Nov 28, 1973 - Soc. Microbiol., p. 243, 1973;. 26). Further evidence supporting the hypothesis .... Microbios 5:247-257. 2. Bikel, Ilan, and P. H. Duesberg. 1969.
JOURNAL
OF
Vol. 13, No. 4 Printed in U.S.A.
VIROLOGY, Apr. 1974, p. 788-800
Copyright 0 1974 American Society for Microbiology
Protein Synthesis in Newcastle Disease Virus-Infected Chicken Embryo Cells LAWRENCE E. HIGHTOWER' AND MICHAEL A. BRATT Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received for publication 28 November 1973
A double-isotopic label difference analysis of polyacrylamide gels has been used to distinguish between cellular and viral protein accumulation in infected cells and to quantify the kinetics of accumulation of viral polypeptides. This technique, coupled with the determination of total radioactive amino acid incorporation in infected cultures, has revealed the following kinetic patterns. Viral polypeptides are first detected in infected cultures 2.0 to 2.5 h postinfection. The rate of accumulation of radioactive amino acids in viral polypeptides increases to a maximum (30 to 35% of the rate of accumulation in uninfected control cultures), whereas the rate of accumulation of radioactive amino acids in host-cell protein decreases to a minimum (20% of the rate of accumulation in uninfected control cultures) by 5 to 6 h postinfection. All of the viral polypeptides detected late in infection are also present at the earlier times, and the major virion structural polypeptides are present in approximately the same (N/G-2, 53K) or slightly increasing (L, G-1, M) relative amounts. One peak area containing a nonstructural glycopeptide with an apparent molecular weight of 66,000 shows significant alterations in rates of accumulation during infection. Inhibition in the rate of radioactive amino acid incorporation into both trichloroacetic acid-soluble and acid-precipitable material during infection has been demonstrated. However, these two inhibition phenomena can be uncoupled temporally by incubating infected cultures at 36 C instead of the usual 40 C, suggesting that they may not be directly related.
Until recently, studies on protein metabolism in paramyxovirus-infected cells have been limited to kinetic analyses of total amino acid incorporation (24, 30, 31). Now, it is possible to resolve viral polypeptides in Newcastle disease virus (NDV)-infected cells on sodium dodecyl sulfate (SDS)-polyacrylamide gels (1, 18, 26). We have used this technique as the basis of a quantitative method for studying protein metabolism in infected cells. This method employs a double-isotopic label difference analysis of SDS-disc polyacrylamide gels. A similar procedure has been employed by Zweerink et al. (32) to study the intracellular synthesis of Reovirusspecific proteins. We have used this analytical technique in the present study to pursue the following objectives: (i) To distinguish between cellular and viral protein accumulation in infected cells and to study the effects of viral infection on host cell protein metabolism. (ii) To monitor the accumulation of individual viral polypeptides.
(iii) To gather information about viral proteins and virus cell interactions of the AV strain of NDV which could be useful in analyzing the process of fusion from within (3, 4) and in characterizing temperature-sensitive mutants of this strain (J. E. Tsipis and M. A. Bratt, Negative strand viruses, presented in Cambridge, England at the Negative Strand Viruses Meeting, 22-27 July, 1973; also, portions of this study were included in another chapter of the same publication by M. A. Bratt et al.)
