells. Ann. Sclavo 5, 412-424. FUJI~ARA, S., and KAPLAN, A. S. (1967). Site of protein synthesis in cells infected with pseudorabies virus. V’Gology 32, 60-68. KAPLAN, A. S., and BEN-P• RAT, T. (1968). Metabolism of animal cells infected with nuclear 22, 427450. DNA viruses. Ann. Rev. Microbial. MORGAN, C., ELLISON, S. A., ROSE, H. M., and MOORE, D. H. (1954). Structure and development of viruses as observed in the electron microscope. I. Herpes simplex virus. J. Exptl. Med. 100, 195-202. OLSHEVSKY, U., LEVITT, J., and BECKER, Y. (1967). Studies on the synthesis of herpes simplex virions. V+oZogy 33, 323-334. REISSIG, M., ~~~MELNICE, J. L. (1955). The cellular changes produced in tissue cultures by herpes B virus correlated with the concurrent multiplication of the virus. J. Exptl. Med. 101, 341351. SHIMONO, H., BEN-P• RAT, T., and KAPLAN, A. S. (1968). Synthesis of proteins in cells infected with herpesvirus. I. Structural viral proteins. Virolow. 37, 49-55. TOURNIER, P., CATHALA, F., and BERNHARD, W. (1957). Ultrastructure et dbveloppement intracellulaire du virus de la varicelle observe au microscope Blectronique. Presse Med. 65, 12291234.