- Email: [email protected]
Protein synthesis in bluetongue virus-infected cells
VIROLOGY
92, 385-396
(1979)
Protein Synthesis
in Bluetongue
HENDRIK
Virus-Infected
Cells
HUISMANS
Biochemistry Section, Veterinary Research Institute, Onderstepoort, South Africa Accepted August 22, 1978 The first virus-specific proteins in bluetongue virus (BTV)-infected cells can be demonThere is a rapid strated 2 to 4 hr after infection (p.i.) at 31” by immune precipitation. increase in the rate of synthesis until 11 hr p.i. after which synthesis remains at about the same level until 26 hr p.i. Apart from the seven BTV capsid polypeptides, two noncapsid polypeptides P5A and P6A, with respective molecular weights of 54,600 and 40,OOOdaltons, were identified in the infected cells. Polypeptide P5A is synthesized at a higher rate than any of the others. It is the main if not only component of a complex with an S value slightly less than 400. There is little evidence for specific regulation of the translation process in BTV-infected cells. With the possible exception of polypeptide Pl, all the others are translated with a relative frequency that is not significantly different from that of the corresponding mRNA species. Pulse-chase experiments indicate that the BTV polypeptides do not accumulate in the soluble fraction in the same ratio in which they are synthesized. Polypeptide P3 is removed from the soluble protein pool much more rapidly than any of the other capsid polypeptides. These pulse-chase experiments provided no evidence for a precursor-product relationship between any of the BTV polypeptides. The possibility that one of the noncapsid polypeptides, P6A, is a modified form of one of the capsid polypeptides can, however, not be excluded. INTRODUCTION
Bluetongue virus (BTV) is an orbivirus and a member of the family Reoviridae (Joklik, 1974). Although it is the best studied orbivirus and regarded as the prototype for this subgroup, little is known about the biochemistry of the virus apart from the characterization of the components of the purified virus itself. BTV comprises 10 double-stranded RNA segments (Verwoerd et al., 1970) surrounded by a protein coat containing seven different polypeptides, two of which form a diffuse protein layer which surrounds a smaller core particle (Martin and Zweerink, 1972; Verwoerd et al., 1972). This outer capsid layer differs morphologically from that of reovirus and rotavirus (Palmer et al., 1977) and is one of the main distinguishing characteristics of the orbiviruses. Protein synthesis in BTV-infected cells has never been studied and little is known about the actual replication process. The only aspect of replication that has been 385
studied is mRNA synthesis (Huismans and Verwoerd, 1973). The replication of reovirus, on the other hand, has been well characterized. Different noncapsid proteins have been identified (Zweerink, et al., 1971) and several studies have been made of subviral particles and their role in the replication process (Zweerink et al., 1972;Morgan and Zweerink, 1974; Morgan and Zweerink, 1975). None of this information is available for orbiviruses although they form a much larger group of viruses than the reoviruses and are clinically far more important. It is therefore warranted to study at least one of the orbiviruses in greater detail and determine in what respect if differs from reovirus. In this paper, experiments are described which were designed to study protein synthesis in BTV-infected cells, especially the synthesis of BTV noncapsid proteins, the kinetics of protein synthesis, and the relative proportion in which polypeptides are synthesized.
0042~6822/‘79/020385-12$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form resewed.
386
HENDRIK
Two new BTV proteins were identified, one of which accumulates in the insoluble protein fraction of infected cells in the form of a protein complex of approximately 400 s. MATERIALS
AND METHODS
Cells. BHK cells and mouse fibroblast L cells were obtained from the American Type Culture Collection. The cells were grown on Roux flasks or roller bottles in modified Eagle’s medium as described by Verwoerd et al. (1967). The medium was supplemented with 5% bovine serum. Virus. Methods for growing BTV type 1OAin BHK cells have been described (Verwoerd et al., 1967). Virus was titered by a plaque method on L cells (Howell, et al., 1967). A BTV stock was prepared as follows. A single plaque was selected from an L cell Petri dish as described by Shipham and de la Rey (1976) and used for inoculation of a monolayer of 2 x lo6 BHK cells. When the fuIl cytopathic effect was noticed, the virus was titered and this material used to infect Roux flasks with BHK cells at an input multiplicity of less than 0.1 PFU/cell. Virus produced by these cells was used as inoculum. It was stored at 4“ and used in an unpurified form because purified virus is very unstable. BHK cells infected with this inoculum at a multiplicity of between 0.1 and 40 PFU/cell gave a yield of at least 1000 PFU/cell. Virus purification. Virus was purified by a modification of the Freon extraction method described by Verwoerd (1969). It was found that after the first extraction, at least four more successive buffer extractions of the Freon phase were necessary to quantitatively dissociate virus from the cellular material to which it is very tightly bound. Two more Freon extractions were carried out on the combined water phases, and the virus was pelleted by centrifugation for 2 hr at 24,000 rpm in an SW 27 rotor through a 5-ml cushion of 40% sucrose in 0.002 M Tris, pH 8.8. The virus was then banded on sucrose gradients as described by Verwoerd (1969) and was considered purified when no more than the seven BTV polypeptides could be detected on electro-
HUISMANS
phoresis of a virus sample. Infection and Labeling of Cells. In labeling experiments, cells were infected with a high multiplicity of infection (40 PFU/celI) in order to obtain a high degree of synchronization of infection. Virus was allowed to adsorb to monolayer cells from a volume of 5 ml inoculum/108 cells. After 15 min at room temperature, the volume was increased to 60 ml by addition of Eagle’s medium and the cells were incubated at 31”. At the indicated times after infection, the cells were rinsed with amino acid (AA)-free Eagle’s medium and then incubated with 5 ml AA-free medium containing 1 or 2 #X/ml “C-protein hydrolysate (Radiochemical Centre, Amersham, England). Labeling was carried out at 37” and a shaking platform was used to ensure even distribution of the isotope-containing medium. At the end of a labeling period, cells were scraped from the glass and collected by centrifugation for 10 min at 1,500 rpm. Preparation of sub-cellular fractions. 1. Cytoplasmic extract: Cells were suspended in cold STM buffer (0.15 M NaCl, 0.005 M MgCh, 0.01 M Tris, pH 7.4) at a concentration of 5 X lo7 cells/ml. Nonidet P40 was added to a final concentration of 0.5% and the cells were incubated in an ice bath for 5 min. Nuclei were pelleted by centrifugation for 5 min at 3,000 rpm, resuspended with a syringe in one-half the original volume STM, and pelleted again. The two supernatants were combined and are referred to as the cytoplasmic extract. Twenty-microliter aliquots of the extract were lyophilized and stored at -20” until used for electrophoresis. 2. Soluble (SlOO) fraction: A suitable portion of the cytoplasmic extract was centrifuged for 2 hr at 45,000 rpm in an SW 50 rotor through 2 ml of a 40% sucrose cushion in 0.15 M STE buffer (0.15 M NaCl, 0.601 M EDTA, 0.001 M Tris, pH 7.4). The supernatant (SlOO fraction) was removed with a syringe. Extreme care was taken to avoid contamination of the fraction with the material in the interphase. Samples for electrophoresis (40 ~1) were lyophilized and stored at -20” before use. Samples for immune precipitations (0.2 or 0.3 ml) were
BTV-INFECTED
CELL
