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The polypeptides of influenza virus
VIROLOCIY
39,
419-425
(1969)
The Polypeptides I. Cytoplasmic J. M. TAYLOR, School
of Microbiology,
of Influenza
Virus
Synthesis and Nuclear
Accumulation
A. W. HAMPSON,
D. 0. WHITE
University of Melbourne, Accepted
June
AND
Pa&de,
Victoria,
Australia
23, 1969
Three major structural peptides were found in radioactive influenza virus type A/Be1 that had been grown in calf kidney cells, purified, and analyzed by acrylamide gel electrophoresis. The same three peptides were found in infected HeLa cells. All proved to be stable; none was a precursor of another. The site of synthesis and subsequent migration of these peptides was investigated by cell fractionation. Evidence is presented which suggests that viral peptides were synthesized in the cytoplasm, and that two of them subsequently underwent significant accumulation into the nucleus. INTRODUCTION
The intracellular localization of influenza antigens has been studied in the past by the technique of immunofluorescence (Schafer, 1959; Hillis et al., 1960; Fraser, 1967; Rott and Scholtissek, 1967). The S-antigen (ribonucleoprotein) has been generally observed to accumulate first in the nucleus, and only later in the cytoplasm. In contrast, the V-antigen (widely assumed to correspond with the hemagglutinin) is detected exclusively in the cytoplasm. These observations have been interpreted by Rott and Scholtissek (1967) as evidence that both the Sand V-peptides may be synthesized in the nucleus, but that the V-peptides do not assumean antigenic configuration until after they have migrated to the cytoplasm. Furthermore, Becht (1969) has presented autoradiographic data suggesting that nuclear protein synthesis may occur in influenzainvected cells. In this communication an attempt is made to determine the site of synthesis of individual influenza peptides by combining the following approaches: (1) brief radioactive pulse-labeling of virus-infected cells, followed by a prolonged “chase”; (2) fractionation of cells into nucleus and cytoplasm; 419
(3) separation of viral peptides by acrylamide gel electrophoresis. The results demonstrate that all three of the major structural peptides of the influenza virion are synthesized in the cytoplasm, and that two of these subsequently migrate to the nucleus. MATERIALS
AND METHODS
Growth and puriJication of 14C-labeled influenza virus. Confluent monolayer cultures of primary embryonic calf kidney cells (Lehmann-Grube, 1965) were rinsed twice with Hanks’ balanced salt solution and replenished with Minimal Essential Medium (MEM) (Eagle, 1959), modified to contain one-tenth the usual amino acid concentrations and supplemented with 0.1% bovine serum albumin. Influenza virus A/Be1 (Burnet and Bull, 1943), grown in the ahantois of developing chicken embryos, was inoculated into the cultures at a multiplicity of infection (m.0.i.) of 0.05 tissue culture IDbOper cell together with 14C-labeled amino acid mixture (Amersham CFB. 104 at 52 mCi/mAtom carbon) to a final concentration of 0.25 &X/ml. The cultures were incubated at 35” for 72 hours, by which time they showed marked cytopathic effects.
420
TAYLOR,
HAMPSON,
Virus was then purified by the following modification of the procedure developed for Newcastle disease virus (Haslam et al., 1969). The supernatant tissue culture fluid was clarified by centrifugation (12,000 g, 10 min), the virus was deposited (64,000 g, 30 min) onto a small cushion of 2.3 M sucrose, resuspended by brief sonic vibration, and purified by rate zonal centrifugation in a l&60% (w/v) sucrose gradient (64,000 g, 120 min), followed by equilibrium centrifugation in a 2040% (w/v) potassium tartrate gradient (174,000 g, 180 min). Fractions were collected dropwise from the bottom of the tube and assayed for hemagglutinin and 14C. Peaks of the two activities coincided and corresponded to the visible band in both gradients. Infection and pulse-labeling of HeLa cells. Suspension cultures of HeLa 53 cells were propagated in MEM supplemented with 5 % fetal calf and 5% bovine serum. For virus infection, cells prelabeled for 48 hours with thymidine-3H (at 0.07 &i/ml and 35.7 mCi/mmole) were washed once and then resuspendedat a concentration of 10’ cells/ ml in serum-free MEM modified to contain one-tenth the usual amino acid concentrations. Influenza A/Be1 propagated in embryonated hen’s eggs and concentrated by centrifugation, was added at an input multiplicity of 300 egg ID60 per cell, and allowed to adsorb for 30 min at room temperature. The cell suspensionwas then diluted lo-fold with prewarmed MEM, lacking amino acids and supplemented with 5% dialyzed fetal calf serum. Cultures were incubated at 37’ with stirring for a further 3.5 hours. The
