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Synthesis of proteins in cells infected with herpesvirus
VIROLOGY49, 102-111 (1972)
Synthesis of Proteins in Cells Infected with Herpesvirus VIII. Absence of Virus-induced Alteration of Nuclear Membrane in Arginine-Deprived Cells1 G E O R G E E. M A R K .2 ANT) ALBEP~T S. K A P L A N
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Departments of Microbiology, *Temple University, School of Medicine, Philadelphia, Pennsylvania, 19140 and **Research Laboratories, Albert Einstein Medical Center, Philadelphia, Pennsylvania 191~1 Accepted March 30, 1972 In an effort to determine the role of arginine in herpesvirus infection, several parameters of infection were measured under conditions of arginine starvation. It was found that whereas infection of rabbit kidney cells in stationary phase results in an increase in the rate of incorporation of 3H-choline into the phospholipids of the nuclear membrane, as well as in a flow of phospholipids from the cytoplasmic membranes to the nuclear membrane, these events do not occur in cells deprived of arginine. Furthermore, even though virus-specific glyeoproteins are synthesized by arginine-deprived cells, the structural viral glyeoproteins are not integrated into the nuclear membrane as they are during the course of normal infection. We conclude from these results that the virus-induced synthesis of nuclear membrane does not occur in arginine-deprived, infected cells and that viral glyeoproteins can only become integrated into areas of the nuclear membrane newly synthesized by the cells after infection. INTRODUCTION
The mechanism controlling the migration of structural viral proteins from their site of synthesis in the cytoplasm to their site of assembly in the nucleus is at present un -i known. Although structural viral proteins are synthesized b y infected cells deprived of arginine, migration of these proteins to the nucleus and their assembly into nucleoeapsids do not take place under these conditions (Courtney et al., 1970 and 1971; Olshevsky and Beeker, 1970; ~/Iark and Kaplan, 1971), indicating t h a t arginine-rieh proteins m a y be directly or indirectly involved in the assembly process. Recent experiments b y Winters and RusThis investigation was supported by a grant sell (1971) suggest t h a t nuclear membranes from the National Institutes of Health (AI-03362). m a y play an important role in the process of s Present address : Department of Biochemistry, McGill University School of iVfedicine, Montreal, assembly of adenoviruses. Since, after infection of cells with pseudorabies (Pr) virus, Quebec, Canada. Present address : Department of Microbiology, there is an increase in the rate of synthesis of Vanderbilt University School of Medicine, Nash- nuclear m e m b r a n e (Ben-Porat and Kaplan, ville, Tennessee 37203. 1971 and 1972) and integration of virus102 Copyright© 1972by AcademicPress, Inc. All rights of reproductionin any formreserved. I t is well-established t h a t the structural proteins of the herpesviruses are synthesized in the cytoplasm and migrate thereafter to the nucleus where they are assembled into nueleoeapsids (Fujiwara and Kaplan, 1967; Olshevsky et at., 1967; Spear and Roizman, 1968; Ben-Porat et aI., 1969). The nueleoeapsids then acquire an envelope b y budding from the inner nuclear membrane of the infected cells (Siegert and Falke, 1966; Shipkey et al., 1967; Darlington and Moss, 1968; Nii et al., 1968), and the mature virus is released into the cytoplasm.
PROTEIN IN CELLS INFECTED WITH HERPESVII~US specific structural glycoproteins into this membrane (Ben-Porat and Kaplan, 1970), we considered the possibility that there may be a correlation between these characteristic alterations in the nuclear membrane and the migration of the viral capsid proteins to the nucleus. The experiments described in this paper were therefore performed to determine whether the alterations in the nuclear membrane normally induced by infection with Pr virus occur under conditions of arginine starvation. MATERIALS AND METHODS
Virus and cell culture The properties of Pr virus and the cultivation of rabbit kidney (RK) monolayer cultures were described previously (Kaplan, 1957 and 1969). Primary R K cells were grown in 90-mm Petri dishes in ELS. The cells were infected with approximately 20 plaque-forming units (PFU)/eell. Media and solutions ELS: Earle's saline containing 0.5% lactalbumin hydrolyzate and 5% bovine serum. EDS: Eagle's synthetic medium (Eagle, 1959), plus 3% dialyzed bovine serum. EDS ( - A r g ) : The same as EDS, except that arginine was omitted. TBSA: a buffer containing the same salts as PBS (Dulbeceo and Vogt, 1954), except that the phosphate was replaced by 0.01 M TrisHC1, pH 7.5, plus 1% crystalline serum albumin. RSB: 0.01 M Tris HC1, pH 7.4, 0.01 M KC1, and 0.0015 M MgCI~ (Warner et al., 1963). Triton-X-100 buffer: 0.14 M NaC1, 0.005 M MgC12, 0.01 M Tris-HC1, ptI 8.5, and 0.5 % Triton-X-100. Chemicals and radiochemicals. Triton X100 was a gift of Rohm and Haas Co. 6-~H-glueosamine (sp act, 3.6 Ci/mM), 114C-glueosamine (sp act, 57 mCi/mM), and 3H-methyleholine (sp act, 100 mCi/mM) were purchased from the New England Nuclear Coporation; 14C-L-leucine (sp act, 312 mCi/mM) was purchased from Schwartz/Matin. Cell fraetionation. This was done essentially according to Penman (1969). R K cells (4-8 X 106/ml) were scraped into RSB and
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allowed to swell for 10 min at 4 °. The cells were homogenized (20 strokes) in ~ tightfitting Dounee homogenizer and the tyroplasmie fraction was separated from the nuclear fraction by centrifugation at 1,000g for 7 rain. To remove the outer nuclear membrane (Holtzmann et al., 1966), the nuclear pellet was resuspended in Triton X-100 buffer, rehomogenized (15 strokes), and centrifuged as above; the resulting supernatant fluid (outer nuclear membrane fraction) was collected. The pellet contained at least 80 % of the nuclei present in the initial cell suspension; the nuclei appeared microscopically free of cytoplasmic tags. Since choline and glucosamine become membrane-associated only (Kornfeld and Ginsburg, 1966; Molnar, 1967; Plagemann, 1968), we refer in this paper to the phospholipids and the glycoproteins extracted from the different fractions (after extensive acid washing) as being the phospholipids and the glycoproteins of the membranes present in each of these fractions. We have shown previously (Ben-Porat and Kaplan, 1971) that the so-called outer nuclear membrane fraction has a different phospholipid composition from the other cellular fractions. We do not know, however, to what degree the outer nuclear membrane fraction is contaminated with cytoplasmic or nuclear components. In some of the experiments we present the results obtained with this fraction; we do so only because we consider it desirable to present the data obtained with all fractions isolated from the cells. For electrophoretic analysis of the glyeoproteins, the cytoplasmic fraction and outer nuclear membrane fraction were combined and are called the cytoplasmic fraction. SDS-polyacrylamide gel electrophoresis. Electrophoresis, fractionation of the gels, and the assay of radioactivity were performed as described previously (Kaplan et al., 1970). Extraction of lipids. The fractions were washed with cold perehloric acid, 0.2 N, the lipids were extracted, and the radioactivity in the lipid fractions was determined as described previously (Ben-Porat and Kaplan, 1971).