routinely used as soon as possible. They were never left at 4’ for longer than 16 hr because insoluble precipitates form on prolonged standing. 3. Particulate fraction: The interphase and sucrose cushion from the preparation of the SlOO fraction were discarded. The pellet was resuspended in 0.01 A4 STE in one-fifth of the original volume of cytoplasmic extract. Fractions of 15 1.11 were lyophilized and stored at -20”. Gel electrophoresis. Gel electrophoresis of proteins was carried out on slab gels as described by Stone et al. (1974). Lyophilized samples for electrophoresis were resuspended in 20 d of a solution consisting of 2 ml of 10 M urea, 0.4 ml 10% SDS, and 0.1 ml mercaptoethanol. To each sample, 5 ~1 of a 4 M NaCl solution was added. The high salt concentration prevented “narrowing” of the bands. In most cases, samples were electrophoresed for 18 hr at 40 V and 50 mA/gel. Gels were stained with Coomassie Brilliant Blue G-250 solution as described (Anderson et al., 1974). Gels were destained in 4% acetic acid at 50” and dried on filter paper under vacuum utilizing a heated gel-drying apparatus. Autoradiography. Cronex No. 4 medical X-ray film was placed on top of the dried gel in a X-ray film holder. Films were developed after the required period of exposure and used for making contact prints or scanned with a Vitatron densitometer. Peak areas were quantitated by an integrator coupled to the same instrument. Preparation of BTV antibodies. Antibodies against purified BTV were prepared by subcutaneous injection into a rabbit of 500 pg of purified virus mixed with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, Mich.). Two booster shots of purified virus only were given at l-week intervals after the first injection. Antibodies against the cytoplasmic extract of BTV-infected BHK cells were made as follows. Cells from eight infected BHK Roux flasks were harvested 20 hr after infection at 31”. A cytoplasmic extract was prepared as described above and one-third of this amount was injected subcutaneously into a rabbit together with an equal volume
PROTEIN
387
SYNTHESIS
of complete Freund’s adjuvant. Two booster shots, with no adjuvant, were given at I-week intervals after the first injection. Antibodies against the cytoplasm of uninfected control cells were made in exactly the same way. Immune precipitation. A volume of 0.2 or 0.3 ml of a SlOO fraction was mixed with 0.2 ml antiserum. This normally represented an excess of antibody. The mixture was left at 4’ for at least 16 hr, diluted to 2 ml with cold STM buffer, and the precipitate was collected by centrifugation at 4,200 rpm for 15 min. Precipitates were washed once with cold STM and were electrophoresed exactly as described for the lyophilized protein samples. RESULTS
Synthesis of BTV Proteins at Different Time Intervals after Infection BTV-infected BHK monolayer cells were pulse-labeled for different 2-hr intervals in the period 2-36 hr p.i. at 31”. Cells were harvested at the end of the labeling period. Cytoplasmic and SlOO protein fractions were analyzed by electrophoresis and autoradiography. The results are shown in Fig. 1, A and B. Until 8 hr p.i., the synthesis of viral polypeptides is almost entirely obscured by that of the host cell. After 11 hr p.i., however, synthesis becomes predominantly virus-specified and in this period all seven BTV capsid polypeptides can be identified in the cytoplasmic extracts. In addition, there is at least one noncapsid polypeptide which is synthesized in very large amounts in infected cells but not in control cells. This polypeptide is called P5A and has a molecular weight of about 54,000 daltons. Most of the virus polypeptides in the cytoplasmic extracts are also present in soluble form. The exception is P5A which is seen only in cytoplasmic extracts. Due to the fact that host cell protein synthesis is not completely suppressed, even late in the infection cycle, it is very difficult to decide whether a polypeptide like P6A, which is seen in both cytoplasmic and soluble extracts, is virus specified. It is also impossible to determine when the first
333
HENDRIK
HUISMANS
(140) (110) ml) (821 (61) (42) (29)
FIG. 1. Autoradiograms of the electrophoretically separated ‘%-labeled polypeptides in the total cytoplasmic (A) and soluble (B) extracts of BTV-infected cells. Monolayers of 10’ BHK cells were incubated with 5 ml AAfree Eagle’s medium containing 1 @/ml ‘%-protein hydrolysate and pulse-labeled for the following 2-hr periods after infection at 31”: 2-4 hr (B), 4-6 hr (C), 6-8 hr (D), 11-13 hr (E), 16-18 hr (F), 20-22 hr (G), 24-26 hr (H), 34-36 hr (I), mock-infected culture (A). Purified BTV (J) was used as a control and molecular weight marker. The molecular weights of the BTV capsid polypeptides in thousands are indicated in brackets and are as determined by Verwoerd et al. (1972). Cytoplasmic extracts were prepared by treatment of the cells, suspended in STM buffer at a concentration of 5 x 10’ cells/ml, with Nonidet P40 to a final concentration of 0.5%. Nuclei were removed by low-speed centrifugation and the SlOO extracts prepared by centrifugation of the cytoplasmic extracts for 2 hr at 45,000 rpm in an SW 50.1 rotor. Lyophilized samples of the cytoplasmic and SlOO extracts were electrophoresed on SDS and urea-containing polyacrylamide slab gels, as described in Materials and Methods.
virus polypeptides are synthesized. Concentration of the viral polypeptides by means of BTV-specific immune precipitation should therefore provide a much better result.
Immune Precipitation of Soluble BTV Proteins at Different Intervals after Infection Antibody against both purified and unpurified BTV was prepared as described.
Initially, different animals such as sheep, guinea pigs, and rabbits were compared in their ability to produce antibodies against BTV. Rabbits were found to be the most satisfactory and were subsequently used for making antisera. Infected cells were labeled for different 2-l-n intervals after infection. Immune precipitation with the soluble protein extracts of these cells were carried out with antisera against both purified BTV and BTV-in-
BTV-INFECTED
CELL
PROTEIN
fected cell extracts. Immune precipitates were analyzed by electrophoresis and autoradiography. The results are shown in Fig. 2, A and B. The largest number of different polypeptides were precipitated by serum against BTV-infected cell extracts (Fig. 2B). As was expected, some precipitation of cellular proteins is, however, also observed, especially in those fractions labeled early in the infection cycle. This contamination is not seen when serum against purified BTV is used (Fig. 2A). The main polypeptides precipitated by BTV antiserum are the four major BTV capsid polypeptides P2, P3, P5, and P7 as well as one polypeptide, P6A, not found in the protein coat of the virus. Precipitation of P6A is very effective if serum against unpurified BTV is used but only trace amounts are precipitated by antiserum against purified BTV. Several experiments were carried out to determine if precipitation of P6A was virus-specific. It was found that P6A was only precipitated from SlOO extracts of BTV-infected cells and not from uninfected cells or cells infected with other viruses like reovirus or epizootic hemorrhagic disease virus. An experiment was also carried out to determine if the precipitation of P6A could not be due to unspecific co-precipitation with the BTV polypeptides. An unlabeled soluble protein extract from BTV-infected cells, harvested 18 hr p.i., was mixed with an equal volume of a labeled soluble protein extract of uninfected cells. When the immune precipitate of this mixture with BTV antiserum was analyzed by electrophoresis the precipitation of the unlabeled virus polypeptides, including P6A, could be visualized by staining. The autoradiogram, however, indicated that there was no label associated with the band in the position of P6A. Lastly, polypeptide P6A is not precipitated from SlOO fractions of virus-infected cells by antiserum made against uninfected cell extracts. It would, therefore, seem that the ~O,CM)Odalton polypeptide P6A, which was first detected in cytoplasmic and soluble extracts of infected cells (Fig. 1, A and B), is indeed a virus-specified, nonstructural protein.
SYNTHESIS
389
As can be seen in Fig. 2, A and B, there is very little if any immune precipitation of P6. A few of the antibody preparations against purified BTV seemed to contain, for no clearly defined reason, a much higher level of antibody against P6. One of these sera was used in a later experiment described in Fig. 4.