AND
cells were then gently centrifuged (250 g, 5 min) and resuspended in amino acid-free medium at a concentration of 4 X lo7 cells/ml. After a lo-min recovery period, 14C-labeledamino acid mixture was added to a final concentration of 10 &i/ml. After labeling for 3 min, a chase in the presenceof unlabeled amino acids was achieved by diluting the cells 40-fold with complete growth medium. At 1 and 60 min, respectively, aliquots of 2 X lo7 cells were diluted into ice-cold phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). These chase conditions inhibit further incorporation of 14C-amino acids into acid-insoluble material (Table 1). Cell fractionation. Cells were washed twice in ice-cold PBS, then swollen for 15 min in 1 ml of hypotonic buffer (0.01 M KCl, 0.002 M MgC12, and 0.01 M Tris at pH 7.4), and disrupted in a Dounce homogenizer (Penman et al., 1963). After centrifugation (1000 g, 5 min), the pellet comprised predominantly nuclei, but also small numbers of unbroken cells, as well as cell debris. Since it was imperative to obtain an uncontaminated preparation of clean nuclei, even at the expense of some loss of nuclei, the specimen was subjected to further purification as follows. The pellet was resuspended in 0.9 ml of cold TKM buffer (0.025 M KU, 0.005 M MgClz, and 0.05 M Tris at pH 7.4) containing 1.5 % by volume of the nonionic detergent Nonidet P40 (Shell Chemicals) (O’Brien, 1964), in order to rupture cytoplasmic membranes and remove cytoplasmic material from around unclean nuclei (Borun et al., 1967). The suspensionwas then mixed
TABLE DEMONSTRATION
OF THE
1
VALIDITY
OF THE
Duration
‘%-amino
0 Virus-infected cells were unlabeled (cold) amino acids, were chilled and precipitated counts given in the table are represent half the maximum
CHASE
of cold amino
PROCEDUREQ
acid chase (min)
1
1.5
30
45
60
185
170 (10)
165 (15)
180
160 (10)
-. TCA-precipitable acid counts
WHITE
(16)
(6)
labeled for 3 min with ‘%-amino acids and then chased by the addition of as described above. At the indicated times, aliquots of cell suspension with an equal volume of 20% trichloroacetic acid. The acid-precipitable the mean of three determinations, and the figures shown in parentheses range of those assays.
SYNTHESIS
AND MIGRATION
with 2.1 ml of 2.3 M sucrosein TKM buffer, underlaid with 1 ml of 2.3 1M sucrose in TKM and centrifuged at 120,000 g for 30 min. The resulting nuclear pellet and the original intact cells were assayed for radioactivity (14Cand 3H) in trichloroacetic acidprecipitable material using an Ansitron scintillation counter. The recovery of nuclei was equated with the recovery of thymidine-3H (usually 20-40 %) . Acrylamide gel electrophoresis.Electrophoresis on 5 % acrylamide gels was performed as described by Maize1 (1966) with the following modifications. Samples were dissociated with 2 % sodium dodecyl sulfate, 0.5 M urea, 0.04 M 2-mercaptoethanol, and 0.1 M dithiodiethanol in 0.01 M phosphate buffer pH 7.2 at 37” for 30 min, then at 100” for 1 min, and finally, made 10% in glacial acetic acid. Mercaptoethanol was omitted from the dialysis buffer.
OF INFLUENZA
PEPTIDES “PI
VP2
421 VP3
400
0
200
CPM 0
RESULTS
The Structural Peptides of InJEuenza Virus Influenza virus, labeled with 14C-amino acids, was grown in calf kidney cells, purified, and analyzed by gel electrophoresis (Fig. la). The virus possesses three major structural peptides, referred to here as VPl, VP2, and VP3. The identification and characterization 40 60 20 of these and minor components of the virion, FRACTION NUMBER such asthe shoulder sometimesdetectable on FIG. 1. Acrylamide gel electrophoresis of Wthe anodal side of VPl, is currently under amino acid-labeled peptides from (a) purified instudy. Viral Peptide Synthesis in Infected Cells Suspensioncultures of HeLa cells infected 4 hours earlier with influenza virus were labeled for 15 min with 14C-amino acids then analyzed by gel electrophoresis (Fig. lb). Fig. lc shows the electropherogram of mock-infected cells. It is apparent that the infected cell produces predominantly the three main structural peptides of the virion.
fluenza virus grown in calf kidney cells, (b) HeLa cells infected for 4 hours with influenza virus then labeled for 15 min, (c) mock-infected HeLa cells. Migration is toward the anode on the right.