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FIG. 1. Incorporation or ~H-eholine into the phospholipids of infected and uninfected RK cells i~ &e presence and absence of arginine. Primary RK monolayers were infected (or mock-infected) and incubated in EDS or EDS (-Arg). At 3 hr postinfection, the culture media were changed to EDS or EDS (-Arg) containing 3It-choline (1.2 ~Ci/ml) and incubation was continued. At various times thereafter the cells were scraped into the overlying media and acidified with perchloric acid (final concentration, 0.2 N). The acidified samples were washed four times with 0.2 N perchloric acid, the lipids were extracted, and the amount of radioactivity associated with the lipids was determined, as described in Materials and Methods. (A) arginine+; (B) arginine-. Closed circles, solid line, infected cells; open circles, broken line, mock-infected cells. RESULTS
Incorporation of 3H-choline into the phospholipids of infected and uninfected cells in the presence and absence of arginine During the course of infection of cells with P r virus, various changes in the metabolism of phospholipids can be detected. One of these changes is an increase, after infection of stationary phase cells, in the rate of incorporation of all-choline into the phospholipids, an increase t h a t is especially marked in the nuclear fraction (Ben-Porat and Kaplan, 1971). The experiments summarized in Fig. 1 and Table 1 were designed to determine whether these virus-induced alterations of phospholipid metabolism will also occur when the cells are deprived of arginine. Figure 1A shows t h a t under conditions of normal infection, i.e., in the presence of arginine, there was a slight inhibition of choline incorporation early in infection. B y 5 hr postinfeetion an increase in the incor-
poration became evident. By 9 hr postinfection, i.e., near the end of the infective process, the rate of incorporation of choline again decreased. Although the early inhibition of choline incorporation, as well as its subsequent activation, are relatively small, these changes were observed consistently when stationary phase cells were infected. Infection of arginine-deprived cultures (Fig. 1B) also resulted in a slight initial decrease in the incorporation of choline; however, in contrast to cells infected under normal conditions, at no time did infected arginine-deprived ceils show an increased rate of choline incorporation. Table 1 shows the distribution of choline between the nuclear and cytoplasmic fractions of infected and uninfected cells in an experiment similar to the one illustrated in Fig. 1. Infection in the presence of arginine gave
PROTEIN IN CELLS INFECTED WITH HERPESVIRUS
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TABLE 1 INCORPORATION OF 3I~I-CHOLINE INTO TIIE PItOSPIIOLIPIDS OF INFECTED AND UNINFECTED R K CELLS IN THE PRESENCE AND ABSENCE OF ARGININE a
Arginine
Fraction
Infected
Present
Cytoplasmic Outer nuclear Inner nuclear Cytoplasmic Outer nuclear Inner nuclear
26.51 b 2.07 3.70 23.26 1.76 2.83
Absent
Uninfected 24.16 2.23 2.96 24.80 1.81 2.93
Infected Uninfected 1.10 0.93 1.25 0.94 0.97 0.98
Primary RK monolayer cultures, 8 days old, were infected (or mock-infected) and incubated in EDS or EDS (-Art), containing aIl-eholine (3 ~Ci/ml) between 3 and 8 hr postinfeetion. The cells were fraetionated, acidified, washed with acid, the lipids extracted, and the amount of radioactivity determined, as described in Materials and Methods. b cpm X 10-~/eulture. results similar to those reported previously (Ben-Porat and Kaplan, 1971). Increases of approximately 10 % in the amount of choline incorporated into the cytoplasmic lipids and 25 % into the lipids of the inner and outer nuclear membranes were obtained. I n the absence of arginine, however, no virusinduced increase of choline incorporation into the phospholipids of the cytoplasm or nuclear membranes was observed.
Flow of phospholipids from the cytoplasm to the nuclear membrane in arginine +- and arginine--infected cells After infection of R K cells with P r virus, in addition to the increase in incorporation of externally supplied choline into the lipids of the nuclear membrane, there is a flow to the nuclear m e m b r a n e of phospholipids preexisting in the cells at the time of infection which most likely represents the synthesis of new nuclear m e m b r a n e (Ben-Porat and Kaplan, 1972). The experiment summarized in Table 2 was performed to determine whether this flow occurs in infected argininedeprived cells. I n this particular experiment, a virus-induced increase of approximately 45 % in the a m o u n t of the phospholipids associated with the inner nuclear m e m b r a n e was obtained. I t has been shown previously (Ben-Porat and Kaplan, 1972) t h a t this increase is not the result of an increased contamination in the infected cells of the nuclear fraction with cytoplasmic membranes, b u t t h a t it reflects the synthesis
TABLE 2 DISTRIBUTION~ AFTER A CHASE, OF THE RADIOACTIVITY DERIVED FROiYi 3~I-CHOLINE AMONG THE CYTOPLASMIC AND NUCLEAR MEMBRANES IN THE PRESENCE AND ABSENCE OF ARGININE a
Arginine
Present Absent
Treatment
Infected Uninfected Infected Uninfected
Membrane Cytoplasmic Outer Inner nuclear nuclear 19,550 18,250 19 450
I 3420 / 3670 IJ 3150
6210 4320 4790 4420
Primary RK monolayer cultures, 3 days old, were incubated for 3 days in EDS eontaining-~Hcholine (0.2 uCi/ml). The cultures were washed several times with Earle's saline and incubated for 4 days in EDS not containing labeled choline. At the end of this incubation period, the specific activities of the phospholipids (cpm/mg phosphorus) in the different cellular fractions were approximately the same. The monolayers were infected (or mock-infected) and incubated in either EDS or EDS(-Arg.) At 7 hr postinfeetion the cells were harvested, fraetionated, and the radioactivity in the phospholipids associated with the various fractions was determined, as described in Materials and Methods. cpm/eulture. of new nuclear membrane. Since the specific activities of the phospholipids in the different cellular fractions were the same at the time of infection (see legend Table 2), these
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MARK AND KAPLAN
results indicate that by 7 hr postinfection the amount of phospholipids of the nuclear membrane in the infected cells had increased by about 45 %. There was also in arginine-deprived cells a slight increase in the amount of phospholipid associated with the inner nuclear membrane, but it was relatively small compared to that found in infected cells supplied with arginine. Thus, the increase in the incorporation of externally supplied choline into the nuclear membrane and the flow of preexisting cellular phospholipids to this membrane normally observed in infected cells are greatly reduced under conditions of arginine starvation. We conclude therefore that little new nuclear membrane is formed in infected, arginine-deprived cells.