Turnover of Polypeptides The possibility that any one of the BTV polypeptides is a precursor or turnover product of another protein was investigated by a pulse-chase experiment. Three infected BHK monolayers were pulse-labeled 16 l-n p.i. at 31” with 2 &i/ml 14C-protein hydrolysate for 1 hr. One of these cultures was harvested immediately, whereas the isotope-containing medium in the others was replaced by Eagle’s medium with 4~ the normal concentration of unlabeled amino acids. The cultures were incubated at 37” for a 3- and 6-hr period, respectively, before harvesting. The cytoplasmic and soluble fractions were analyzed by electrophoresis and autoradiography. Autoradiograms were scanned with a densitometer and peak areas quantitated by integration. The scans are shown in Fig. 3. The result of the 6-hr chase was indistinguishable from that of the 3-hr chase and is not shown. The chase has little effect on the relative amount of the labeled polypeptides in the cytoplasmic extract but it does affect the soluble polypeptides ratio. Polypeptide P3 has almost disappeared from the soluble protein fraction after the chase. The peak areas in the densitometer tracings of Fig. 3 were used to calculate the relative amount of the polypeptides in the SlOO and cytoplasmic extracts before and after the chase. The results are shown in Table 1. In the SlOO fraction, there is at least a lo-fold reduction in the relative amount of P3 and a much smaller reduction in the amount of P7. The relative amount of the others remain more or less the same as do the polypeptides in the total cytoplasmic extract. The soluble protein extracts in the pulsechase experiment were further analyzed by immune precipitation. The results are illustrated in Fig. 4. Also shown are the immune
390
HENDRIK
HUISMANS
mP6 -P6A -P7
FIG. 2. Autoradiograms of the W-labeled polypeptides in immune precipitates of SlOO fractions of cells labeled during the following 2-hr intervals pi.: 2-4 hr (B), 4-6 hr (C), 8-10 hr (D), 12-14 hr (E), 16-18 br (F), 20-22 hr (G), 26-28 hr (H), and mock-infected cells (A). Labeled purified BTV control (I). The different soluble extracts were prepared as described in the legend to Fig. 1 and 0.2 ml of each extract was precipitated with an equal volume antisera against both purified BTV (A) and cytoplasmic extracts of BTV-infected cells (B). Immune precipitates were left at 4O for 16 hr, collected by centrifugation, and were electrophoresed and prepared for autoradiography as described in Materials and Methods.
precipitates obtained from SlOO extracts of infected cells labeled 16 hr p.i. for a 20-min and a 4-hr period, respectively. The result confirms the large reduction in the relative amount of polypeptide P3 in the soluble fraction after a chase. This reduction was already evident 1 hr after the labeling period (results not shown). It is also indicated that the ratio of labeled polypeptides in the soluble protein extracts is not significantly affected by an increase in the length of the pulse period from 20 min to 4 hr, provided a sufficient supply of labeled precursor amino acids is available. In the experiment in Fig. 4, a serum was used that contained a high level of antibody against P6. The purpose of this was to
investigate the earlier-mentioned possibility that the nonstructural polypeptide P6A could be a modified form of one of the capsid polypeptides such as P6. However, neither the variation in pulse length or the cold amino acid chase seemed to have a significant effect on the P6/P6A ratio in the soluble protein fraction. There is therefore no evidence that there is a turnover of soluble P6 to P6A or vice versa. The same applies to the other soluble proteins. Accumulation of P5A in the Particulate Protein Fraction Polypeptide P5A is removed from the soluble protein fraction almost instantaneously. This was indicated by labeling of infected cells for a brief 20min period.
BTV-INFECTED
CELL
There was no labeled P5A in the soluble protein extract but, in the cytoplasmic extract, it was present in the largest relative amount (result not shown). This raises the question of what happened to the polypeptide after it had been synthesized. A comparison of the particulate fraction of infected cells labeled for a 20-min, 1 hr-, and 4-hr. period after 16 hr p.i. at 31’ is shown in Fig. 5. PI
w
I
Ps
p5.A Pe P6A
P7
.:
A
L
A
B
ch
JJAA
PROTEIN
391
SYNTHESIS
After a 2Omin labeling period, the particulate fraction contained predominantly P5A and small amounts of the labeled virus capsid polypeptides. After 4 hr, P5A was still in excess but the proportion of capsid polypeptides had increased significantly. The assembly of capsid polypeptides into virus or subvirus particles is obviously a slower process than incorporation of P5A into a high-molecular-weight complex. To investigate the nature of this complex, the particulate fractions obtained after a lhr labeling period were analyzed by centrifugation on sucrose gradients. Also analyzed was the particulate fraction of a similarly labeled mock-infected cell culture. Labeled, purified reovirus and bluetongue virus with respective values of 630 and 550 S (Martin and Zweerink, 1972) were centrifuged under the same conditions. The results are shown in Fig. 6. These indicate that a large amount of labeled material sediments in a heterogenous peak in the region 300-500 S with an average of just less than 400 S. The main isotope-containing fractions in Fig. 7 (fractions 10 to 16) were pooled and TABLE
0
w FIG. 3. Densitometer tracing of an autoradiogram of the ‘%-labeled polypeptides in the cytoplasmic and SlOO extracts of BTV-infected cells obtained in a pulse-chase experiment. Two BHK monolayer cultures were infected with BTV and pulsed for a I-hr period with 2 &X/ml “C-protein hydrolysate, 16 hr p.i. at 31”. One culture was harvested immediately while the isotope medium in the other one was replaced with Eagle’s medium containing 4~ the normal concentration of unlabeled amino acids and the cells were incubated for 3 hr at 37’. Cytoplasmic and SlOO fractions were prepared from both cultures as described in Fig. 1 and were analyzed by electrophoresis. Cytoplasmic extracts before the chase (A) and after the chase (B). SlOO extracts before the chase (C) and after the chase (D).
1
RELATIVE AMOUNT OF LABELED BTV POLYPEPTIDES IN THE SOLUBLE AND TOTAL CYTOPLASMIC EXTRACTS BEFORE AND AFTER 3-HR COLD AMINO ACID CHASE OF INFECTED CELLS LABELED FOR A PERIOD OF 1 HR, STARTING 16 HR POSTINFECTION AT 31” PolyRelative amount, Relative amount, peptide SlOO” cytoplasmic”
Pl P2 P3 P4 P5 P5A P6 P6A P7
Before chase
After chase
Before chase
After chase
3.7 12.5 11.3 9.8 17.0 14.0 20.4 11.3
3.7 16.8 1.2 12.1 20.1
2.9 9.3 8.6 8.6 12.9 26.1 6.3 17.3 8.0
3.3 11.4 7.5 8.8 12.0 25.0 6.3 18.4 7.3
16.3 22.6 7.0
n Relative amounts were calculated as foIIows. The densitometer tracings of the results shown in Fig. 3, A, B, C, and D, were used to calculate the peak areas representative of the different polypeptides. The integration values, obtained from an integrator coupled to the densitometer, were normalized to a percentage of the total.
392
HENDRIK
-PI -P2 -P3 ‘P4 ,EA -P6 -P6A -P7 FIG. 4. An autoradiogram of the electrophoretitally separated polypeptides in immune precipitates of SlOO fractions of infected cells labeled as described in Fig. 3 for respective periods of 20 min (A), 1 hr (B), and 4 hr (C). Also shown are the immune precipitates of two SlO6 fractions obtained from BTV-infected cells labeled for a 1-hr period, and then chased with unlabeled amino acids for a 3-hr (D) and a 6-hr (El period as described in Fig. 3. SlO6 fractions were prepared as described in the legend to Fig. 1 and immune precipitations were carried out with antisera against purified BTV as described in Fig. 2. BTV control (F).
analyzed by gel electrophoresis. A picture of the stained gel is shown in Fig. 7. It shows a single polypeptide that electrophoreses in the position of the noncapsid polypeptide P5A. There are only trace amounts of other components. An autoradiogram prepared from the same gel confirmed that almost all of the label is associated with this polypeptide.