acids for 3 min, chasedwith unlabeled amino acids for 1 or 60 min, then separated into nuclear and cytoplasmic fractions and analyzed by gel electrophoresis. A chase of at least 1 min was necessary to ensure the completion of most of the peptides still attached to polyribosomes at the termination Nuclear-Cytoplasmic Fractionation of Infected of the pulse (Shapiro et al., 1966) In Fig. Cells 2a, the electropherogram of the nuclei is In order to determine the site of synthesis compared with that of the whole cells after of these viral peptides, cells infected 3.8 the 1-min chase.The three viral peptides can hours earlier were labeled with r4C-amino be clearly seenin the whole cell; at the same
5oc
1
I tb)
25C ,-
CPM
. 0
0 FRACTION
FIG. 2. Acrylamide gel electrophoresis infection, cells were labeled for 3 min amino acids for (a) 1 min, or (b) 60 represent whole cells and the broken equivalent number of whole cells or
60
40
of peptides in influenza virus-infected cells. At 3.8 hours after with ‘%-amino acids and then chased in the presence of unlabeled min. Migration is toward the anode on the right. The solid lines lines, the nuclei. The data have been normalized to represent an nuclei as described in Table 2. TABLE
DETERMINATION
20
NUMBER
OF THE
2
NORMALIZATION
FACTOR
USED
FIG.
IN
2Q
Duration of cold amino acid chase (mm)
l*C-amino Whole Nuclei Hence
Activity Whole Nuclei
acid/sH-thymidine cells
ratio
per
aliquot
1
60
0.995
0.984 0.279
of: 6.185
fraction
of labeled
(1% cpm) cells
Hence normalization nuclear material
layered
protein
in nucleus
in electrophoresis
of cell
=
0.185 0.995
scale
0.984
= 0.186
= 0.283
3996
3250 3654
of: 1944
factor for ordinate in Fig. 2 =
0.279
of electropherograms
of 3996
1944-O.
186
= 0.382
3250 -.0.283 3654 = 0.252
D This normalization was applied because of variation in the recovery of nuclei in the extraction procedure and also because of the different amounts of radioactive sample used in each electrophoresis. Note, however, that the use of the above raC/aH ratios, obtained with an Ansitron scintillation counter, eliminates the need to derive nuclear recoveries in the deduction of the normalization factors. Actually, in the above experiment the nuclear recoveries, as obtained using 3H-thymidine data, were 40.5 and 20.4%, respectively. 422
SYNTHESIS
AND
MIGRATION
OF
INFLUENZA
PEPTIDES
423
Furthermore, the high multiplicities of infection necessary for meaningful biochemical studies would result in the production of incomplete virus even in a permissive cell Duration line (von Magnus, 1954). Nevertheless, it is of cold VP3 amino acid VP1 VP2 apparent from the experiments reported in chase this paper that HeLa cells infected at high (min) m.o.i. synthesize three major peptides which comigrate in acrylamide gel electrophoresis 36.6 1 2.2 9.0 47.0 64.1 with the three major structural peptides of 60 2.7 purified virus grown in another cell system, a Expressed as a percentage of the amount of namely calf kidney cells. the corresponding peptide in the whole cells. The pulse-chaseexperiment, the results of Data are derived from Fig. 2. which are depicted in Fig. 2 and expressed quantitatively in Table 3, demonstrates time, the nuclear fraction contains only a three main facts. (1) After a 3min label and minor portion of them. a 1-min chase, only a minor portion of each Figure 2b shows the results obtained when of the three newly synthesized viral peptides the period of chase in unlabeled amino acids is in the nuclear fraction. (2) During the was increased from 1 min to 60 min. The 60-min period of chase, two of the viral data indicate clearly that all three viral peptides, VP2 and VP3, then show signifipeptides are stable; none is a short-lived cant accumulation into the nuclear fraction. precursor of the type encountered in polio(3) The remaining peptide VPl, does not virus-infected cells (Maize1 and Summers, accumulate in the nuclear fraction. Similar 1968). The most striking result, however, is results have been obtained in several comthat VP2 and VP3 have been chased into parable experiments. the nuclear fraction. In contrast, VP1 has The obvious and most interesting interstill not left the cytoplasm. pretation of these data is that the three The data contained in Fig. 2 is expressed major structural peptides of the virus are quantitatively in Table 3, which gives the synthesized in the cytoplasm, and that percentage of each viral peptide from the eventually two of them, VP2 and VP3, whole cell that is present in the nuclear accumulate in significant amounts in the fraction at the two chase times. nucleus, but the third, VPl, doesnot. Before discussing this interpretation further, it is DISCUSSION essential to consider possible alternative Acrylamide gel electrophoresis of purified explanations. influenza virus type A/Be1 has demonstrated As yet there is no cell fractionation prothree major structural peptides. Duesberg cedure that can guarantee absolute purity as and Robinson (1967) previously reported well as integrity of mammalian cell nuclei. two or three peptides in purified influenza Attempts to “clean” isolated nuclei of adtype A/PR8, analyzed by a somewhat difherent cytoplasmic contamination may lead ferent electrophoretic technique. to loss of nuclear material in the process. The primary purpose of this communicaHence, the possibility cannot be entirely distion has been to demonstrate certain aspects counted that influenza peptides may be synthesized in the nucleus then leak out of of the viral infection cycle in a line of HeLa cells conveniently handled in suspension culthe nucleus during cell fractionation. Following a 60 min chase, VP2 and VP3 may have ture. It should be noted that this experibecome incorporated into a structure too mental system is one in which the virus undergoes an abortive cycle of multiplicalarge to leak out in this way, e.g. nucleocapsid. Conversely, the presence of VP2 and tion, not leading to any significant production of infectious virus (Henle et al., 1955). VP3 in the nuclear fraction is unlikely to be TABLE
PERCENTAGE
3
OF EACH VIRAL PEPTIDE THE NUCLEUSQ
FOUND
IN
424
TAYLOR,
HAMPSON,
due to contamination with cytoplasmic material, as VP1 is not found in the nuclear fraction at all, while the content of VP2 and VP3 increases markedly with the time of chase. Nevertheless, the possibility cannot be entirely discounted that VP2 and VP3 become associated in a time-dependent fashion with some structure which cosediments with nuclei. Perhaps these viral peptides enter the nucleus in the course of simple diffusion around the cell. The smaller the molecule the more rapidly it may be expected to enter the nucleus. Work is proceeding to complete the identification of these three viral peptides. Preliminary data suggest that VP1 is the peptide of the viral hemagglutinin, and VP2 that of the ribonucleoprotein. The older fluorescent antibody work can now be reinterpreted in the light of the results contained in this paper. The presence of S-antigen in the nucleus and V-antigen in the cytoplasm should not be taken to mean that their constituent peptides are made in those sites. Both the ribonucleoprotein and the hemagglutinin peptides are synthesized in the cytoplasm, then one of them migrates to the nucleus. The identity of the peptide, VP3, perhaps corresponding to the viral neuraminidase, and the reason for its migration to the nucleus now become matters of particular interest. ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and the Australian Research Grant.s Committee. A. W. Hampson is on leave from, and supported by, the Commonwealth Serum Laboratories. REFERENCES BECHT, H. (1969). Induction of an argine-rich component during infection with influenza virus. J. Gen. Viral. 4, 215-220. BORUN, T. W., SCHARFF, M. D., and ROBBINS, E. (1967). Preparation of mammalian polyribosomes with the detergent Nonidet P-40. Biochim. Biophys. Acta 149, 302-304. BURNET, F. M., and BULL, D. R. (1943). Changes in influenza virus associated with adaption to
AND
WHITE
passage in chick embryos. d&ralian J. G’.~cp(l. Biol. Med. Sci. 21, 55-69. D~~s~ERG, P. H., aud RO~INSOX, W. S. (1967). On the structure and replication of influenza virus. J. Mol. Biol. 25, 383-405. DUI,HICCCO, R., and VOGT, M. (1954). Plaqrle formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99, 167-182. EAGTX. II. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. FRASER, K. B. (1967). Immunofluorescence of abortive and complete infections by influenza A virus in hamster BHK21 cells and mouse L cells. J. Gen. Viral. 1, 1-12. HASLAM, E. A., CHEYNF,, I. M., and WHITE, D. 0. (1969). The structural proteins of Newcastle disease virus. Virology 39, 118-129. HENLE, G.. GIRARDI, A., and HENLE, W. (1955). A non-transmissible cyt,opathogenic effect, of influenza virus in tissue culture accompanied by formation of non-infectious hemagglut.inins. J. Exptl. Med. 101, 25-41. HILLIS, W. D., MOFFA~~, M. A. J., and HOLTERMANN, 0. A. (1960). The development of soluble and viral antigens of influenza A virus in tissue culture as studied by the fluorescent antibody technique. 3. Studies on t,he abortive cycle of replication in HeLa cells. Actu Pathol. Microbial. &and. 50, 419-429. LEHMANN-GRUBE, F. (1965). Influenza viruses in cell cultures. III. Use of calf kidney cells for quanta1 assay. Arch. Ges. Virusjorsch. 17, 534548. MAIZEL, J. V., JR. (1966). Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science 151,98& 990. MAIZEL, J. V., JR., and SUMMERS, D. F. (1968). Evidence for difference in size and composition of the poliovirus specific polypeptides in infected HeLa cells. Virology 36, 48-54. O’BRIEN, B. R. A. (1964). The partial cytolysis of the amphibian erythrocyte and liver parenchyma cell by a non-ionogenic surface active agent. J. Cell Biol. 20, 521-525. PENMAN, S., S~HERRER, K.. BECKER, Y., and DARNELL, J. E. (1963). Polyribosomes in normal and poliovirus-infected HeLa cells and their relationship to messenger-RNA. Proc. Natl. Acad. Sci. U.S. 49, 654-662. ROTT, R., and SCHOLTISSEK, C. (1967). Multiplication of myxoviruses. In “Modern Trends in Medical Virology” (R. B. Heath and A. P. Waterson, eds.), Vol. 1, pp. 25-48. Butterworths, London.