Gel electrophoretic analysis of the glycoproteins synthesized in infected, arginine-deprived cells The envelope of Pr virus is composed of virus-specific glycoproteins which are identical to those associated with the nuclear membrane of infected cells; cell-specific proteins do not become part of the virions (Ben-Porat et al., 1970; Ben-Porat and Kaplan, 1970). The mechanism which ensures the presence of only virus-specific proteins in the viral envelope is not yet understood completely. However, since after infection there is a considerable increase in the amount of phospholipids in the nuclear membrane which represents de novo synthesis of this membrane (Ben-Porat and Kaplan, 1971 and 1972; see also Fig. 1, Tables 1 and 2 of this paper), and since the infected cells synthesize exclusively virus-specific proteins, only viral proteins presumably would be integrated into the newly assembled nuclear membrane. It is not clear, however, whether viral glycoproteins become associated with newly synthesized nuclear membrane only, or whether they also become integrated into areas of the nuclear membrane preexisting in the cells at the time of infection. In Pr virus-infected arginine-deprived cells, most of the structural viral proteins are synthesized (Mark and Kaplan, 1971), but, as shown above, little new nuclear
TABLE 3 EFFECT OF ARGININE STARVATION ON THE SYNTHESIS OF PROTEINS AND GLYCOFROTEINS IN VIRUS-INFECTED CELI~S a
Inhibition Arginine+ Arginine- (percent) Leucine 68b 25 63 Glucosamine 28 11 60 Primary monolayers of RK cells were infected and incubated in EDS or EDS (-Arg). At 4 hr postinfeetion, the medium was replaced either with EDS containing one-half the usual amount of leucine or with EDS (-Arg) also containing this amount of leueine; both contained 6-3H-glueosamine (10 ~Ci/ml) and uC-leueine (2 ttCi/ml). At 8 hr postinfection, the cells were scraped into the overlying medium and the amount of radioactivity incorporated into aeid-precipitable material was determined as described in Materials and Methods. b epm X 10-3/culture. membrane is formed. It was interesting, therefore, to determine whether (1) structural viral glycoproteins are synthesized in arginine-deprived cells, and, if so, (2) whether these glycoproteins become part of the nuclear membrane, despite the lack of synthesis of new nuclear membrane. Table 3 shows that starving the infected cells of arginine reduced the synthesis of glyeoproteins to about the same degree as the synthesis of all other proteins. Thus, glycoproteins are synthesized in the infected cell in the absence of arginine. During the course of normal infection, R K cells synthesize structural and nonstructural viral glycoproteins. Most of the glycoproteins synthesized by the infected cells do not become associated with mature virions. These nonstructural glycoproteins are associated with the cytoplasmic fraction only and migrate as one peak, designated peak 3a. Four structural glycoproteins are also synthesized by the infected cells; most of these become associated with the nuclear membrane and become part of the viral envelope. When all the glycoproteins synthesized by the infected cells are analyzed by gel eleetrophoresis, the nonstruetural glycoproteins migrating as peak 3a obscure the structural proteins migrating as peaks
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FIG. 2. Electropherograms of the glycoproteins synthesized by infected cells in the presence and absence of
arginine. RK cells were infected and incubated in EDS or EDS (-Arg). At 5 hr postinfection 3H-choline (10 t,Ci/ml) was added to the culture media, and the cultures were incubated further for 2 hr. The cells were then harvested and the proteins electrophoresed as described ill Materials and Methods. (A) arginine+; (B) arginine-. 3 and 4. The latter glycoproteins are clearly resolved only when purified nuclear or viral preparations are eleetrophoresed (Ben-Porat and Kaplan, 1970). Figure 2 shows the eleetrophoretic pattern of migration in aerylamide gels of the glycoproteins synthesized by infected cells incubated in the presence and absence of arginine. The electrophoretic profiles of the glyeoproteins synthesized by infected, arginine+ cells and by arginine- cells were similar, although some differences, which will be discussed below, were found between the two. Figure 3 illustrates an experiment in which the electrophoretic patterns of the glycoproteins associated with the nuclear and cytoplasmic fractions in arginine+ and arginine- cells were analyzed. Figure 4 shows the results obtained when the glycoproteins of similarly treated uninfected cells were analyzed. Since most glycoproteins (approximately 90 %) are associated with the cytoplasmic fraction, the electrophoretie pattern of the glycoproteins from this fraction of the cells (Fig. 3A) is similar to that of the glycoproteins from unfractionated cells (Fig. 2). A relatively larger proportion of the glycoproteins synthesized by arginine- cells than by arginine+ cells remained at the origin, and a peak (1) which was barely detectable in the electrophoregrams of the glyeoproteins from arginine+-infeeted ceils appeared
in those from arginine- cells. The large amount of glycoproteins remaining at the origin and the peak 1 glyeoproteins also appeared in the profiles of the glycoproteins from uninfected cells (see Fig. 4). Otherwise, the cytoplasmic glycoproteins synthesized by arginine--infected cells migrated coincidentally with those synthesized by arginine +infected cells, indicating that the same glycoproteins were synthesized by both. However, a larger proportion of the total glyeoproteins from infected arginine--ce]Is than from arginine + cells migrated as peaks 4a and 6. Furthermore, although the glyeoproteins migrating as peaks 3 and 4 are, in major part, obscured by the glyeoproteins migrating as peak 3a, it appears from the shoulders in peak 3a that the cytoplasmic fraction from arginine- cells is also richer than that from arginine+ cells in glycoproteins in peaks 3 and 4. The larger amounts of peaks 3, 4, 4a and 6 relative to peak 3a glyeoproteins in the cytoplasmic fraction from arginine- cells may be due to the lack of integration of the structural proteins (3, 4, 4a and 6) into the nuclear membrane of these cells (see below), leading to their accumulation in the cytoplasm. This is probably a factor, but Fig. 2 indicates that relative to the glycoproteins migrating as peak 3a, the glycoproteins in peak 4a and 6 seem to be synthesized in larger amounts by arginine- than by arginine + ceils. We have
108
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FIG. 3. Electropherograms of the glycoproteins associated with the cytoplasmic and inner nuclear membranes of infected cells incubated in the presence or absence of arginine. I~K cells were infected and incubated in EDS or E D S (-Arg). At 5 hr post infection ttC-glucosamine (4 ~Ci/ml) was added to the arginine + cultures and ~H-glucosamine (20 ttCi/ml) to the arginine- cultures. The cultures were further incubated for 2 hr, and the t4C-labeled arginine + cells and ~H-labeled arginine- cells were harvested and mixed. The cytoplasmic and nuclear fractions were isolated and the proteins electrophoresed, as described in Materials and Methods. (A) Cytoplasmic fraction; (B) Nuclear fraction. Closed circles, solid lines, arginine + cells ; open circles, broken lines, arginine- cells. I
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FIG. 4. Electropherogram of the glycoproteins associated with the cytoplasmic and inner nuclear membranes of uninfected cells. Uninfected I~K cells were incubated in EDS with 3tt-glucosamine (8 t~Ci/ml) for 2 hr. The cells were then harvested, the cytoplasmic fraction separated from the nuclear fraction, and the proteins electrophoresed as described in Materials and Methods. (A) Cytoplasmic fraction; (B) Nuclear fraction.
no explanation at present for this phenomenon. The electrophoretic patterns of the glyeoproteins associated with the nuclear fractions from arginine+ and arginine- cells were different. The characteristic pattern of migration of nuclear glycoproteins synthesized by infected cells (which is also that
of mature virions) was not observed in the arginine-deprived ceils. Instead, the pattern obtained was similar to that of the glyeoproteins from the nuclei of uninfected cells (Fig. 4). We conclude from these results that (1) the major structural viral glycoproteins are synthesized in arginine--infeeted cells and