MATERIALS AND METHODS Cell culture. Primary and secondary chicken embryo cell cultures were prepared and grown in a 5% CO2 atmosphere at 40 C in Eagle minimal essential medium (MEM), supplemented with 2.5% calf serum, 2.5% tryptose phosphate broth, and 0.07% NaHCO, (standard medium) as previously described (3). The secondary cultures were plated at 2.0 x 106 cells per 60-mm tissue culture plate and used after 48 h of incubation at 40 C. At this time, the cell layers (2.6 x 106 cells) appeared to be confluent. Virus purification and assay. NDV-AV (AusIPresent address: Department of Biochemistry and Mo- tralia-Victoria, 1932) was grown in embryonated eggs lecular Biology, Harvard University, Cambridge, Mass. 02138. and purified by a procedure similar to that previously 788
VOL. 13, 1974
NDV PROTEIN SYNTHESIS
described (8). Virus was concentrated by centrifugation of infected allantoic fluids at 30,000 rpm (78,000 x g) in a Spinco type 30 rotor for 1 h. The concentrated virus was then centrifuged for 1 h at 24,000 rpm (59,500 x g) through a 20% sucrose layer onto a 65% sucrose-D2O pad in a Spinco model SW25.1 rotor. The buffer used in all sucrose solutions described (standard buffer) is 0.1 M NaCl, 0.01 M Tris, pH 7.4, and 0.002 M EDTA. The virus band at the interface of the sucrose layers was collected and infectivity titers (PFU per milliliter) were determined as previously described (3). Rate of virus production. The amount of infectious virus released from infected cells was measured in the following way. Virus (MOI = 5 PFU/ml) was allowed to adsorb to cells at 40 C for 45 min. Cultures were then washed five times with 2 ml of prewarmed standard medium. A 2-ml amount of prewarmed standard medium was added (time zero) and cultures were incubated at 40 C, in 5% CO2, with medium changes every 90 min. These medium samples were stored at 4 C for subsequent assay by plaque titration. Chemicals and isotopes. Mixtures of 15 3H-amino acids (1 to 10 Ci/mmol) or "4C-amino acids (approximately 0.10 Ci/mmol) and Omnifluor were purchased from New England Nuclear Corp. Acrylamide and N,N'-methylenebisacrylamide were purchased from Eastman Organic. N, N, N',N'-tetramethylethylenediamine, ammonium persulfate, and glycine were purchased from Matheson, Coleman, and Bell. Sodium lauryl sulfate (specially pure) was purchased from Gallard-Schlesinger Corp. Bovine serum albumin which had been recrystallized three times was a gift from R. W. Hastings, and aldolase (dog muscle), glyceraldehyde-3-phosphate-dehydrogenase, and lactate-dehydrogenase were gifts from D. G. Fraenkel. Preparation of radioactive virus. Cell cultures were infected at a multiplicity of 10 to 20 PFU/cell and incubated as described above. After the adsorption period, 2 ml per 100-mm tissue culture plate of label medium consisting of 200 uCi of 3H-amino acid mixture per ml, 2% dialyzed calf serum, and MEM containing 5% the normal concentration of amino acids was added to the infected cultures. The medium containing virus was collected 17 h later and purified as follows. After centrifugation at 27,000 x g for 10 min to remove cell debris, the supernatant was collected and sedimented at 24,000 rpm (60,000 x g) for 1 h in a Beckman SW25.1 rotor at 4 C through a 20% sucrose layer onto a 65% sucrose-D2O pad. The virus band at the interface of the sucrose layers was then collected, diluted threefold with cold standard buffer, and layered on a 20 to 65% sucrose-D2O linear gradient. Centrifugation was carried out in the Beckman SW41 rotor at 38,000 rpm (173,000 x g) for 4 h. The peak of radioactivity was pooled and stored at -20 C. Preparation of UV-irradiated virus. Virus was irradiated for 10 min at a distance of 55 cm from a G15T 8 Sylvania UV-lamp as previously described
(7).
Preparation of radioactive culture samples. Determination of total radioactivity. Cultures were infected as described above. At various times after infection, the medium was removed and the cultures
789
were washed with 2 ml of Hanks balanced salts (pH 7.2). A 1-ml amount per plate of 3H-amino acid mixture in prewarmed label medium was then added. After a 30-min labeling period, the label medium was removed, and the cultures were washed three times at 4 C with 2 ml of Tris-saline (0.15 M NaCl, 5 x 10-3M KCl, 0.025 M Tris, pH 7.3, 1 g of glucose per liter). To extract soluble radioactivity, 2 ml per plate of cold 5% trichloroacetic acid was added and allowed to sit ovemight at 4 C. After removal of the 5% acid (acidsoluble radioactivity), 2 ml of 1 N NaOH was added and the cultures were incubated at 40 C for 30 min. Samples (acid-precipitable radioactivity) were removed from the plates, boiled for 2 min, and 