HUISMANS
this respect, BTV differs from reovirus in which at least one of the capsid polypeptides is derived by cleavage of a precursor (Zweerink and Joklik, 1970). Evidence for the virus-specific origin of polypeptides P5A and P6A is as follows. No synthesis of P5A is detected in control cells or very early in the infection cycle. After 12 hr pi., it is the polypeptide synthesized in the largest relative amount. The protein is also synthesized in BTV-infected mouse fibroblast L cells (H. Huismans, unpublished observation) but not in L cells or BHK cells infected with reovirus. However, in cells infected with viruses much more closely related to BTV such as African horse sickness virus and epizootic hemorrhagic disease virus, a noncapsid polypeptide of similar size is synthesized (Huismans and Els, 1979). The other polypeptide, P6A, co-electrophoreses with a major cellular protein. Evidence for virus specificity is therefore based on the result with immune serum. There remains some doubt whether P6A is a true nonstructural polypeptide or a mod-
A
B
C
‘1 -P2 -P3
DISCUSSION
A study was made of protein synthesis in BTV-infected cells. Synthesis of the first virus-specific polypeptides can be detected between 2 and 4 hr p.i. at 31” but the rate of synthesis is highest in the period 10 to 26 hr p.i. Synthesis declines after 32 hr p.i. During this infection period, at least nine different virus-specific polypeptides are synthesized. Two of these are noncapsid polypeptides, P5A and P6A, with respective molecular weights of 54,000 and 40,000. Pulse-chase experiments did not demonstrate a precursor-product relationship between any of these nine polypeptides. In
-P7 FIG. 5. Autoradiogram of the electrophoretic separation of the ‘%-labeled polypeptides in the particulate fractions of BTV-infected cells labeled with 2 $.X/ml “C-protein hydrolysate for respective periods of 20 min (A), 1 hr (B), and 4 hr (Cl starting 16 hr pi. at 31’. Particulate fractions were prepared and electrophoresed as described in Materials and Methods.
BTV-INFECTED
CELL
PROTEIN
SYNTHESIS
393
FRACTION NUMBER FIG. 6. Sucrose gradient sedimentation profiles of particulate fractions obtained from BTV-infected (0) and mock-infected (A) cell cultures. Cells were labeled for a 1-hr period as described in the legend to Fig. 5. Particulate fractions were prepared as described in Materials and Methods, layered onto preformed 10-3041, sucrose gradients in 0.15 M STE buffer, and centrifuged for 40 min at 34,600 rpm in a SW 50.1 rotor. Fractions were collected from the bottom of the tube and the radioactivity in 10 ~1 of each fraction determined. The position of 630 and 556 S markers are those of purified reovirus and BTV, respectively. Fractions lo-16 were pooled and centrifuged for 3 hr at 35,000 rpm.
A
B
-m -P2 -P3 ,
m
-P4 -P5 -P5A _ eP6
FIG. 7. Electrophoretic analysis of the polypeptides in the 400 S complex, isolated and concentrated as described in the legend to Fig. 6. After electrophoresis on a polyacrylamide slab gel, the gel was stained with Coomassie Brilliant Blue and photographed. Polypeptides in the complex (A) and in a BTV control (B) are shown.
ified (e.g., phosphorylated) form of one of the capsid polypeptides. It has been shown by Lamb and Choppin (1977) that phosphorylation can have a very significant effect on the migration of polypeptides in polyacrylamide gels. The only evidence of a possible relationship between P6A and one of the capsid polypeptides is that there are P6A antibodies in antiserum against purified BTV. Other explanations for this can be advanced, however, and the matter remains to be investigated. It is not known whether the BTV mRNA species are translated with equal frequency. This translation frequency is best calculated from densitometer tracings of the labeled virus polypeptides in the cytoplasmic extracts of infected cells in the late period of infection such as shown in Fig. 3. The actual calculation is as described in the legend to Table 2. The result depicted in Table 2 is the average of eight independent calculations using different cytoplasmic extracts. The molar ratio of corresponding mRNA species is as determined by Huismans and Verwoerd (1973) and the coding assignments are as proposed by Verwoerd et al. (1972) and Martin and Zweerink (1972) with polypeptides P5A and P6A assigned positions of best fit. In most cases, the ratio of translation to transcription frequency does not
394
HENDRIK
HUISMANS
TABLE 2 A COMPARISON OF THE MOLAR RATIO IN WHICH THE BTV POLYPEPTIDES AND THE BTV mRNA SPECIES ARE SYNTHESIZED IN INFECTED CELLS Molar ratio BTV polypeptides mRNA Relative molar amount ““;iFe;pMW X 10m3” Relative amount (?I#’ Relative molar (Wd amount’ molar ratio mRNA Pl P2 P3 P4 P5 P5A P6 P6A P7
140 110 101 82 61 54 42 40 29
2.9 f 10.5 f 8.7 f 8.5 f 12.3 f 25.0 k 6.3 k 18.0 f 7.0 *
0.4 1.5 1.3 0.8 1.4 2.5 0.7 1.5 0.9
1.1 5.2 4.7 5.6 10.9 25.2 a.2 24.4 14.6
3.8 5.3 4.2 8.0 8.1 18.2 12.8 16.5 12.0
0.29 0.98 1.12 0.70 1.34 1.38 0.64 1.47 1.22
a Molecular weight values of capsid polypeptides are from Verwoerd et al. (1972). ‘Calculated as described in Table 1 from densitometer tracings of the ?labeled polypeptides in the cytoplssmic extract of BTV-infected cells such as shown in Fig. 3, A and B. The value shown is the mean of eight different calculations using different cytoplasmic extracts labeled for a I-hr period with 2 @i/ml 14Cprotein hydrolysate. Also shown is the calculated SD. ’ Relative amount divided by corresponding molecular weight and normalized to a percentage. d From H&mans and Verwoerd (1973).