PROTEIN IN CELLS INFECTED WITH HERPESVII:~US (2) that these proteins remain associated with the cytoplasmic fraction and are not incorporated into the nuclear membrane as in arginine-+infected cells. It seems, therefore, that structural viral glycoproteins cannot be integrated into the nuclear membrane preexisting in the cells at the time of infection but become associated only with new regions of the nuclear membrane, the synthesis of which is normally stimulated by infection. DISCUSSION The results in this paper show that the de novo assembly of nuclear membrane that normally occurs in Pr virus-infected R K cells is greatly reduced when the cells are deprived of arginine. Thus, in the absence of arginine, infection with Pr virus does not result in the usual increase in choline incorporation into the phospholipids of stationary R K cells. The relatively large increase in the incorporation of phospholipids into the nuclear membrane of infected cells is also not observed (Table 1, Fig. 1). In addition, the virus-induced flow from cytoplasmic to nuclear membranes of phospholipids preexisting in the cells at the time of infection is greatly reduced in the absence of arginine (Table 2). Since the increased rate of incorporation of externally supplied choline, as well as the virus-induced flow of pre-formed phospholipids to the nuclear membrane, probably represents de novo synthesis (reduplication) of that membrane, we conclude that in the absence of arginine, little new nuclear membrane is formed. This conclusion has been reinforced by electron microscopic observations which show that the convolutions of the nuclear membranes normally seen in cells infected with the herpesviruses does not occur in argininedeprived infected cells (unpublished results). The electrophoregram of the glyeoproteins synthesized by arginine-deprived, infected cells is similar to that of the glycoproteins synthesized under conditions of normal infection; on the basis of their electrophoretie profiles in acrylamide gels we concude that both the nonstructural and the structural viral glyeoproteins are synthesized by the arginine-deprived cells. However, the structural viral glyeoproteins do not become part
109
of the nuclear membrane in arginine-infected cells; instead, these proteins remain associated with the cytoplasmic fraction. We do not know at present the nature of the glycoproteins synthesized by arginine-infected cells that become associated with the nuclear membranes. These glycoproteins may be of viral origin and may have been abnormally glyeosylated or abnormally cleaved as a consequence of argin]ne deprivation; on the other hand, they may be cellspecific. Since the pattern of eleetrophoretic migration of these glyeoproteins is similar to that of the nuclear glyeoproteins from uninfected cells, the second alternative is more likely. This would imply that whereas most host protein synthesis is inhibited in arginine-infeeted cells (Mark and Kaplan, 1971), some species of cell-specific proteins are still synthesized by such cells. From previous data (Ben-Porat and Kaplan, 1971 and 1972), we think that the mechanism which ensures that the viral envelope is composed of virus-specific proteins only is as follows: new regions of the nuclear membrane are synthesized after infection into which viral proteins (the only proteins synthesized by the infected cells) are integrated; the viral particles bud only from these newly synthesized areas of the nuclear membrane. The experiments described in this paper show that when new nuclear membrane is not synthesized, the viral glycoproteins do not become part of the nuclear membrane. Thus, the nuclear membrane preexisting in the cell at the time of infection cannot accept viral giycoproreins. We conclude, therefore, that during the course of infection, the viral giycoproreins do not become associated with the nuclear membrane in a random manner but become associated only with the newly synthesized areas of this membrane. We have shown previously that viral DNA and proteins are synthesized in arginine-deprived, infected cells. However, the structural viral proteins which normally migrate from the cytoplasm (the site of their synthesis) to the nucleus (the site of assembly of the nucleoeapsids) remain associated with the cytoplasm under conditions of arginine starvation (Mark and Kaplan,
110
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1971; C o u r t n e y et al., 1971). Thus, the conditions favoring the migration of the structural viral proteins to the nucleus do not exist in arginine-deprived cells. W e consider t h a t the assembly of the structural viral proteins into capsids within the nucleus, the latter thus serving as a diffusion sink, is likely to be responsible for the continued migration of the proteins to the nucleus. I t has been shown recently t h a t nuclear m e m b r a n e structures are i m p o r t a n t for the assembly of adenovirus (Winters and Russell, 1970). If this were also the case for herpesvirus-infected cells, the lack of assembly of viral particles in arginine-deprived cells and the consequent failure of migration of viral proteins to the nucleus m a y be related to the lack of characteristic virusinduced alterations of the nuclear m e m b r a n e in these cells. REFERENCES BEN-PonA% T. SHI~rONO, H., and KAPL~N, A. S. (1969). Synthesis of proteins in cells infected with herpesvirus. II. Flow of structural viral proteins froln cytoplasm to nucleus. Virology 37, 56-61. BEN-PoI~AT, T. SHIMONO,H., and KAPLAN, A. S. (1970). Synthesis of proteins in cells infected with herpesvirus. IV. Analysis of the proteins in viral particles isolated from the cytoplasm and the nucleus. Virology 41,256-264. BEN-PoI~AT,T., and KAPLAN,A. S. (1970). Synthesis of proteins in cells infected with herpesvirus. V. Viral glycoproteins. Viroiogy 41,265-273. BEN-PoRAT, T., and KAPLAN, A. S. (1971). Phospholipid metabolism of herpesvirus-infected and uninfected rabbit kidney cells. Virology 45,252264. BEN-PoI~AT,T., and KA~'LAN,A. S. (1972). Studies on the biogenesis of herpesvirus envelope. Nalure (London) 235, 165-166, COURTNEY,R. J., MeCoMBs, R. iV[., and BENYESItMELNICK, M. (1970). Antigens specified by herpesviruses, I. Effect of arginine deprivation on antigen synthesis. Virology 4[!, 379-386. COUI~TNE¥,R. J., MeCoMBS, R. M., and BENYESHMELNICK, M. (1971). Antigens specified by herpesviruses. II. Effect of arginine deprivation on the synthesis of cytoplasmic and nuclear proteins. Virology 43,356-365. DARLINGTON, R. W., and Moss, L. H. (1968). Herpesvirus envelopment. J. Virol. 2, 48-55. DULBECCO,R., and VOGT,M. (1954). Plaque formation and isolation of pure lines of poliomyelitis virus. J. Exp. Med. 99,183 199.
EAGLE,H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. FVJIWAR.a, S., and KAPSAN, A. S. (1967). Site of protein synthesis in cells infected with pseudorabies virus. Virology 32, 60-68. HOLTZMAN,E., SMITH, I., and PENMAN, S. (1966). Electron microscopic studies of detergent treated nuclei. J. Mol. Biol. ]7,131-135. KAPLAN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell system by the plaque technique. Virology 4, 435-457. KAPL~N, A. S. (1969). Herpes simplex and pseudorabies viruses. Virology Monographs, Vol. 5 (Gard, S., Hallauer, C., and Meyer, K. F., eds.) Springer, New York. KAPLAN,A. S., and BEN-PORAT,T. (1970). Synthesis of proteins in cells infected with herpesvirus. VI. Characterization of the proteins of the viral membrane. Proc. Nat. Acad. Sci. U.S.A. 66, 799-806. KAPLAN, A. S., SHIMONO,~-~., and BEN-PORAT,T. (1970). Synthesis of proteins in cells infected with herpesvirus. III. Relative amino acid content of various proteins formed after infection. Virology 40, 90-101. KORNFELD, S., and GINSBURG, V. (1966). The metabolism of glucosamine by tissue culture cells. Exp. Cell Res. 41,592-600. ~AR~:, G. E., and KAPLAN,A. S. (1971). Synthesis of proteins in cells infected with herpesvirus. VII. Lack of migration of structural viral proteins to the nucleus of arginine-deprived cells. Virology 45, 53-60. MOLNAR, J. (1967). Glycoproteins of Ehrlich aseites carcinoma cells. Incorporation of (14C) glucosamine and (14C) sialic acid into membrane proteins. Biochemistry 5, 3064-3076. NII, S., MORGAN, C., and ROSE, H. M. (1968). Electron microscopy of herpes simplex virus. II. Sequence of development. J. Virol. 2, 517-536. OLSHF~VSKY,U., and BEEPER, Y. (1970). Synthesis of herpes simplex virus structural proteins in arginine-deprived cells. Nature (London) 226, 851-853. OLSHEVSKY, U., LEVITT, J., and BECKER, Y. (1967). Studies on the synthesis of herpes simplex virions. Virology 33,323-334. PENMAN, S. (1969). Preparation of purified nuclei and nucleoli from mammalian cells. In "Fundamental Techniques in Virology" (K. Habel and N. P. S~lzman, eds.), pp. 35-48. Academic Press, New York. PLAGEMANN,P. G. W. (1968). Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. I. Incorporation of choline into phosphatidylcholine of mitochondria and other membranous structures and
PROTEIN IN CELLS INFECTED WITH HERPESVIRUS effect of metabolic inhibitors. Arch. Biochem. Biophys. 128, 70-87. SHIPKEY, F. H., ERLANDSON,1~. t . , BAIUEY,R. B., B~BCOCK, V. I., and SOUT~AM, C. M., (1967). Virus biographies. II. Growth of herpes simplex virus in tissue culture. Exp. Mol. Pathol. 6, 3967. SIEGERT, R., and FALKE, D. (1966), Elektronenmikroskopische Untersuehungen fiber die Entwicklung des Herpesvirus hominis in Kulturzellen. Arch. Gesamte. Virusforsch. 19, 230-249.
l ll
SP~AR, P. G., and ROIZMAN,B. (1988). 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. WANNER, J. R., KNOPF, P. M., and RICH, A. (1963). A multiple ribosomal structure in protein synthesis. Proc. Nat. Acad. Sci. U.S.A. 49,122-129. WINSE~S, W., and RUSSELL,W. C. (1971). Studies on the assembly of adenovirus in vitro. J. Gen. Virol. 10, 181-194.