0.25-ml samples were taken for Lowry protein assays (19). The remaining samples were mixed with an equal volume of 25% acid and 0.25 mg of bovine serum albumin carrier. After 30 min at 4 C, precipitates were collected on membrane filters (0.45 um pore size; Millipore Corp.), dried, and counted in 5 ml of tolueneOmnifluor in a Packard liquid scintillation counter. For determinations of acid-soluble radioactivity, 0.5 ml of the acid-soluble fraction was diluted with 10 ml of modified Brays solution (2 parts toluene, 2 parts dioxane, 1 part methanol, 5 g of Omnifluor per liter, 73 g of naphthalene per liter; Kaplan and Moskowitz, unpublished data) and counted. Lowry protein determinations showed that the amount of protein in 48-h cultures was constant at 200 4 20 gg/60-mm plate (2.6 4 0.2 x 106 cells), and that in infected cultures, no loss of cells from the plate occurred up to 10 h postinfection. Consequently, Lowry assays were not routinely done. Polyacrylamide gel samples. Cultures were infected and incubated as described above. Infected cultures were labeled with 3H-amino acid mixture (uninfected control cultures were labeled with "Camino acid mixture) in prewarmed label medium for 30-min periods at various times after infection. Cultures were washed three times with cold Tris-saline and taken up in 0.3 ml of gel sample buffer (0.05 M Tris, pH 6.7, 1% mercaptoethanol, 1% SDS). Samples were stored at -20 C. Gel electrophoresis. Electrophoresis on SDS-disc polyacrylamide gels was carried out according to the method of Laemmli (17). In all experiments, 8.5% resolving gels (0.6 by 12 cm) with 0.3 ml of 3% spacer gels were used. Appropriate 3H-labeled infected culture samples and "IC-labeled uninfected culture samples were mixed, made 20% (vol/vol) in glycerol and 0.1% in bromphenol blue, boiled for 2 min, and layered on gels. A 120 to 130-jAg amount of protein in 0.3 ml was applied to each gel. Electrophoresis was carried out at 100 V constant voltage for 5 h. Gels were removed, frozen on dry ice, and stored at - 20 C until fractionated. Gels were fractionated on a Savant autogel divider, allowed to elute overnight in 0.2 ml of distilled water per vial, and then 4 ml of modified Brays counting fluid was added per vial. Gels were counted on a Packard liquid scintillation counter with a maximum of 5% standard error on double-label setting. 'H spillover in the 14C channel was approximately 1%, whereas '4C spillover in the 3H channel was 12%. Autoradiography. Confluent chicken embryo sec-
790
J. VIROL.
HIGHTOWER AND BRATT
used to emphasize the fact that the contributions of the processes of protein synthesis and protein degradation have not been evaluated separately and that for NDV-infected cells, the extent of reutilization of amino acids is unknown.) This is indicated by the dramatic change from the roughly continuous distribution of polypeptides observed in gels of uninfected cultures to the small number of welldefined peaks observed at late times in gels of infected cultures. Figure 2 shows that the major peaks observed in gel patterns of infected cultures late in infection correspond to the major virion polypeptides. Here the polypeptides of purified virions grown in chicken embryo cell cultures in the presence of 3H-amino acid mixture have been co-electrophoresed with an infected culture sample labeled with "C-amino acid mixture. The peak areas designated L (large), G-1 (glycopeptide), N (nucleocapsid), G-2 (glycopeptide), M (membrane), and 53K (the shoulder on the leading edge of the N/G-2 peak) correspond to the major virion structural polypeptides. In addition, a peak designated 66K and often a peak at 36K are found in patterns from infected cultures. The latter appear to constitute the major nonvirion polypeptides. The 66K peak contains a glycopeptide, as demonstrated by 3H-glucosamine labeling of
ondary cell cultures were infected as described above. After 90 min of incubation at 40 C cells were removed from the plate by trypsinization (0.1 % Trypsin in Tris-saline) and replated at one half the normal concentration. At 4 h postinfection, 5 jig of actinomycin D (courtesy of Merck, Sharp, & Dohme, Rahway, N.J.) per ml was added, and 30 min later, 25 gCi of 3H-uridine per ml was added per culture. At 6.5 h postinfection, cells were washed with 5 x 2 ml per plate of ice-cold Tris-saline, followed by 3 x 2 ml of citric acid-phosphate buffer, pH 3.0 (20), and fixed with 95% methanol. The tissue culture plates were then cut into sections, each section mounted on a glass slide, and autoradiography was performed as described by Perry (23) by using Ilford G-5 emulsion. Slides were stained with Giemsa prior to counting.