SYNTHESIS
AND
MIGRATION
SCH:~FEH, W. (1959). Some observations concerning the reproduction of RNA-containing animal viruses. Symp. Sot. Gen. Microbial. 9, 61-81. SHAPIRO, A. L., SCHARFF, M. D., MAIZEL, J. V., JR., and UHR, J. W. (1966). Polyribosomal syn-
OF
INFLUENZA
PEPTIDES
thesis and assembly of the H and L chains gamma globulin. Proc. Natl. Acad. Sci. U.S. 216-221. VON MAGNUS, P. (1954). Incomplete forms influenza virus. .4dvan. Virus Res. 2, 59-79.
425 of 56, of
39,
419-425
(1969)
The Polypeptides I. Cytoplasmic J. M. TAYLOR, School
of Microbiology,
of Influenza
Virus
Synthesis and Nuclear
Accumulation
A. W. HAMPSON,
D. 0. WHITE
University of Melbourne, Accepted
June
AND
Pa&de,
Victoria,
Australia
23, 1969
Three major structural peptides were found in radioactive influenza virus type A/Be1 that had been grown in calf kidney cells, purified, and analyzed by acrylamide gel electrophoresis. The same three peptides were found in infected HeLa cells. All proved to be stable; none was a precursor of another. The site of synthesis and subsequent migration of these peptides was investigated by cell fractionation. Evidence is presented which suggests that viral peptides were synthesized in the cytoplasm, and that two of them subsequently underwent significant accumulation into the nucleus. INTRODUCTION
The intracellular localization of influenza antigens has been studied in the past by the technique of immunofluorescence (Schafer, 1959; Hillis et al., 1960; Fraser, 1967; Rott and Scholtissek, 1967). The S-antigen (ribonucleoprotein) has been generally observed to accumulate first in the nucleus, and only later in the cytoplasm. In contrast, the V-antigen (widely assumed to correspond with the hemagglutinin) is detected exclusively in the cytoplasm. These observations have been interpreted by Rott and Scholtissek (1967) as evidence that both the Sand V-peptides may be synthesized in the nucleus, but that the V-peptides do not assumean antigenic configuration until after they have migrated to the cytoplasm. Furthermore, Becht (1969) has presented autoradiographic data suggesting that nuclear protein synthesis may occur in influenzainvected cells. In this communication an attempt is made to determine the site of synthesis of individual influenza peptides by combining the following approaches: (1) brief radioactive pulse-labeling of virus-infected cells, followed by a prolonged “chase”; (2) fractionation of cells into nucleus and cytoplasm; 419
(3) separation of viral peptides by acrylamide gel electrophoresis. The results demonstrate that all three of the major structural peptides of the influenza virion are synthesized in the cytoplasm, and that two of these subsequently migrate to the nucleus. MATERIALS
AND METHODS
Growth and puriJication of 14C-labeled influenza virus. Confluent monolayer cultures of primary embryonic calf kidney cells (Lehmann-Grube, 1965) were rinsed twice with Hanks’ balanced salt solution and replenished with Minimal Essential Medium (MEM) (Eagle, 1959), modified to contain one-tenth the usual amino acid concentrations and supplemented with 0.1% bovine serum albumin. Influenza virus A/Be1 (Burnet and Bull, 1943), grown in the ahantois of developing chicken embryos, was inoculated into the cultures at a multiplicity of infection (m.0.i.) of 0.05 tissue culture IDbOper cell together with 14C-labeled amino acid mixture (Amersham CFB. 104 at 52 mCi/mAtom carbon) to a final concentration of 0.25 &X/ml. The cultures were incubated at 35” for 72 hours, by which time they showed marked cytopathic effects.