Synthesis of Proteins in Cells Infected with Herpesvirus VIII. Absence of Virus-induced Alteration of Nuclear Membrane in Arginine-Deprived Cells1 G E O R G E E. M A R K .2 ANT) ALBEP~T S. K A P L A N
TM
Departments of Microbiology, *Temple University, School of Medicine, Philadelphia, Pennsylvania, 19140 and **Research Laboratories, Albert Einstein Medical Center, Philadelphia, Pennsylvania 191~1 Accepted March 30, 1972 In an effort to determine the role of arginine in herpesvirus infection, several parameters of infection were measured under conditions of arginine starvation. It was found that whereas infection of rabbit kidney cells in stationary phase results in an increase in the rate of incorporation of 3H-choline into the phospholipids of the nuclear membrane, as well as in a flow of phospholipids from the cytoplasmic membranes to the nuclear membrane, these events do not occur in cells deprived of arginine. Furthermore, even though virus-specific glyeoproteins are synthesized by arginine-deprived cells, the structural viral glyeoproteins are not integrated into the nuclear membrane as they are during the course of normal infection. We conclude from these results that the virus-induced synthesis of nuclear membrane does not occur in arginine-deprived, infected cells and that viral glyeoproteins can only become integrated into areas of the nuclear membrane newly synthesized by the cells after infection. INTRODUCTION
The mechanism controlling the migration of structural viral proteins from their site of synthesis in the cytoplasm to their site of assembly in the nucleus is at present un -i known. Although structural viral proteins are synthesized b y infected cells deprived of arginine, migration of these proteins to the nucleus and their assembly into nucleoeapsids do not take place under these conditions (Courtney et al., 1970 and 1971; Olshevsky and Beeker, 1970; ~/Iark and Kaplan, 1971), indicating t h a t arginine-rieh proteins m a y be directly or indirectly involved in the assembly process. Recent experiments b y Winters and RusThis investigation was supported by a grant sell (1971) suggest t h a t nuclear membranes from the National Institutes of Health (AI-03362). m a y play an important role in the process of s Present address : Department of Biochemistry, McGill University School of iVfedicine, Montreal, assembly of adenoviruses. Since, after infection of cells with pseudorabies (Pr) virus, Quebec, Canada. Present address : Department of Microbiology, there is an increase in the rate of synthesis of Vanderbilt University School of Medicine, Nash- nuclear m e m b r a n e (Ben-Porat and Kaplan, ville, Tennessee 37203. 1971 and 1972) and integration of virus102 Copyright© 1972by AcademicPress, Inc. All rights of reproductionin any formreserved. I t is well-established t h a t the structural proteins of the herpesviruses are synthesized in the cytoplasm and migrate thereafter to the nucleus where they are assembled into nueleoeapsids (Fujiwara and Kaplan, 1967; Olshevsky et at., 1967; Spear and Roizman, 1968; Ben-Porat et aI., 1969). The nueleoeapsids then acquire an envelope b y budding from the inner nuclear membrane of the infected cells (Siegert and Falke, 1966; Shipkey et al., 1967; Darlington and Moss, 1968; Nii et al., 1968), and the mature virus is released into the cytoplasm.
PROTEIN IN CELLS INFECTED WITH HERPESVII~US specific structural glycoproteins into this membrane (Ben-Porat and Kaplan, 1970), we considered the possibility that there may be a correlation between these characteristic alterations in the nuclear membrane and the migration of the viral capsid proteins to the nucleus. The experiments described in this paper were therefore performed to determine whether the alterations in the nuclear membrane normally induced by infection with Pr virus occur under conditions of arginine starvation. MATERIALS AND METHODS
Virus and cell culture The properties of Pr virus and the cultivation of rabbit kidney (RK) monolayer cultures were described previously (Kaplan, 1957 and 1969). Primary R K cells were grown in 90-mm Petri dishes in ELS. The cells were infected with approximately 20 plaque-forming units (PFU)/eell. Media and solutions ELS: Earle's saline containing 0.5% lactalbumin hydrolyzate and 5% bovine serum. EDS: Eagle's synthetic medium (Eagle, 1959), plus 3% dialyzed bovine serum. EDS ( - A r g ) : The same as EDS, except that arginine was omitted. TBSA: a buffer containing the same salts as PBS (Dulbeceo and Vogt, 1954), except that the phosphate was replaced by 0.01 M TrisHC1, pH 7.5, plus 1% crystalline serum albumin. RSB: 0.01 M Tris HC1, pH 7.4, 0.01 M KC1, and 0.0015 M MgCI~ (Warner et al., 1963). Triton-X-100 buffer: 0.14 M NaC1, 0.005 M MgC12, 0.01 M Tris-HC1, ptI 8.5, and 0.5 % Triton-X-100. Chemicals and radiochemicals. Triton X100 was a gift of Rohm and Haas Co. 6-~H-glueosamine (sp act, 3.6 Ci/mM), 114C-glueosamine (sp act, 57 mCi/mM), and 3H-methyleholine (sp act, 100 mCi/mM) were purchased from the New England Nuclear Coporation; 14C-L-leucine (sp act, 312 mCi/mM) was purchased from Schwartz/Matin. Cell fraetionation. This was done essentially according to Penman (1969). R K cells (4-8 X 106/ml) were scraped into RSB and
103
allowed to swell for 10 min at 4 °. The cells were homogenized (20 strokes) in ~ tightfitting Dounee homogenizer and the tyroplasmie fraction was separated from the nuclear fraction by centrifugation at 1,000g for 7 rain. To remove the outer nuclear membrane (Holtzmann et al., 1966), the nuclear pellet was resuspended in Triton X-100 buffer, rehomogenized (15 strokes), and centrifuged as above; the resulting supernatant fluid (outer nuclear membrane fraction) was collected. The pellet contained at least 80 % of the nuclei present in the initial cell suspension; the nuclei appeared microscopically free of cytoplasmic tags. Since choline and glucosamine become membrane-associated only (Kornfeld and Ginsburg, 1966; Molnar, 1967; Plagemann, 1968), we refer in this paper to the phospholipids and the glycoproteins extracted from the different fractions (after extensive acid washing) as being the phospholipids and the glycoproteins of the membranes present in each of these fractions. We have shown previously (Ben-Porat and Kaplan, 1971) that the so-called outer nuclear membrane fraction has a different phospholipid composition from the other cellular fractions. We do not know, however, to what degree the outer nuclear membrane fraction is contaminated with cytoplasmic or nuclear components. In some of the experiments we present the results obtained with this fraction; we do so only because we consider it desirable to present the data obtained with all fractions isolated from the cells. For electrophoretic analysis of the glyeoproteins, the cytoplasmic fraction and outer nuclear membrane fraction were combined and are called the cytoplasmic fraction. SDS-polyacrylamide gel electrophoresis. Electrophoresis, fractionation of the gels, and the assay of radioactivity were performed as described previously (Kaplan et al., 1970). Extraction of lipids. The fractions were washed with cold perehloric acid, 0.2 N, the lipids were extracted, and the radioactivity in the lipid fractions was determined as described previously (Ben-Porat and Kaplan, 1971).