RESULTS Comparison of polypeptide patterns from infected and uninfected cultures and virions. Figure 1 shows an SDS-disc gel electropherogram of differentially labeled polypeptides from infected and uninfected cultures. The areas under the curves represent the relative extent of radioactive amino acid incorporation into polypeptides in uninfected and infected cultures, 9 to 9.5 h postinfection. Accumulation of radioactivity in host-cell polypeptides is substantially reduced in infected cultures and at least partially replaced by accumulation in viral polypeptides. (The term "accumulation" has been T
III
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FRACTION FIG. 1. Comparison of the polypeptides of infected and uninfected cultures by co-electrophoresis. Cultures were infected at a multiplicity of 5 PFUIcell. At 9 h postinfection, label medium containing either 10 4Ci of 3H-amino acid mixture per ml (infected, *) or 1 qCi of 14C-amino acid mixture per ml (uninfected, 0) was added to cultures for 30 min. Samples were prepared and processed for electrophoresis. The same patterns were obtained when the radioisotopes were reversed. Scales were chosen so that the integrated area under the curve of the infected culture pattern represents 55% of the integrated area under the curve of the uninfected culture pattern. As shown in Fig. 6, the rate of accumulation of acid-precipitable radioactivity in infected cultures at 9 to 9.5 h postinfection is 55% of the uninfected control rate.
791
NDV PROTEIN SYNTHESIS
VOL. 13, 1974
3
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0 z
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FRACTION FIG. 2. Comparison of the polypeptides from virions and infected cultures by coelectrophoresis. Infected cultures (0) were labeled 6 to 6.5 h postinfection with label medium containing 20 uCi of "C-amino acid mixture per ml. Samples were prepared and co-electrophoresed with 'H-amino acid labeled purified virions (0).
infected cells (J. Kaplan and M. A. Bratt, Abstracts Amer. Soc. Microbiol., p. 243, 1973). The presence of glycopeptides in infected cultures corresponding in mobility to the virion G-1 and G-2 was similarly shown. Difference analysis of double-label polyacrylamide gel electropherograms. Having established that peaks corresponding to virion polypeptides and two additional polypeptides can be detected in NDV-infected cultures, we required a technique which would allow for quantification of the kinetics of accumulation of viral polypeptides as well as the kinetics of inhibition of host-cell protein accumulation in infected cultures. A data analysis was selected which compares differentially labeled polypeptides from infected and uninfected cultures co-electrophoresed on SDS-disc polyacrylamide gels. Figure 3 illustrates this double-label difference analysis. 'H-amino acid-labeled infected cultures are co-electrophoresed with "C-amino acid-labeled uninfected cultures. As shown in Fig. 3B, a region of normalization (in all cases, gel fractions 20 to 30, corresponding to the molecular weight range 120 to 150K) which contains no apparent viral polypeptides is chosen. The total 'H radioactivity in fractions 20 to 30 is divided by the total "C radioactivity in the same fractions in order to obtain an average 'H/"C ratio. The "IC radioactivity in each fraction is then multiplied by this ratio. The normalized "IC radioactivity is operationally defined as host-cell background, and the difference be-
tween 'H radioactivity in the infected cell pattern and the normalized "C radioactivity is defined as virus-specific radioactivity (Fig. 3A). A mathematical summary of the difference analysis is given in the Appendix. The large peak co-migrating with the marker dye in Fig. 3B is mainly due to radioactively labeled free amino acids in the undialyzed samples (since most of the peak disappears after dialysis) (L. E. Hightower, unpublished data). Since this region is not resolved into discrete peaks on higher percentage gels (L. E. Hightower, unpublished data) and never represents more than 8% of the total radioactivity in polypeptides, it was not included in quantitative gel analysis. In the difference plot shown in Fig. 3A, the negative deviation in the lower molecular weight region is only observed early in infection and disappears as a function of time during infection. This deviation probably represents a perturbation in the distribution of cellular peptides in the infected culture. This perturbation may be associated with the inhibition in protein accumulation in infected cells described in the following section. Inhibition of host-cell protein accumulation. The following experiment was designed to monitor the reduction of host-cell polypeptide accumulation in infected cultures. Infected and mock-infected cultures were labeled for 30 min with 'H-and "C-amino acids, respectively, at various times after infection. The appropriate
HIGHTOWER AND BRATT
792
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FRACTION FIG. 3. Difference analysis. Cultures were infected, labeled 3 to 3.5 h postinfection with label medium containing 10 UiCi of 3H-amino acid mixture per ml (infected) or 1 uCi of 'IC-amino acid mixture per ml (uninfected), and processed for electrophoresis. (A) Plot of the difference between the normalized polypeptide patterns from infected and uninfected cultures. (B) Plot of the polypeptide patterns from infected (0) and uninfected (0) cultures after normalization.