420
TAYLOR,
HAMPSON,
Virus was then purified by the following modification of the procedure developed for Newcastle disease virus (Haslam et al., 1969). The supernatant tissue culture fluid was clarified by centrifugation (12,000 g, 10 min), the virus was deposited (64,000 g, 30 min) onto a small cushion of 2.3 M sucrose, resuspended by brief sonic vibration, and purified by rate zonal centrifugation in a l&60% (w/v) sucrose gradient (64,000 g, 120 min), followed by equilibrium centrifugation in a 2040% (w/v) potassium tartrate gradient (174,000 g, 180 min). Fractions were collected dropwise from the bottom of the tube and assayed for hemagglutinin and 14C. Peaks of the two activities coincided and corresponded to the visible band in both gradients. Infection and pulse-labeling of HeLa cells. Suspension cultures of HeLa 53 cells were propagated in MEM supplemented with 5 % fetal calf and 5% bovine serum. For virus infection, cells prelabeled for 48 hours with thymidine-3H (at 0.07 &i/ml and 35.7 mCi/mmole) were washed once and then resuspendedat a concentration of 10’ cells/ ml in serum-free MEM modified to contain one-tenth the usual amino acid concentrations. Influenza A/Be1 propagated in embryonated hen’s eggs and concentrated by centrifugation, was added at an input multiplicity of 300 egg ID60 per cell, and allowed to adsorb for 30 min at room temperature. The cell suspensionwas then diluted lo-fold with prewarmed MEM, lacking amino acids and supplemented with 5% dialyzed fetal calf serum. Cultures were incubated at 37’ with stirring for a further 3.5 hours. The
AND
cells were then gently centrifuged (250 g, 5 min) and resuspended in amino acid-free medium at a concentration of 4 X lo7 cells/ml. After a lo-min recovery period, 14C-labeledamino acid mixture was added to a final concentration of 10 &i/ml. After labeling for 3 min, a chase in the presenceof unlabeled amino acids was achieved by diluting the cells 40-fold with complete growth medium. At 1 and 60 min, respectively, aliquots of 2 X lo7 cells were diluted into ice-cold phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). These chase conditions inhibit further incorporation of 14C-amino acids into acid-insoluble material (Table 1). Cell fractionation. Cells were washed twice in ice-cold PBS, then swollen for 15 min in 1 ml of hypotonic buffer (0.01 M KCl, 0.002 M MgC12, and 0.01 M Tris at pH 7.4), and disrupted in a Dounce homogenizer (Penman et al., 1963). After centrifugation (1000 g, 5 min), the pellet comprised predominantly nuclei, but also small numbers of unbroken cells, as well as cell debris. Since it was imperative to obtain an uncontaminated preparation of clean nuclei, even at the expense of some loss of nuclei, the specimen was subjected to further purification as follows. The pellet was resuspended in 0.9 ml of cold TKM buffer (0.025 M KU, 0.005 M MgClz, and 0.05 M Tris at pH 7.4) containing 1.5 % by volume of the nonionic detergent Nonidet P40 (Shell Chemicals) (O’Brien, 1964), in order to rupture cytoplasmic membranes and remove cytoplasmic material from around unclean nuclei (Borun et al., 1967). The suspensionwas then mixed
TABLE DEMONSTRATION
OF THE
1
VALIDITY
OF THE
Duration
‘%-amino
0 Virus-infected cells were unlabeled (cold) amino acids, were chilled and precipitated counts given in the table are represent half the maximum
CHASE
of cold amino
PROCEDUREQ
acid chase (min)
1
1.5
30
45
60
185
170 (10)
165 (15)
180
160 (10)
-. TCA-precipitable acid counts
WHITE
(16)
(6)
labeled for 3 min with ‘%-amino acids and then chased by the addition of as described above. At the indicated times, aliquots of cell suspension with an equal volume of 20% trichloroacetic acid. The acid-precipitable the mean of three determinations, and the figures shown in parentheses range of those assays.
SYNTHESIS
AND MIGRATION
with 2.1 ml of 2.3 M sucrosein TKM buffer, underlaid with 1 ml of 2.3 1M sucrose in TKM and centrifuged at 120,000 g for 30 min. The resulting nuclear pellet and the original intact cells were assayed for radioactivity (14Cand 3H) in trichloroacetic acidprecipitable material using an Ansitron scintillation counter. The recovery of nuclei was equated with the recovery of thymidine-3H (usually 20-40 %) . Acrylamide gel electrophoresis.Electrophoresis on 5 % acrylamide gels was performed as described by Maize1 (1966) with the following modifications. Samples were dissociated with 2 % sodium dodecyl sulfate, 0.5 M urea, 0.04 M 2-mercaptoethanol, and 0.1 M dithiodiethanol in 0.01 M phosphate buffer pH 7.2 at 37” for 30 min, then at 100” for 1 min, and finally, made 10% in glacial acetic acid. Mercaptoethanol was omitted from the dialysis buffer.