104
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FIG. 1. Incorporation or ~H-eholine into the phospholipids of infected and uninfected RK cells i~ &e presence and absence of arginine. Primary RK monolayers were infected (or mock-infected) and incubated in EDS or EDS (-Arg). At 3 hr postinfection, the culture media were changed to EDS or EDS (-Arg) containing 3It-choline (1.2 ~Ci/ml) and incubation was continued. At various times thereafter the cells were scraped into the overlying media and acidified with perchloric acid (final concentration, 0.2 N). The acidified samples were washed four times with 0.2 N perchloric acid, the lipids were extracted, and the amount of radioactivity associated with the lipids was determined, as described in Materials and Methods. (A) arginine+; (B) arginine-. Closed circles, solid line, infected cells; open circles, broken line, mock-infected cells. RESULTS
Incorporation of 3H-choline into the phospholipids of infected and uninfected cells in the presence and absence of arginine During the course of infection of cells with P r virus, various changes in the metabolism of phospholipids can be detected. One of these changes is an increase, after infection of stationary phase cells, in the rate of incorporation of all-choline into the phospholipids, an increase t h a t is especially marked in the nuclear fraction (Ben-Porat and Kaplan, 1971). The experiments summarized in Fig. 1 and Table 1 were designed to determine whether these virus-induced alterations of phospholipid metabolism will also occur when the cells are deprived of arginine. Figure 1A shows t h a t under conditions of normal infection, i.e., in the presence of arginine, there was a slight inhibition of choline incorporation early in infection. B y 5 hr postinfeetion an increase in the incor-
poration became evident. By 9 hr postinfection, i.e., near the end of the infective process, the rate of incorporation of choline again decreased. Although the early inhibition of choline incorporation, as well as its subsequent activation, are relatively small, these changes were observed consistently when stationary phase cells were infected. Infection of arginine-deprived cultures (Fig. 1B) also resulted in a slight initial decrease in the incorporation of choline; however, in contrast to cells infected under normal conditions, at no time did infected arginine-deprived ceils show an increased rate of choline incorporation. Table 1 shows the distribution of choline between the nuclear and cytoplasmic fractions of infected and uninfected cells in an experiment similar to the one illustrated in Fig. 1. Infection in the presence of arginine gave
PROTEIN IN CELLS INFECTED WITH HERPESVIRUS
105
TABLE 1 INCORPORATION OF 3I~I-CHOLINE INTO TIIE PItOSPIIOLIPIDS OF INFECTED AND UNINFECTED R K CELLS IN THE PRESENCE AND ABSENCE OF ARGININE a
Arginine
Fraction
Infected
Present
Cytoplasmic Outer nuclear Inner nuclear Cytoplasmic Outer nuclear Inner nuclear
26.51 b 2.07 3.70 23.26 1.76 2.83
Absent
Uninfected 24.16 2.23 2.96 24.80 1.81 2.93
Infected Uninfected 1.10 0.93 1.25 0.94 0.97 0.98
Primary RK monolayer cultures, 8 days old, were infected (or mock-infected) and incubated in EDS or EDS (-Art), containing aIl-eholine (3 ~Ci/ml) between 3 and 8 hr postinfeetion. The cells were fraetionated, acidified, washed with acid, the lipids extracted, and the amount of radioactivity determined, as described in Materials and Methods. b cpm X 10-~/eulture. results similar to those reported previously (Ben-Porat and Kaplan, 1971). Increases of approximately 10 % in the amount of choline incorporated into the cytoplasmic lipids and 25 % into the lipids of the inner and outer nuclear membranes were obtained. I n the absence of arginine, however, no virusinduced increase of choline incorporation into the phospholipids of the cytoplasm or nuclear membranes was observed.
Flow of phospholipids from the cytoplasm to the nuclear membrane in arginine +- and arginine--infected cells After infection of R K cells with P r virus, in addition to the increase in incorporation of externally supplied choline into the lipids of the nuclear membrane, there is a flow to the nuclear m e m b r a n e of phospholipids preexisting in the cells at the time of infection which most likely represents the synthesis of new nuclear m e m b r a n e (Ben-Porat and Kaplan, 1972). The experiment summarized in Table 2 was performed to determine whether this flow occurs in infected argininedeprived cells. I n this particular experiment, a virus-induced increase of approximately 45 % in the a m o u n t of the phospholipids associated with the inner nuclear m e m b r a n e was obtained. I t has been shown previously (Ben-Porat and Kaplan, 1972) t h a t this increase is not the result of an increased contamination in the infected cells of the nuclear fraction with cytoplasmic membranes, b u t t h a t it reflects the synthesis
TABLE 2 DISTRIBUTION~ AFTER A CHASE, OF THE RADIOACTIVITY DERIVED FROiYi 3~I-CHOLINE AMONG THE CYTOPLASMIC AND NUCLEAR MEMBRANES IN THE PRESENCE AND ABSENCE OF ARGININE a
Arginine
Present Absent
Treatment
Infected Uninfected Infected Uninfected
Membrane Cytoplasmic Outer Inner nuclear nuclear 19,550 18,250 19 450
I 3420 / 3670 IJ 3150
6210 4320 4790 4420
Primary RK monolayer cultures, 3 days old, were incubated for 3 days in EDS eontaining-~Hcholine (0.2 uCi/ml). The cultures were washed several times with Earle's saline and incubated for 4 days in EDS not containing labeled choline. At the end of this incubation period, the specific activities of the phospholipids (cpm/mg phosphorus) in the different cellular fractions were approximately the same. The monolayers were infected (or mock-infected) and incubated in either EDS or EDS(-Arg.) At 7 hr postinfeetion the cells were harvested, fraetionated, and the radioactivity in the phospholipids associated with the various fractions was determined, as described in Materials and Methods. cpm/eulture. of new nuclear membrane. Since the specific activities of the phospholipids in the different cellular fractions were the same at the time of infection (see legend Table 2), these
106
MARK AND KAPLAN
results indicate that by 7 hr postinfection the amount of phospholipids of the nuclear membrane in the infected cells had increased by about 45 %. There was also in arginine-deprived cells a slight increase in the amount of phospholipid associated with the inner nuclear membrane, but it was relatively small compared to that found in infected cells supplied with arginine. Thus, the increase in the incorporation of externally supplied choline into the nuclear membrane and the flow of preexisting cellular phospholipids to this membrane normally observed in infected cells are greatly reduced under conditions of arginine starvation. We conclude therefore that little new nuclear membrane is formed in infected, arginine-deprived cells.