the fact that it is the largest of the mRNA species and possibly translated less efficiently than the others. This result differs markedly from that obtained in reovirus-infected cells in which PolyRelative amount (%) of polypeptides at difcase more than a 30-fold difference between peptide ferent 2-hr intervals p.i. at 31”” the translation and the transcription ratios of some mRNA species have been reported E-10 12-14 16-18 20-22 26-28 (Zweerink and Joklik, 1970; Zweerink et al., 2.0 1.8 1.9 1.9 2.2 1.6 1971). 24.5 22.2 23.9 24.7 25.5 20.1 A question that remains is whether the a.2 7.0 a.2 9.2 9.2 12.5 ratio in which polypeptides are synthesized 4.4 4.1 4.8 3.7 3.3 2.0 changes during the course of infection. In 9.5 11.1 15.2 12.9 13.5 13.0 29.6 29.1 25.1 22.1 21.3 18.3 the early period of infection, this ratio can 24.6 20.9 25.4 25.0 32.5 only be calculated by immune precipitation. The result in Fig. 2 indicates that the a Relative amounts were calculated from densitomsame virus polypeptides are synthesized eter tracings of the results in Fig. 2B as described in the legend to Table 1. both early and late in the infection cycle. The relative amount also appears to remain deviate more than f0.4 from unity. This the same. This result was verified by quanwould indicate that these mRNA species tit&ion of the relative amounts from denare translated with much the same fre- sitometer tracings of the result in Fig. 2B. quency as that with which they are synthe- The results are shown in Table 3. There sized. The only exception is possibly Pl were only minor differences in relative which is translated with a lower frequency amounts and no significant changes during than that predicted from the relative abun- the course of infection. However, immune dance of mRNA species number 1. It is precipitation is not a satisfactory method doubtful, however, whether this is due to a for quantitating relative amounts, as was specific regulatory mechanism or merely to pointed out by Cross and Fields (1976); TABLE 3 RELATIVE AMOUNT OF LABELED POLYPEPTIDES IN THE IMMUNE PRECIPITATES OF SOLUBLE PROTEIN FRACTIONS OF BTV-INFECTED CELLS LABELED AT DIFFERENT INTERVALS POSTINFECTION AT 31”
BTV-INFECTED
CELL PROTEIN
therefore, one must evaluate these results with caution. The ratio in which the BTV polypeptides are synthesized is not identical to that in which they are found in the soluble fraction. Certain polypeptides are removed from the soluble fraction much more rapidly than others. For example, polypeptides P2 and P3 are synthesized at approximately the same rate (Table 2). They are also present in mature virions in almost the same ratio (Verwoerd et al., 1972). Nevertheless, pulse-chase experiments indicate that, whereas labeled P3 is rapidly removed from the soluble fraction, labeled P2 is chased into mature vii-ions very slowly. A much smaller, but reproducible, reduction in the relative amount of polypeptide P7 in the soluble pool was also observed (Table 1). This reduction in P3 and P7 could be related to the fact that both are major polypeptides of the virus core particle (Verwoerd et al., 1972). Whatever the explanation, it does appear as if the soluble protein pool size of P3 is much smaller than that of the other capsid polypeptides. Although polypeptide P5A is synthesized in a larger relative amount than any of the other virus polypeptides, only very small amounts (if any) are found in the soluble protein fraction. It is converted very rapidly into a high-molecular-weight complex of about 400 S. This complex contains P5A as probably the only protein component. The characteristics of this complex are the subject of a separate communication (Huismans and Els, 1979). It appears to be quite different from the noncapsid polypeptide complex described in reovirus-infected cells (Huismans and Joklik, 1976) and is composed of hollow tubular structures of indeterminate length. ACKNOWLEDGMENTS I would like to thank Mr. Peter Carter for excellent technical assistance, Mr. P. A. M. Wege for the provision of cell cultures and the Department of Agricultural Technical Services for the facilities provided. REFERENCES ANDERSON,M., CAWSTQN,T., and CHESEMAN,G. C. (1974). Molecular weight estimates of milk-fat-glob-
SYNTHESIS
395
ule membrane protein-sodium sulphate complexes by electrophoresis in gradient acrylamide gels. Biothem. J. 139,653-669. CROSS, R. K., and FIELDS, B. N. (1976). Reovirusspecific polypeptides: Analysis using discontinuous gel electrophoresis. J. Viral. 19, 162-173. HOWELL,P. G., VERWOERD,D. W., and OELLERMANN, R. A. (1967). Plaque formation by bluetongue virus. Onderstepoort J. Vet. Res. 34, 317-332. HUISMANS, H., and ELS, H. J. (1979). Characterization of the tubules associated with the replication of three different orbiviruses. Virology, 92, 397-406. HUISMANS, H., and JOKLIR, W. K. (1976). Reoviruscoded polypeptides in infected cells: Isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively. Virology 70, 411-424. HUISMANS, H., and VERWOERD, D. W. (1973). Control of transcription during the expression of the bluetongue virus genome. Virology 52,81-88. JOKLIK, W. K. (1974). Reproduction of Reoviridae. In “Comprehensive Virology 2” (H. Fraenkel-Conrat and R. R. Wagner, eds.), pp. 231-334. Plenum Press, New York. LAMB, R. A., and CHOPPIN, P. W. (1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology 81, 382-397. MARTIN, S. A., and ZWEERINK, H. J. (1972). Isolation and characterization of two types of bluetongue virus particles. Virology 60,495~506. MORGAN, E. M., and ZWEERINK, H. J. (1974). Reovirus morphogenesis. Corelike particles in cells infected at 39” with wild-type reovirus and temperature-sensitive mutants of groups B and G. Virology 59, 556-569. MORGAN, E. M., and ZWEERINK, H. J. (1975). Characterization of transcriptase and replicase particles isolated from reovirus-infected cells. Virology 68, 455-466. PALMER, E. L., MARTIN, M. L., and MURPHY, F. A.
(1977). Morphology and stability of infantile gastroenteritis virus: Comparison with reovirus and bluetongue virus. J. Gen. Virol. 35,403-414. SHIPHAM, S. O., and DE LA REY, M. (1976). The isolation and preliminary genetic classification of temperature-sensitive mutants of bluetongue virus. Onderstepoort J. Vet. Res. 43, 189-192. STONE, K. R., SMITH, R. E., and JOKLIK, W. K. (1974). Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses. Virology 58, 86-109. VERWOERD,D. W. (1969). Purification and characterization of bluetongue virus. Virology 38, 203-212. VERWOERD,D. W., Louw, H., and OELLERMANN, R. A. (1970). Characterization of bluetongue virus ribonucleic acid. J. Virol5, 1-7.
396
HENDRIK
VERWOERD, D. W., Em, H. J., DE VILLIERS, E. M., and HUISMANS, H. (1972). Structure of the bluetongue virus capsid. J. Virol. 10, 733-794. VERWOEF~D,D. W., OELLERMANN, R. A., BROEKMAN, J., and WEISS, K. E. (1967).The serological relation-
ship of South African bovine enterovirus strains (Echo SA-I and II) and the growth characteristics iu ceU culture of the prototype strain (Echo SA-I). Onderstepoort J. Vet. Res. 34,41-52.
HUISMANS ZWEERINK, H. J., and JOKLIK, W. K. (1970). Studies
on the intracellular synthesis of reovirus-specified proteius. Virology 41,501-518. ZWEERINK, H. J., ITO, Y., and MATSUHISA, T. (1972).
Synthesis of reovirus double-stranded RNA within viriouhke particles. Virology 50,349-358. ZWEERINK, H. J., MCDOWELL, M. J. and JOKLIK, W.