OF INFLUENZA
PEPTIDES “PI
VP2
421 VP3
400
0
200
CPM 0
RESULTS
The Structural Peptides of InJEuenza Virus Influenza virus, labeled with 14C-amino acids, was grown in calf kidney cells, purified, and analyzed by gel electrophoresis (Fig. la). The virus possesses three major structural peptides, referred to here as VPl, VP2, and VP3. The identification and characterization 40 60 20 of these and minor components of the virion, FRACTION NUMBER such asthe shoulder sometimesdetectable on FIG. 1. Acrylamide gel electrophoresis of Wthe anodal side of VPl, is currently under amino acid-labeled peptides from (a) purified instudy. Viral Peptide Synthesis in Infected Cells Suspensioncultures of HeLa cells infected 4 hours earlier with influenza virus were labeled for 15 min with 14C-amino acids then analyzed by gel electrophoresis (Fig. lb). Fig. lc shows the electropherogram of mock-infected cells. It is apparent that the infected cell produces predominantly the three main structural peptides of the virion.
fluenza virus grown in calf kidney cells, (b) HeLa cells infected for 4 hours with influenza virus then labeled for 15 min, (c) mock-infected HeLa cells. Migration is toward the anode on the right.
acids for 3 min, chasedwith unlabeled amino acids for 1 or 60 min, then separated into nuclear and cytoplasmic fractions and analyzed by gel electrophoresis. A chase of at least 1 min was necessary to ensure the completion of most of the peptides still attached to polyribosomes at the termination Nuclear-Cytoplasmic Fractionation of Infected of the pulse (Shapiro et al., 1966) In Fig. Cells 2a, the electropherogram of the nuclei is In order to determine the site of synthesis compared with that of the whole cells after of these viral peptides, cells infected 3.8 the 1-min chase.The three viral peptides can hours earlier were labeled with r4C-amino be clearly seenin the whole cell; at the same
5oc
1
I tb)
25C ,-
CPM
. 0
0 FRACTION
FIG. 2. Acrylamide gel electrophoresis infection, cells were labeled for 3 min amino acids for (a) 1 min, or (b) 60 represent whole cells and the broken equivalent number of whole cells or
60
40
of peptides in influenza virus-infected cells. At 3.8 hours after with ‘%-amino acids and then chased in the presence of unlabeled min. Migration is toward the anode on the right. The solid lines lines, the nuclei. The data have been normalized to represent an nuclei as described in Table 2. TABLE
DETERMINATION
20
NUMBER
OF THE
2
NORMALIZATION
FACTOR
USED
FIG.
IN
2Q
Duration of cold amino acid chase (mm)
l*C-amino Whole Nuclei Hence
Activity Whole Nuclei
acid/sH-thymidine cells
ratio
per
aliquot
1
60
0.995
0.984 0.279
of: 6.185
fraction
of labeled
(1% cpm) cells
Hence normalization nuclear material
layered
protein
in nucleus
in electrophoresis
of cell
=
0.185 0.995
scale
0.984
= 0.186
= 0.283
3996
3250 3654
of: 1944
factor for ordinate in Fig. 2 =
0.279
of electropherograms
of 3996
1944-O.
186
= 0.382
3250 -.0.283 3654 = 0.252
D This normalization was applied because of variation in the recovery of nuclei in the extraction procedure and also because of the different amounts of radioactive sample used in each electrophoresis. Note, however, that the use of the above raC/aH ratios, obtained with an Ansitron scintillation counter, eliminates the need to derive nuclear recoveries in the deduction of the normalization factors. Actually, in the above experiment the nuclear recoveries, as obtained using 3H-thymidine data, were 40.5 and 20.4%, respectively. 422
SYNTHESIS
AND
MIGRATION
OF
INFLUENZA
PEPTIDES
423
Furthermore, the high multiplicities of infection necessary for meaningful biochemical studies would result in the production of incomplete virus even in a permissive cell Duration line (von Magnus, 1954). Nevertheless, it is of cold VP3 amino acid VP1 VP2 apparent from the experiments reported in chase this paper that HeLa cells infected at high (min) m.o.i. synthesize three major peptides which comigrate in acrylamide gel electrophoresis 36.6 1 2.2 9.0 47.0 64.1 with the three major structural peptides of 60 2.7 purified virus grown in another cell system, a Expressed as a percentage of the amount of namely calf kidney cells. the corresponding peptide in the whole cells. The pulse-chaseexperiment, the results of Data are derived from Fig. 2. which are depicted in Fig. 2 and expressed quantitatively in Table 3, demonstrates time, the nuclear fraction contains only a three main facts. (1) After a 3min label and minor portion of them. a 1-min chase, only a minor portion of each Figure 2b shows the results obtained when of the three newly synthesized viral peptides the period of chase in unlabeled amino acids is in the nuclear fraction. (2) During the was increased from 1 min to 60 min. The 60-min period of chase, two of the viral data indicate clearly