Gel electrophoretic analysis of the glycoproteins synthesized in infected, arginine-deprived cells The envelope of Pr virus is composed of virus-specific glycoproteins which are identical to those associated with the nuclear membrane of infected cells; cell-specific proteins do not become part of the virions (Ben-Porat et al., 1970; Ben-Porat and Kaplan, 1970). The mechanism which ensures the presence of only virus-specific proteins in the viral envelope is not yet understood completely. However, since after infection there is a considerable increase in the amount of phospholipids in the nuclear membrane which represents de novo synthesis of this membrane (Ben-Porat and Kaplan, 1971 and 1972; see also Fig. 1, Tables 1 and 2 of this paper), and since the infected cells synthesize exclusively virus-specific proteins, only viral proteins presumably would be integrated into the newly assembled nuclear membrane. It is not clear, however, whether viral glycoproteins become associated with newly synthesized nuclear membrane only, or whether they also become integrated into areas of the nuclear membrane preexisting in the cells at the time of infection. In Pr virus-infected arginine-deprived cells, most of the structural viral proteins are synthesized (Mark and Kaplan, 1971), but, as shown above, little new nuclear
TABLE 3 EFFECT OF ARGININE STARVATION ON THE SYNTHESIS OF PROTEINS AND GLYCOFROTEINS IN VIRUS-INFECTED CELI~S a
Inhibition Arginine+ Arginine- (percent) Leucine 68b 25 63 Glucosamine 28 11 60 Primary monolayers of RK cells were infected and incubated in EDS or EDS (-Arg). At 4 hr postinfeetion, the medium was replaced either with EDS containing one-half the usual amount of leucine or with EDS (-Arg) also containing this amount of leueine; both contained 6-3H-glueosamine (10 ~Ci/ml) and uC-leueine (2 ttCi/ml). At 8 hr postinfection, the cells were scraped into the overlying medium and the amount of radioactivity incorporated into aeid-precipitable material was determined as described in Materials and Methods. b epm X 10-3/culture. membrane is formed. It was interesting, therefore, to determine whether (1) structural viral glycoproteins are synthesized in arginine-deprived cells, and, if so, (2) whether these glycoproteins become part of the nuclear membrane, despite the lack of synthesis of new nuclear membrane. Table 3 shows that starving the infected cells of arginine reduced the synthesis of glyeoproteins to about the same degree as the synthesis of all other proteins. Thus, glycoproteins are synthesized in the infected cell in the absence of arginine. During the course of normal infection, R K cells synthesize structural and nonstructural viral glycoproteins. Most of the glycoproteins synthesized by the infected cells do not become associated with mature virions. These nonstructural glycoproteins are associated with the cytoplasmic fraction only and migrate as one peak, designated peak 3a. Four structural glycoproteins are also synthesized by the infected cells; most of these become associated with the nuclear membrane and become part of the viral envelope. When all the glycoproteins synthesized by the infected cells are analyzed by gel eleetrophoresis, the nonstruetural glycoproteins migrating as peak 3a obscure the structural proteins migrating as peaks
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arginine. RK cells were infected and incubated in EDS or EDS (-Arg). At 5 hr postinfection 3H-choline (10 t,Ci/ml) was added to the culture media, and the cultures were incubated further for 2 hr. The cells were then harvested and the proteins electrophoresed as described ill Materials and Methods. (A) arginine+; (B) arginine-. 3 and 4. The latter glycoproteins are clearly resolved only when purified nuclear or viral preparations are eleetrophoresed (Ben-Porat and Kaplan, 1970). Figure 2 shows the eleetrophoretic pattern of migration in aerylamide gels of the glycoproteins synthesized by infected cells incubated in the presence and absence of arginine. The electrophoretic profiles of the glyeoproteins synthesized by infected, arginine+ cells and by arginine- cells were similar, although some differences, which will be discussed below, were found between the two. Figure 3 illustrates an experiment in which the electrophoretic patterns of the glycoproteins associated with the nuclear and cytoplasmic fractions in arginine+ and arginine- cells were analyzed. Figure 4 shows the results obtained when the glycoproteins of similarly treated uninfected cells were analyzed. Since most glycoproteins (approximately 90 %) are associated with the cytoplasmic fraction, the electrophoretie pattern of the glycoproteins from this fraction of the cells (Fig. 3A) is similar to that of the glycoproteins from unfractionated cells (Fig. 2). A relatively larger proportion of the glycoproteins synthesized by arginine- cells than by arginine+ cells remained at the origin, and a peak (1) which was barely detectable in the electrophoregrams of the glyeoproteins from arginine+-infeeted ceils appeared
in those from arginine- cells. The large amount of glycoproteins remaining at the origin and the peak 1 glyeoproteins also appeared in the profiles of the glycoproteins from uninfected cells (see Fig. 4). Otherwise, the cytoplasmic glycoproteins synthesized by arginine--infected cells migrated coincidentally with those synthesized by arginine +infected cells, indicating that the same glycoproteins were synthesized by both. However, a larger proportion of the total glyeoproteins from infected arginine--ce]Is than from arginine + cells migrated as peaks 4a and 6. Furthermore, although the glyeoproteins migrating as peaks 3 and 4 are, in major part, obscured by the glyeoproteins migrating as peak 3a, it appears from the shoulders in peak 3a that the cytoplasmic fraction from arginine- cells is also richer than that from arginine+ cells in glycoproteins in peaks 3 and 4. The larger amounts of peaks 3, 4, 4a and 6 relative to peak 3a glyeoproteins in the cytoplasmic fraction from arginine- cells may be due to the lack of integration of the structural proteins (3, 4, 4a and 6) into the nuclear membrane of these cells (see below), leading to their accumulation in the cytoplasm. This is probably a factor, but Fig. 2 indicates that relative to the glycoproteins migrating as peak 3a, the glycoproteins in peak 4a and 6 seem to be synthesized in larger amounts by arginine- than by arginine + ceils. We have
108
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FIG. 3. Electropherograms of the glycoproteins associated with the cytoplasmic and inner nuclear membranes of infected cells incubated in the presence or absence of arginine. I~K cells were infected and incubated in EDS or E D S (-Arg). At 5 hr post infection ttC-glucosamine (4 ~Ci/ml) was added to the arginine + cultures and ~H-glucosamine (20 ttCi/ml) to the arginine- cultures. The cultures were further incubated for 2 hr, and the t4C-labeled arginine + cells and ~H-labeled arginine- cells were harvested and mixed. The cytoplasmic and nuclear fractions were isolated and the proteins electrophoresed, as described in Materials and Methods. (A) Cytoplasmic fraction; (B) Nuclear fraction. Closed circles, solid lines, arginine + cells ; open circles, broken lines, arginine- cells. I
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FIG. 4. Electropherogram of the glycoproteins associated with the cytoplasmic and inner nuclear membranes of uninfected cells. Uninfected I~K cells were incubated in EDS with 3tt-glucosamine (8 t~Ci/ml) for 2 hr. The cells were then harvested, the cytoplasmic fraction separated from the nuclear fraction, and the proteins electrophoresed as described in Materials and Methods. (A) Cytoplasmic fraction; (B) Nuclear fraction.
no explanation at present for this phenomenon. The electrophoretic patterns of the glyeoproteins associated with the nuclear fractions from arginine+ and arginine- cells were different. The characteristic pattern of migration of nuclear glycoproteins synthesized by infected cells (which is also that
of mature virions) was not observed in the arginine-deprived ceils. Instead, the pattern obtained was similar to that of the glyeoproteins from the nuclei of uninfected cells (Fig. 4). We conclude from these results that (1) the major structural viral glycoproteins are synthesized in arginine--infeeted cells and