samples of solubilized infected and uninfected cultures were mixed and co-electrophoresed. Representative difference plots are shown in Fig. 4. The percentage of total counts in the gels in virus-specific and cellular polypeptides at each time point was determined (as described in the Appendix, equations 3 and 4) and is summarized in Fig. 5B. By 5 to 6 h postinfection, 60% of the total radioactivity accumulating in a 30min labeling period is in virus-specific polypeptides, and 40% is in cellular polypeptides. For comparison, the rate of release of infectious virus was determined by measuring the amount of virus released into the medium during 90-min intervals. Figure 5A shows the usual logarithmic increase in rate of release of virus until 6 h postinfection. Between 6 and 10 h postinfection, the rate of virus release and the rates of accumulation of viral and cellular polypeptides are all constant. To determine the rates of total accumulation of radioactive amino acids into polypeptides, infected and mock-infected cultures were la-
beled with 3H-amino acids for 30 min at various times after infection. Acid-precipitable and acid-soluble radioactivity was determined and is shown in Fig. 6. By 5 to 6 h postinfection, the rate of accumulation of acid-soluble radioactivity decreased by 30% and the rate of accumulation of acid-precipitable radioactivity decreased by 45% in infected cultures. Using this data on the rates of accumulation of acid-precipitable radioactivity and the gel data summarized in Fig. 5, it is possible to calculate the absolute rates of accumulation of radioactive amino acids in total virus-specific polypeptides and in cellular background in infected cultures (see Appendix, equations 5 and 6). By 5 to 6 h postinfection, the rate of accumulation of viral polypeptides has reached a maximum (30 to 35% of the total rate of accumulation in uninfected controls), whereas the rate of host-cell polypeptide accumulation has decreased to 20% of the level in the uninfected control cultures. Three additional aspects of the inhibition of
VOL. 13, 1974
793
NDV PROTEIN SYNTHESIS
accumulation of host cell proteins in this system have also been studied. First, we wanted to determine whether there is a causal relationship between the inhibitions of rates of accumulation in acid-soluble and acid-precipitable radioactivity as is suggested by their temporal coincidence at 40 C (as shown in Fig. 6). When the rate of accumulation of acid-soluble and acidprecipitable radioactivity was measured at 36 C instead of the usual 40 C, a striking alteration in the time course of the two inhibition phenomena was observed (Table 1). At the lower temperature, the rate of accumulation of radioactivity in acid-precipitable material in infected cultures is only slightly inhibited, whereas that of the acid-soluble pool undergoes a 40 to 50% reduction. This finding demonstrates that the inhibition in the rate of accumulation of acid-soluble radioactivity is not sufficient to cause the inhibition of acid-precipitable radioactivity. Next, we wanted to determine whether the inhibition of accumulation of either acid-solu-
ble or acid-precipitable radioactivity is a function of intracellular events in productive infection or merely a cellular response to the virus particles in the initial inoculum. Cultures were infected under the usual conditions of infection with equivalent multiplicities of either UVirradiated or unirradiated virus. As in previous experiments, acid-soluble and precipitable radioactivity were measured after 30-min labeling periods at various times after infection. Table 2 shows that no inhibition of either acid-soluble or acid-precipitable radioactivity occurs in cultures infected with UV-irradiated virus. Since it is known that virus irradiated for even much longer periods is capable of initiating infection as indicated by synthesis of limited amounts of virus-specific RNA fragments (7), we conclude that the inhibitory phenomena are a consequence of intracellular events in productive infection. Finally, we wanted to determine whether the residual rate of host cell protein accumulation which persists late in infection represents the
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FIG. 4. Difference analysis of infected cell cultures. Conditions of infection and-radioactive labeling are the same as described in Fig. 3. The arrows in part (A) mark the positions of the major virion structural polypeptides as determined in gels run in parallel. Note the scale change in part (E). Labeling times (hours postinfection): (A) 1 to 1.5; (B) 2 to 2.5; (C) 3 to 3.5; (D) 4 to 4.5 (E) 9 to 9.5.