that all three viral peptides, VP2 and VP3, then show signifipeptides are stable; none is a short-lived cant accumulation into the nuclear fraction. precursor of the type encountered in polio(3) The remaining peptide VPl, does not virus-infected cells (Maize1 and Summers, accumulate in the nuclear fraction. Similar 1968). The most striking result, however, is results have been obtained in several comthat VP2 and VP3 have been chased into parable experiments. the nuclear fraction. In contrast, VP1 has The obvious and most interesting interstill not left the cytoplasm. pretation of these data is that the three The data contained in Fig. 2 is expressed major structural peptides of the virus are quantitatively in Table 3, which gives the synthesized in the cytoplasm, and that percentage of each viral peptide from the eventually two of them, VP2 and VP3, whole cell that is present in the nuclear accumulate in significant amounts in the fraction at the two chase times. nucleus, but the third, VPl, doesnot. Before discussing this interpretation further, it is DISCUSSION essential to consider possible alternative Acrylamide gel electrophoresis of purified explanations. influenza virus type A/Be1 has demonstrated As yet there is no cell fractionation prothree major structural peptides. Duesberg cedure that can guarantee absolute purity as and Robinson (1967) previously reported well as integrity of mammalian cell nuclei. two or three peptides in purified influenza Attempts to “clean” isolated nuclei of adtype A/PR8, analyzed by a somewhat difherent cytoplasmic contamination may lead ferent electrophoretic technique. to loss of nuclear material in the process. The primary purpose of this communicaHence, the possibility cannot be entirely distion has been to demonstrate certain aspects counted that influenza peptides may be synthesized in the nucleus then leak out of of the viral infection cycle in a line of HeLa cells conveniently handled in suspension culthe nucleus during cell fractionation. Following a 60 min chase, VP2 and VP3 may have ture. It should be noted that this experibecome incorporated into a structure too mental system is one in which the virus undergoes an abortive cycle of multiplicalarge to leak out in this way, e.g. nucleocapsid. Conversely, the presence of VP2 and tion, not leading to any significant production of infectious virus (Henle et al., 1955). VP3 in the nuclear fraction is unlikely to be TABLE
PERCENTAGE
3
OF EACH VIRAL PEPTIDE THE NUCLEUSQ
FOUND
IN
424
TAYLOR,
HAMPSON,
due to contamination with cytoplasmic material, as VP1 is not found in the nuclear fraction at all, while the content of VP2 and VP3 increases markedly with the time of chase. Nevertheless, the possibility cannot be entirely discounted that VP2 and VP3 become associated in a time-dependent fashion with some structure which cosediments with nuclei. Perhaps these viral peptides enter the nucleus in the course of simple diffusion around the cell. The smaller the molecule the more rapidly it may be expected to enter the nucleus. Work is proceeding to complete the identification of these three viral peptides. Preliminary data suggest that VP1 is the peptide of the viral hemagglutinin, and VP2 that of the ribonucleoprotein. The older fluorescent antibody work can now be reinterpreted in the light of the results contained in this paper. The presence of S-antigen in the nucleus and V-antigen in the cytoplasm should not be taken to mean that their constituent peptides are made in those sites. Both the ribonucleoprotein and the hemagglutinin peptides are synthesized in the cytoplasm, then one of them migrates to the nucleus. The identity of the peptide, VP3, perhaps corresponding to the viral neuraminidase, and the reason for its migration to the nucleus now become matters of particular interest. ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and the Australian Research Grant.s Committee. A. W. Hampson is on leave from, and supported by, the Commonwealth Serum Laboratories. REFERENCES BECHT, H. (1969). Induction of an argine-rich component during infection with influenza virus. J. Gen. Viral. 4, 215-220. BORUN, T. W., SCHARFF, M. D., and ROBBINS, E. (1967). Preparation of mammalian polyribosomes with the detergent Nonidet P-40. Biochim. Biophys. Acta 149, 302-304. BURNET, F. M., and BULL, D. R. (1943). Changes in influenza virus associated with adaption to
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SYNTHESIS
AND
MIGRATION
SCH:~FEH, W. (1959). Some observations concerning the reproduction of RNA-containing animal viruses. Symp. Sot. Gen. Microbial. 9, 61-81. SHAPIRO, A. L., SCHARFF, M. D., MAIZEL, J. V., JR., and UHR, J. W. (1966). Polyribosomal syn-
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PEPTIDES
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