K. (1971). EsaentiaI and nonessential noncapsid reovirus proteins. Virology 45,71fI-723.
92, 385-396
(1979)
Protein Synthesis
in Bluetongue
HENDRIK
Virus-Infected
Cells
HUISMANS
Biochemistry Section, Veterinary Research Institute, Onderstepoort, South Africa Accepted August 22, 1978 The first virus-specific proteins in bluetongue virus (BTV)-infected cells can be demonThere is a rapid strated 2 to 4 hr after infection (p.i.) at 31” by immune precipitation. increase in the rate of synthesis until 11 hr p.i. after which synthesis remains at about the same level until 26 hr p.i. Apart from the seven BTV capsid polypeptides, two noncapsid polypeptides P5A and P6A, with respective molecular weights of 54,600 and 40,OOOdaltons, were identified in the infected cells. Polypeptide P5A is synthesized at a higher rate than any of the others. It is the main if not only component of a complex with an S value slightly less than 400. There is little evidence for specific regulation of the translation process in BTV-infected cells. With the possible exception of polypeptide Pl, all the others are translated with a relative frequency that is not significantly different from that of the corresponding mRNA species. Pulse-chase experiments indicate that the BTV polypeptides do not accumulate in the soluble fraction in the same ratio in which they are synthesized. Polypeptide P3 is removed from the soluble protein pool much more rapidly than any of the other capsid polypeptides. These pulse-chase experiments provided no evidence for a precursor-product relationship between any of the BTV polypeptides. The possibility that one of the noncapsid polypeptides, P6A, is a modified form of one of the capsid polypeptides can, however, not be excluded. INTRODUCTION
Bluetongue virus (BTV) is an orbivirus and a member of the family Reoviridae (Joklik, 1974). Although it is the best studied orbivirus and regarded as the prototype for this subgroup, little is known about the biochemistry of the virus apart from the characterization of the components of the purified virus itself. BTV comprises 10 double-stranded RNA segments (Verwoerd et al., 1970) surrounded by a protein coat containing seven different polypeptides, two of which form a diffuse protein layer which surrounds a smaller core particle (Martin and Zweerink, 1972; Verwoerd et al., 1972). This outer capsid layer differs morphologically from that of reovirus and rotavirus (Palmer et al., 1977) and is one of the main distinguishing characteristics of the orbiviruses. Protein synthesis in BTV-infected cells has never been studied and little is known about the actual replication process. The only aspect of replication that has been 385
studied is mRNA synthesis (Huismans and Verwoerd, 1973). The replication of reovirus, on the other hand, has been well characterized. Different noncapsid proteins have been identified (Zweerink, et al., 1971) and several studies have been made of subviral particles and their role in the replication process (Zweerink et al., 1972;Morgan and Zweerink, 1974; Morgan and Zweerink, 1975). None of this information is available for orbiviruses although they form a much larger group of viruses than the reoviruses and are clinically far more important. It is therefore warranted to study at least one of the orbiviruses in greater detail and determine in what respect if differs from reovirus. In this paper, experiments are described which were designed to study protein synthesis in BTV-infected cells, especially the synthesis of BTV noncapsid proteins, the kinetics of protein synthesis, and the relative proportion in which polypeptides are synthesized.
0042~6822/‘79/020385-12$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form resewed.
386
HENDRIK
Two new BTV proteins were identified, one of which accumulates in the insoluble protein fraction of infected cells in the form of a protein complex of approximately 400 s. MATERIALS
AND METHODS
Cells. BHK cells and mouse fibroblast L cells were obtained from the American Type Culture Collection. The cells were grown on Roux flasks or roller bottles in modified Eagle’s medium as described by Verwoerd et al. (1967). The medium was supplemented with 5% bovine serum. Virus. Methods for growing BTV type 1OAin BHK cells have been described (Verwoerd et al., 1967). Virus was titered by a plaque method on L cells (Howell, et al., 1967). A BTV stock was prepared as follows. A single plaque was selected from an L cell Petri dish as described by Shipham and de la Rey (1976) and used for inoculation of a monolayer of 2 x lo6 BHK cells. When the fuIl cytopathic effect was noticed, the virus was titered and this material used to infect Roux flasks with BHK cells at an input multiplicity of less than 0.1 PFU/cell. Virus produced by these cells was used as inoculum. It was stored at 4“ and used in an unpurified form because purified virus is very unstable. BHK cells infected with this inoculum at a multiplicity of between 0.1 and 40 PFU/cell gave a yield of at least 1000 PFU/cell. Virus purification. Virus was purified by a modification of the Freon extraction method described by Verwoerd (1969). It was found that after the first extraction, at least four more successive buffer extractions of the Freon phase were necessary to quantitatively dissociate virus from the cellular material to which it is very tightly bound. Two more Freon extractions were carried out on the combined water phases, and the virus was pelleted by centrifugation for 2 hr at 24,000 rpm in an SW 27 rotor through a 5-ml cushion of 40% sucrose in 0.002 M Tris, pH 8.8. The virus was then banded on sucrose gradients as described by Verwoerd (1969) and was considered purified when no more than the seven BTV polypeptides could be detected on electro-
HUISMANS
phoresis of a virus sample. Infection and Labeling of Cells. In labeling experiments, cells were infected with a high multiplicity of infection (40 PFU/celI) in order to obtain a high degree of synchronization of infection. Virus was allowed to adsorb to monolayer cells from a volume of 5 ml inoculum/108 cells. After 15 min at room temperature, the volume was increased to 60 ml by addition of Eagle’s medium and the cells were incubated at 31”. At the indicated times after infection, the cells were rinsed with amino acid (AA)-free Eagle’s medium and then incubated with 5 ml AA-free medium containing 1 or 2 #X/ml “C-protein hydrolysate (Radiochemical Centre, Amersham, England). Labeling was carried out at 37” and a shaking platform was used to ensure even distribution of the isotope-containing medium. At the end of a labeling period, cells were scraped from the glass and collected by centrifugation for 10 min at 1,500 rpm. Preparation of sub-cellular fractions. 1. Cytoplasmic extract: Cells were suspended in cold STM buffer (0.15 M NaCl, 0.005 M MgCh, 0.01 M Tris, pH 7.4) at a concentration of 5 X lo7 cells/ml. Nonidet P40 was added to a final concentration of 0.5% and the cells were incubated in an ice bath for 5 min. Nuclei were pelleted by centrifugation for 5 min at 3,000 rpm, resuspended with a syringe in one-half the original volume STM, and pelleted again. The two supernatants were combined and are referred to as the cytoplasmic extract. Twenty-microliter aliquots of the extract were lyophilized and stored at -20” until used for electrophoresis. 2. Soluble (SlOO) fraction: A suitable portion of the cytoplasmic extract was centrifuged for 2 hr at 45,000 rpm in an SW 50 rotor through 2 ml of a 40% sucrose cushion in 0.15 M STE buffer (0.15 M NaCl, 0.601 M EDTA, 0.001 M Tris, pH 7.4). The supernatant (SlOO fraction) was removed with a syringe. Extreme care was taken to avoid contamination of the fraction with the material in the interphase. Samples for electrophoresis (40 ~1) were lyophilized and stored at -20” before use. Samples for immune precipitations (0.2 or 0.3 ml) were
BTV-INFECTED
CELL
routinely used as soon as possible. They were never left at 4’ for longer than 16 hr because insoluble precipitates form on prolonged standing. 3. Particulate fraction: The interphase and sucrose cushion from the preparation of the SlOO fraction were discarded. The pellet was resuspended in 0.01 A4 STE in one-fifth of the original volume of cytoplasmic extract. Fractions of 15 1.11 were lyophilized and stored at -20”. Gel electrophoresis. Gel electrophoresis of proteins was carried out on slab gels as described by Stone et al. (1974). Lyophilized samples for electrophoresis were resuspended in 20 d of a solution consisting of 2 ml of 10 M urea, 0.4 ml 10% SDS, and 0.1 ml mercaptoethanol. To each sample, 5 ~1 of a 4 M NaCl solution was added. The high salt concentration prevented “narrowing” of the bands. In most cases, samples were electrophoresed for 18 hr at 40 V and 50 mA/gel. Gels were stained with Coomassie Brilliant Blue G-250 solution as described (Anderson et al., 1974). Gels were destained in 4% acetic acid at 50” and dried on filter paper under vacuum utilizing a heated gel-drying apparatus. Autoradiography. Cronex No. 4 medical X-ray film was placed on top of the dried gel in a X-ray film holder. Films were developed after the required period of exposure and used for making contact prints or scanned with a Vitatron densitometer. Peak areas were quantitated by an integrator coupled to the same instrument. Preparation of BTV antibodies. Antibodies against purified BTV were prepared by subcutaneous injection into a rabbit of 500 pg of purified virus mixed with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, Mich.). Two booster shots of purified virus only were given at l-week intervals after the first injection. Antibodies against the cytoplasmic extract of BTV-infected BHK cells were made as follows. Cells from eight infected BHK Roux flasks were harvested 20 hr after infection at 31”. A cytoplasmic extract was prepared as described above and one-third of this amount was injected subcutaneously into a rabbit together with an equal volume