PROTEIN IN CELLS INFECTED WITH HERPESVII:~US (2) that these proteins remain associated with the cytoplasmic fraction and are not incorporated into the nuclear membrane as in arginine-+infected cells. It seems, therefore, that structural viral glycoproteins cannot be integrated into the nuclear membrane preexisting in the cells at the time of infection but become associated only with new regions of the nuclear membrane, the synthesis of which is normally stimulated by infection. DISCUSSION The results in this paper show that the de novo assembly of nuclear membrane that normally occurs in Pr virus-infected R K cells is greatly reduced when the cells are deprived of arginine. Thus, in the absence of arginine, infection with Pr virus does not result in the usual increase in choline incorporation into the phospholipids of stationary R K cells. The relatively large increase in the incorporation of phospholipids into the nuclear membrane of infected cells is also not observed (Table 1, Fig. 1). In addition, the virus-induced flow from cytoplasmic to nuclear membranes of phospholipids preexisting in the cells at the time of infection is greatly reduced in the absence of arginine (Table 2). Since the increased rate of incorporation of externally supplied choline, as well as the virus-induced flow of pre-formed phospholipids to the nuclear membrane, probably represents de novo synthesis (reduplication) of that membrane, we conclude that in the absence of arginine, little new nuclear membrane is formed. This conclusion has been reinforced by electron microscopic observations which show that the convolutions of the nuclear membranes normally seen in cells infected with the herpesviruses does not occur in argininedeprived infected cells (unpublished results). The electrophoregram of the glyeoproteins synthesized by arginine-deprived, infected cells is similar to that of the glycoproteins synthesized under conditions of normal infection; on the basis of their electrophoretie profiles in acrylamide gels we concude that both the nonstructural and the structural viral glyeoproteins are synthesized by the arginine-deprived cells. However, the structural viral glyeoproteins do not become part
109
of the nuclear membrane in arginine-infected cells; instead, these proteins remain associated with the cytoplasmic fraction. We do not know at present the nature of the glycoproteins synthesized by arginine-infected cells that become associated with the nuclear membranes. These glycoproteins may be of viral origin and may have been abnormally glyeosylated or abnormally cleaved as a consequence of argin]ne deprivation; on the other hand, they may be cellspecific. Since the pattern of eleetrophoretic migration of these glyeoproteins is similar to that of the nuclear glyeoproteins from uninfected cells, the second alternative is more likely. This would imply that whereas most host protein synthesis is inhibited in arginine-infeeted cells (Mark and Kaplan, 1971), some species of cell-specific proteins are still synthesized by such cells. From previous data (Ben-Porat and Kaplan, 1971 and 1972), we think that the mechanism which ensures that the viral envelope is composed of virus-specific proteins only is as follows: new regions of the nuclear membrane are synthesized after infection into which viral proteins (the only proteins synthesized by the infected cells) are integrated; the viral particles bud only from these newly synthesized areas of the nuclear membrane. The experiments described in this paper show that when new nuclear membrane is not synthesized, the viral glycoproteins do not become part of the nuclear membrane. Thus, the nuclear membrane preexisting in the cell at the time of infection cannot accept viral giycoproreins. We conclude, therefore, that during the course of infection, the viral giycoproreins do not become associated with the nuclear membrane in a random manner but become associated only with the newly synthesized areas of this membrane. We have shown previously that viral DNA and proteins are synthesized in arginine-deprived, infected cells. However, the structural viral proteins which normally migrate from the cytoplasm (the site of their synthesis) to the nucleus (the site of assembly of the nucleoeapsids) remain associated with the cytoplasm under conditions of arginine starvation (Mark and Kaplan,
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MARK AND KAPLAN
1971; C o u r t n e y et al., 1971). Thus, the conditions favoring the migration of the structural viral proteins to the nucleus do not exist in arginine-deprived cells. W e consider t h a t the assembly of the structural viral proteins into capsids within the nucleus, the latter thus serving as a diffusion sink, is likely to be responsible for the continued migration of the proteins to the nucleus. I t has been shown recently t h a t nuclear m e m b r a n e structures are i m p o r t a n t for the assembly of adenovirus (Winters and Russell, 1970). If this were also the case for herpesvirus-infected cells, the lack of assembly of viral particles in arginine-deprived cells and the consequent failure of migration of viral proteins to the nucleus m a y be related to the lack of characteristic virusinduced alterations of the nuclear m e m b r a n e in these cells. REFERENCES BEN-PonA% T. SHI~rONO, H., and KAPL~N, A. S. (1969). Synthesis of proteins in cells infected with herpesvirus. II. Flow of structural viral proteins froln cytoplasm to nucleus. Virology 37, 56-61. BEN-PoI~AT, T. SHIMONO,H., and KAPLAN, A. S. (1970). Synthesis of proteins in cells infected with herpesvirus. IV. Analysis of the proteins in viral particles isolated from the cytoplasm and the nucleus. Virology 41,256-264. BEN-PoI~AT,T., and KAPLAN,A. S. (1970). Synthesis of proteins in cells infected with herpesvirus. V. Viral glycoproteins. Viroiogy 41,265-273. BEN-PoRAT, T., and KAPLAN, A. S. (1971). Phospholipid metabolism of herpesvirus-infected and uninfected rabbit kidney cells. Virology 45,252264. BEN-PoI~AT,T., and KA~'LAN,A. S. (1972). Studies on the biogenesis of herpesvirus envelope. Nalure (London) 235, 165-166, COURTNEY,R. J., MeCoMBs, R. iV[., and BENYESItMELNICK, M. (1970). Antigens specified by herpesviruses, I. Effect of arginine deprivation on antigen synthesis. Virology 4[!, 379-386. COUI~TNE¥,R. J., MeCoMBS, R. M., and BENYESHMELNICK, M. (1971). Antigens specified by herpesviruses. II. Effect of arginine deprivation on the synthesis of cytoplasmic and nuclear proteins. Virology 43,356-365. DARLINGTON, R. W., and Moss, L. H. (1968). Herpesvirus envelopment. J. Virol. 2, 48-55. DULBECCO,R., and VOGT,M. (1954). Plaque formation and isolation of pure lines of poliomyelitis virus. J. Exp. Med. 99,183 199.
EAGLE,H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. FVJIWAR.a, S., and KAPSAN, A. S. (1967). Site of protein synthesis in cells infected with pseudorabies virus. Virology 32, 60-68. HOLTZMAN,E., SMITH, I., and PENMAN, S. (1966). Electron microscopic studies of detergent treated nuclei. J. Mol. Biol. ]7,131-135. KAPLAN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell system by the plaque technique. Virology 4, 435-457. KAPL~N, A. S. (1969). Herpes simplex and pseudorabies viruses. Virology Monographs, Vol. 5 (Gard, S., Hallauer, C., and Meyer, K. F., eds.) Springer, New York. KAPLAN,A. S., and BEN-PORAT,T. (1970). Synthesis of proteins in cells infected with herpesvirus. VI. Characterization of the proteins of the viral membrane. Proc. Nat. Acad. Sci. U.S.A. 66, 799-806. KAPLAN, A. S., SHIMONO,~-~., and BEN-PORAT,T. (1970). Synthesis of proteins in cells infected with herpesvirus. III. Relative amino acid content of various proteins formed after infection. Virology 40, 90-101. KORNFELD, S., and GINSBURG, V. (1966). The metabolism of glucosamine by tissue culture cells. Exp. Cell Res. 41,592-600. ~AR~:, G. E., and KAPLAN,A. S. (1971). Synthesis of proteins in cells infected with herpesvirus. VII. Lack of migration of structural viral proteins to the nucleus of arginine-deprived cells. Virology 45, 53-60. MOLNAR, J. (1967). Glycoproteins of Ehrlich aseites carcinoma cells. Incorporation of (14C) glucosamine and (14C) sialic acid into membrane proteins. Biochemistry 5, 3064-3076. NII, S., MORGAN, C., and ROSE, H. M. (1968). Electron microscopy of herpes simplex virus. II. Sequence of development. J. Virol. 2, 517-536. OLSHF~VSKY,U., and BEEPER, Y. (1970). Synthesis of herpes simplex virus structural proteins in arginine-deprived cells. Nature (London) 226, 851-853. OLSHEVSKY, U., LEVITT, J., and BECKER, Y. (1967). Studies on the synthesis of herpes simplex virions. Virology 33,323-334. PENMAN, S. (1969). Preparation of purified nuclei and nucleoli from mammalian cells. In "Fundamental Techniques in Virology" (K. Habel and N. P. S~lzman, eds.), pp. 35-48. Academic Press, New York. PLAGEMANN,P. G. W. (1968). Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. I. Incorporation of choline into phosphatidylcholine of mitochondria and other membranous structures and
PROTEIN IN CELLS INFECTED WITH HERPESVIRUS effect of metabolic inhibitors. Arch. Biochem. Biophys. 128, 70-87. SHIPKEY, F. H., ERLANDSON,1~. t . , BAIUEY,R. B., B~BCOCK, V. I., and SOUT~AM, C. M., (1967). Virus biographies. II. Growth of herpes simplex virus in tissue culture. Exp. Mol. Pathol. 6, 3967. SIEGERT, R., and FALKE, D. (1966), Elektronenmikroskopische Untersuehungen fiber die Entwicklung des Herpesvirus hominis in Kulturzellen. Arch. Gesamte. Virusforsch. 19, 230-249.
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SP~AR, P. G., and ROIZMAN,B. (1988). 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. WANNER, J. R., KNOPF, P. M., and RICH, A. (1963). A multiple ribosomal structure in protein synthesis. Proc. Nat. Acad. Sci. U.S.A. 49,122-129. WINSE~S, W., and RUSSELL,W. C. (1971). Studies on the assembly of adenovirus in vitro. J. Gen. Virol. 10, 181-194.