794 5.
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data by the method of England et al. (12) indicates that at least 95% of the cells are infected. We therefore conclude that the residual rate of host cell protein accumulation primarily represents incomplete inhibition of host cell protein accumulation in infected cells. (Actually, the value calculated by the method of England et al. was 100%. This is in accord with the prediction from the Poisson distribution that over 99% of the cells will be infected at a multiplicity of infection of 5 PFU per cell. However, the data in this experiment are complicated by the fact that a certain proportion of the cells have fused with each other [3] by the time the infected cultures are fixed. Because of the possibility that infected cells might preferentially fuse with uninfected cells [25], one must consider the possibility that multinucleate cells tend to mask cells which were not originally infected. Nuclei (10%) in these cultures were in multinucleate cells, suggesting that a maximum of 5% of the original cells could have been uninfected. The possibility that in-
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J. VIROL.
HIGHTOWER AND BRATT
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FIG. 5. Comparison of the rate of virus release and the percentage of total radioactivity in viral and cellular polypeptides. (A) The determination of the rate of virus release is described in the Materials and Methods. (B) Percent of total radioactivity in viral (-) and cellular (0) polypeptides was obtained from the data in Fig. 4 as described in the Appendix, equations 3 and 4. Determinations were based on the average values from duplicate gels.
contribution of a population of uninfected cells in the infected cultures, or alternatively, incomplete inhibition of host cell protein accumulation in infected cells. The percentage of infected cells in the infected cultures was determined by autoradiography. 3H-uridine was added to actinomycin D-treated infected and uninfected cultures, and cytoplasmic grain counts were determined. The histogram in Fig. 7 summarizes the grain distribution in these cultures. In contrast to the uninfected culture which gave average grain counts of less than 0.2 grains per cell, the infected cultures showed an average of 10 grains per cell. Statistical analysis of this
PRECIPITABLE
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FIG. 6. Kinetics of protein accumulation in infected cultures. Cultures were infected, labeled with 2 ACi of 3H-amino acid mixture per ml for 30 min at the times shown, and acid-precipitable (V) and acid-soluble (A) radioactivity was determined. All points are the average of duplicate determinations. The absolute rates of accumulation of radioactivity in viral (0) and host cell (0) polypeptides were determined as described in the Appendix, equations 5 and 6, by using the data summarized in Fig. 5. For convenience of comparison with the rates of total accumulation, the absolute rates of accumulation have been expressed relative to the uninfected control rates.
795
NDV PROTEIN SYNTHESIS
VOL. 13, 1974
TABLE 1. Rate of protein accumulation in infected cultures at 36 Ca Acid precipitable
Acid soluble
postinfetn |Uninfected| cultures
postinfection
(h)
Infected" cultures
Infected/uninfected x 100
Uninfected| cultures
Infected/unin-
Infected' cultures
fected x 100
2 4
3,454c
3,012
87
6,613
5,863
89
3,444
2,135
62
7,175
7,363
103
6 8
3,468
2,059 1,880
59 53
6,552 6,450
5,248 5,932
80 92
3,544
a Cultures were infected, labeled with 2 ACi of 3H-amino acid mixture per ml for 30 min at various times after infection and processed for acid-soluble and acid-precipitable radioactivity as described in the Materials and Methods. b Counts per minute, average of triplicate cultures. c Average standard deviation is less than 5% of mean.
TABLE 2. Effect of UW-irradiated virus on rate of cellular protein accumulationa Acid precipitable
Acid soluble
Time
postinfection
Uninfectedb
Infectedc
Infectedc
cultures
cultures
(UV-virus)
Uninfectedb cultures
Infectedc cultures
Infectedc (UV-virus)
2
1,587d
94
102
93
1,652
80
93
47
89
6 8
1,760 2,124
60 69
101 127
8,484 10,393 10,754 8,788
98
4
52 55
87 122
j
a Cultures were infected at a multiplicity of 5 infectious units per cell of unirradiated virus or the equivalent of UV-irradiated virus. Labeling conditions and radioactivity determinations were the same as described in Table 1. b Counts per minute, average of triplicate cultures. c Expressed as percentage of uninfected control. dAverage standard deviation is less than 5% of the mean.