PROTEIN
387
SYNTHESIS
of complete Freund’s adjuvant. Two booster shots, with no adjuvant, were given at I-week intervals after the first injection. Antibodies against the cytoplasm of uninfected control cells were made in exactly the same way. Immune precipitation. A volume of 0.2 or 0.3 ml of a SlOO fraction was mixed with 0.2 ml antiserum. This normally represented an excess of antibody. The mixture was left at 4’ for at least 16 hr, diluted to 2 ml with cold STM buffer, and the precipitate was collected by centrifugation at 4,200 rpm for 15 min. Precipitates were washed once with cold STM and were electrophoresed exactly as described for the lyophilized protein samples. RESULTS
Synthesis of BTV Proteins at Different Time Intervals after Infection BTV-infected BHK monolayer cells were pulse-labeled for different 2-hr intervals in the period 2-36 hr p.i. at 31”. Cells were harvested at the end of the labeling period. Cytoplasmic and SlOO protein fractions were analyzed by electrophoresis and autoradiography. The results are shown in Fig. 1, A and B. Until 8 hr p.i., the synthesis of viral polypeptides is almost entirely obscured by that of the host cell. After 11 hr p.i., however, synthesis becomes predominantly virus-specified and in this period all seven BTV capsid polypeptides can be identified in the cytoplasmic extracts. In addition, there is at least one noncapsid polypeptide which is synthesized in very large amounts in infected cells but not in control cells. This polypeptide is called P5A and has a molecular weight of about 54,000 daltons. Most of the virus polypeptides in the cytoplasmic extracts are also present in soluble form. The exception is P5A which is seen only in cytoplasmic extracts. Due to the fact that host cell protein synthesis is not completely suppressed, even late in the infection cycle, it is very difficult to decide whether a polypeptide like P6A, which is seen in both cytoplasmic and soluble extracts, is virus specified. It is also impossible to determine when the first
333
HENDRIK
HUISMANS
(140) (110) ml) (821 (61) (42) (29)
FIG. 1. Autoradiograms of the electrophoretically separated ‘%-labeled polypeptides in the total cytoplasmic (A) and soluble (B) extracts of BTV-infected cells. Monolayers of 10’ BHK cells were incubated with 5 ml AAfree Eagle’s medium containing 1 @/ml ‘%-protein hydrolysate and pulse-labeled for the following 2-hr periods after infection at 31”: 2-4 hr (B), 4-6 hr (C), 6-8 hr (D), 11-13 hr (E), 16-18 hr (F), 20-22 hr (G), 24-26 hr (H), 34-36 hr (I), mock-infected culture (A). Purified BTV (J) was used as a control and molecular weight marker. The molecular weights of the BTV capsid polypeptides in thousands are indicated in brackets and are as determined by Verwoerd et al. (1972). Cytoplasmic extracts were prepared by treatment of the cells, suspended in STM buffer at a concentration of 5 x 10’ cells/ml, with Nonidet P40 to a final concentration of 0.5%. Nuclei were removed by low-speed centrifugation and the SlOO extracts prepared by centrifugation of the cytoplasmic extracts for 2 hr at 45,000 rpm in an SW 50.1 rotor. Lyophilized samples of the cytoplasmic and SlOO extracts were electrophoresed on SDS and urea-containing polyacrylamide slab gels, as described in Materials and Methods.
virus polypeptides are synthesized. Concentration of the viral polypeptides by means of BTV-specific immune precipitation should therefore provide a much better result.
Immune Precipitation of Soluble BTV Proteins at Different Intervals after Infection Antibody against both purified and unpurified BTV was prepared as described.
Initially, different animals such as sheep, guinea pigs, and rabbits were compared in their ability to produce antibodies against BTV. Rabbits were found to be the most satisfactory and were subsequently used for making antisera. Infected cells were labeled for different 2-l-n intervals after infection. Immune precipitation with the soluble protein extracts of these cells were carried out with antisera against both purified BTV and BTV-in-
BTV-INFECTED
CELL
PROTEIN
fected cell extracts. Immune precipitates were analyzed by electrophoresis and autoradiography. The results are shown in Fig. 2, A and B. The largest number of different polypeptides were precipitated by serum against BTV-infected cell extracts (Fig. 2B). As was expected, some precipitation of cellular proteins is, however, also observed, especially in those fractions labeled early in the infection cycle. This contamination is not seen when serum against purified BTV is used (Fig. 2A). The main polypeptides precipitated by BTV antiserum are the four major BTV capsid polypeptides P2, P3, P5, and P7 as well as one polypeptide, P6A, not found in the protein coat of the virus. Precipitation of P6A is very effective if serum against unpurified BTV is used but only trace amounts are precipitated by antiserum against purified BTV. Several experiments were carried out to determine if precipitation of P6A was virus-specific. It was found that P6A was only precipitated from SlOO extracts of BTV-infected cells and not from uninfected cells or cells infected with other viruses like reovirus or epizootic hemorrhagic disease virus. An experiment was also carried out to determine if the precipitation of P6A could not be due to unspecific co-precipitation with the BTV polypeptides. An unlabeled soluble protein extract from BTV-infected cells, harvested 18 hr p.i., was mixed with an equal volume of a labeled soluble protein extract of uninfected cells. When the immune precipitate of this mixture with BTV antiserum was analyzed by electrophoresis the precipitation of the unlabeled virus polypeptides, including P6A, could be visualized by staining. The autoradiogram, however, indicated that there was no label associated with the band in the position of P6A. Lastly, polypeptide P6A is not precipitated from SlOO fractions of virus-infected cells by antiserum made against uninfected cell extracts. It would, therefore, seem that the ~O,CM)Odalton polypeptide P6A, which was first detected in cytoplasmic and soluble extracts of infected cells (Fig. 1, A and B), is indeed a virus-specified, nonstructural protein.
SYNTHESIS
389
As can be seen in Fig. 2, A and B, there is very little if any immune precipitation of P6. A few of the antibody preparations against purified BTV seemed to contain, for no clearly defined reason, a much higher level of antibody against P6. One of these sera was used in a later experiment described in Fig. 4.
Turnover of Polypeptides The possibility that any one of the BTV polypeptides is a precursor or turnover product of another protein was investigated by a pulse-chase experiment. Three infected BHK monolayers were pulse-labeled 16 l-n p.i. at 31” with 2 &i/ml 14C-protein hydrolysate for 1 hr. One of these cultures was harvested immediately, whereas the isotope-containing medium in the others was replaced by Eagle’s medium with 4~ the normal concentration of unlabeled amino acids. The cultures were incubated at 37” for a 3- and 6-hr period, respectively, before harvesting. The cytoplasmic and soluble fractions were analyzed by electrophoresis and autoradiography. Autoradiograms were scanned with a densitometer and peak areas quantitated by integration. The scans are shown in Fig. 3. The result of the 6-hr chase was indistinguishable from that of the 3-hr chase and is not shown. The chase has little effect on the relative amount of the labeled polypeptides in the cytoplasmic extract but it does affect the soluble polypeptides ratio. Polypeptide P3 has almost disappeared from the soluble protein fraction after the chase. The peak areas in the densitometer tracings of Fig. 3 were used to calculate the relative amount of the polypeptides in the SlOO and cytoplasmic extracts before and after the chase. The results are shown in Table 1. In the SlOO fraction, there is at least a lo-fold reduction in the relative amount of P3 and a much smaller reduction in the amount of P7. The relative amount of the others remain more or less the same as do the polypeptides in the total cytoplasmic extract. The soluble protein extracts in the pulsechase experiment were further analyzed by immune precipitation. The results are illustrated in Fig. 4. Also shown are the immune
390
HENDRIK
HUISMANS
mP6 -P6A -P7
FIG. 2. Autoradiograms of the W-labeled polypeptides in immune precipitates of SlOO fractions of cells labeled during the following 2-hr intervals pi.: 2-4 hr (B), 4-6 hr (C), 8-10 hr (D), 12-14 hr (E), 16-18 br (F), 20-22 hr (G), 26-28 hr (H), and mock-infected cells (A). Labeled purified BTV control (I). The different soluble extracts were prepared as described in the legend to Fig. 1 and 0.2 ml of each extract was precipitated with an equal volume antisera against both purified BTV (A) and cytoplasmic extracts of BTV-infected cells (B). Immune precipitates were left at 4O for 16 hr, collected by centrifugation, and were electrophoresed and prepared for autoradiography as described in Materials and Methods.