fected and uninfected cells may indeed have 240 fused to form the multinucleate cells is sugAVERAGE GRAINS CELL 4\ gested by the finding that the average grain count over the multinucleate cells [dashed lines 24b in Fig. 7] is approximately one half of that C) 20 found in mononucleate cells. It is therefore LJ 16 possible that approximately 5% of the cells in the cultures were not originally infected.) 12 Temporal appearance of viral polypeptides. The electropherograms shown in Fig. 4 also aI rn ,i provide information on the accumulation of viral 4 polypeptides at various times after infection. Ij -1njNo peaks at the positions of the major virion 24 40-60 16 20 32 36 4 2 28 8 0 structural polypeptides (indicated by arrows in NUMBER GRAI NS / C E L L Fig. 4A) are detected above background in 1.0 FIG. 7. Determination of the percentage of infected to 1.5-h postinfection labeling period. Since the cells. Cultures were infected, labeled, and prepared distribution of polypeptides in infected and un- for The solid bars represent the infected cultures at this time is similar, this pat- grainautoradiography. distribution in uninfected cultures. The bars tern is indicative of variations inherent in the bounded by the solid line represent the grain distribudifference analysis and provides a measure of its tion in infected cultures, whereas the bars bounded by accuracy. Viral polypeptides are clearly seen the dotted line represent the distribution only in above background at 2.0 to 2.5 h postinfection multinucleate cells. A total of 200 infected and 200 (Fig. 4B). By 3.0 to 3.5 h (Fig. 4C) the intracell- uninfected cells were counted. /
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HIGHTOWER AND BRATT
796
ular viral pattern is firmly established. The 6.0to 6.5-h pattern is similar to the 4- to 4.5- (Fig. 4D) and 9- to 9.5- (Fig. 4E) h patterns, and therefore is not shown. Figure 8A illustrates the relative distribution of radioactivity in viral polypeptides during a 30-min labeling period at the indicated times during infection. It is apparent that the relative proportions of the major viral structural polypeptides are either constant (N/G-2, 53K) or increase (L, G-1, M, 36K) to varying degrees throughout the period of virus production. The major exception is the 66K peak which decreases in proportion to the other viral polypeptides by a factor of two between 2 and 6 h after infection. The absolute rates of accumulation of radioactive amino acids in viral polypeptides are summarized in Fig. 8B. These rates are derived from the rates of accumulation of total virusspecific protein (Fig. 6) and from the relative rates of accumulation of individual peak areas (Fig. 8A) (see Appendix, equations 5, 7, and 8). The rates of accumulation of all of the viral polypeptides increase during infection, attaining maximum or nearly maximum rates by 6 h postinfection. However, the magnitudes of the changes in the absolute rate of accumulation illustrated in Fig. 8B vary considerably among the polypeptides. The absolute rate of accumulation of radioactivity in L increases 14-fold, the largest change, whereas the 66K peak exhibits only a 2.8-fold increase, the smallest increase in
absolute rate of all the polypeptides. This accounts for the decrease in relative proportion of the 66K peak previously shown in Fig. 8A. The remaining polypeptides exhibit intermediate rate increases of 4.2-fold for N/G-2, and 6.4to 7.4-fold for G-1, M, and 36K. DISCUSSION The virion polypeptide patterns of strain AV presented in this communication are in general accord with published gel patterns for various NDV strains (2, 13, 14, 21). In addition, we have made several further observations on the behavior of NDV virion polypeptides on gels. We have consistently found high molecular weight material (150 to 200K) well separated from the gel origin in both virion and infected-cell samples of strain AV. However, the relative amount of this material (designated L) in virion samples varies among different preparations and may represent aggregates or host cell material as well as viral polypeptides. Two nonstructural polypeptides which probably correspond to the 66 K and 36 K peaks reported here have been detected in NDVinfected chicken embryo cultures by others (1, 18). These polypeptides were shown to vary considerably in relative proportion among different strains (18, 26). We have also observed strain variation in the relative proportions of the 66K peak and in the intracellular L peak as well. Cells infected by NDV-AV contain sub-
RATE OF ACCUMULATION OF VIRUS-SPECIFIC PROTEINS A 50
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0. 0
T
z