precipitates obtained from SlOO extracts of infected cells labeled 16 hr p.i. for a 20-min and a 4-hr period, respectively. The result confirms the large reduction in the relative amount of polypeptide P3 in the soluble fraction after a chase. This reduction was already evident 1 hr after the labeling period (results not shown). It is also indicated that the ratio of labeled polypeptides in the soluble protein extracts is not significantly affected by an increase in the length of the pulse period from 20 min to 4 hr, provided a sufficient supply of labeled precursor amino acids is available. In the experiment in Fig. 4, a serum was used that contained a high level of antibody against P6. The purpose of this was to
investigate the earlier-mentioned possibility that the nonstructural polypeptide P6A could be a modified form of one of the capsid polypeptides such as P6. However, neither the variation in pulse length or the cold amino acid chase seemed to have a significant effect on the P6/P6A ratio in the soluble protein fraction. There is therefore no evidence that there is a turnover of soluble P6 to P6A or vice versa. The same applies to the other soluble proteins. Accumulation of P5A in the Particulate Protein Fraction Polypeptide P5A is removed from the soluble protein fraction almost instantaneously. This was indicated by labeling of infected cells for a brief 20min period.
BTV-INFECTED
CELL
There was no labeled P5A in the soluble protein extract but, in the cytoplasmic extract, it was present in the largest relative amount (result not shown). This raises the question of what happened to the polypeptide after it had been synthesized. A comparison of the particulate fraction of infected cells labeled for a 20-min, 1 hr-, and 4-hr. period after 16 hr p.i. at 31’ is shown in Fig. 5. PI
w
I
Ps
p5.A Pe P6A
P7
.:
A
L
A
B
ch
JJAA
PROTEIN
391
SYNTHESIS
After a 2Omin labeling period, the particulate fraction contained predominantly P5A and small amounts of the labeled virus capsid polypeptides. After 4 hr, P5A was still in excess but the proportion of capsid polypeptides had increased significantly. The assembly of capsid polypeptides into virus or subvirus particles is obviously a slower process than incorporation of P5A into a high-molecular-weight complex. To investigate the nature of this complex, the particulate fractions obtained after a lhr labeling period were analyzed by centrifugation on sucrose gradients. Also analyzed was the particulate fraction of a similarly labeled mock-infected cell culture. Labeled, purified reovirus and bluetongue virus with respective values of 630 and 550 S (Martin and Zweerink, 1972) were centrifuged under the same conditions. The results are shown in Fig. 6. These indicate that a large amount of labeled material sediments in a heterogenous peak in the region 300-500 S with an average of just less than 400 S. The main isotope-containing fractions in Fig. 7 (fractions 10 to 16) were pooled and TABLE
0
w FIG. 3. Densitometer tracing of an autoradiogram of the ‘%-labeled polypeptides in the cytoplasmic and SlOO extracts of BTV-infected cells obtained in a pulse-chase experiment. Two BHK monolayer cultures were infected with BTV and pulsed for a I-hr period with 2 &X/ml “C-protein hydrolysate, 16 hr p.i. at 31”. One culture was harvested immediately while the isotope medium in the other one was replaced with Eagle’s medium containing 4~ the normal concentration of unlabeled amino acids and the cells were incubated for 3 hr at 37’. Cytoplasmic and SlOO fractions were prepared from both cultures as described in Fig. 1 and were analyzed by electrophoresis. Cytoplasmic extracts before the chase (A) and after the chase (B). SlOO extracts before the chase (C) and after the chase (D).
1
RELATIVE AMOUNT OF LABELED BTV POLYPEPTIDES IN THE SOLUBLE AND TOTAL CYTOPLASMIC EXTRACTS BEFORE AND AFTER 3-HR COLD AMINO ACID CHASE OF INFECTED CELLS LABELED FOR A PERIOD OF 1 HR, STARTING 16 HR POSTINFECTION AT 31” PolyRelative amount, Relative amount, peptide SlOO” cytoplasmic”
Pl P2 P3 P4 P5 P5A P6 P6A P7
Before chase
After chase
Before chase
After chase
3.7 12.5 11.3 9.8 17.0 14.0 20.4 11.3
3.7 16.8 1.2 12.1 20.1
2.9 9.3 8.6 8.6 12.9 26.1 6.3 17.3 8.0
3.3 11.4 7.5 8.8 12.0 25.0 6.3 18.4 7.3
16.3 22.6 7.0
n Relative amounts were calculated as foIIows. The densitometer tracings of the results shown in Fig. 3, A, B, C, and D, were used to calculate the peak areas representative of the different polypeptides. The integration values, obtained from an integrator coupled to the densitometer, were normalized to a percentage of the total.
392
HENDRIK
-PI -P2 -P3 ‘P4 ,EA -P6 -P6A -P7 FIG. 4. An autoradiogram of the electrophoretitally separated polypeptides in immune precipitates of SlOO fractions of infected cells labeled as described in Fig. 3 for respective periods of 20 min (A), 1 hr (B), and 4 hr (C). Also shown are the immune precipitates of two SlO6 fractions obtained from BTV-infected cells labeled for a 1-hr period, and then chased with unlabeled amino acids for a 3-hr (D) and a 6-hr (El period as described in Fig. 3. SlO6 fractions were prepared as described in the legend to Fig. 1 and immune precipitations were carried out with antisera against purified BTV as described in Fig. 2. BTV control (F).
analyzed by gel electrophoresis. A picture of the stained gel is shown in Fig. 7. It shows a single polypeptide that electrophoreses in the position of the noncapsid polypeptide P5A. There are only trace amounts of other components. An autoradiogram prepared from the same gel confirmed that almost all of the label is associated with this polypeptide.
HUISMANS
this respect, BTV differs from reovirus in which at least one of the capsid polypeptides is derived by cleavage of a precursor (Zweerink and Joklik, 1970). Evidence for the virus-specific origin of polypeptides P5A and P6A is as follows. No synthesis of P5A is detected in control cells or very early in the infection cycle. After 12 hr pi., it is the polypeptide synthesized in the largest relative amount. The protein is also synthesized in BTV-infected mouse fibroblast L cells (H. Huismans, unpublished observation) but not in L cells or BHK cells infected with reovirus. However, in cells infected with viruses much more closely related to BTV such as African horse sickness virus and epizootic hemorrhagic disease virus, a noncapsid polypeptide of similar size is synthesized (Huismans and Els, 1979). The other polypeptide, P6A, co-electrophoreses with a major cellular protein. Evidence for virus specificity is therefore based on the result with immune serum. There remains some doubt whether P6A is a true nonstructural polypeptide or a mod-
A
B
C
‘1 -P2 -P3
DISCUSSION
A study was made of protein synthesis in BTV-infected cells. Synthesis of the first virus-specific polypeptides can be detected between 2 and 4 hr p.i. at 31” but the rate of synthesis is highest in the period 10 to 26 hr p.i. Synthesis declines after 32 hr p.i. During this infection period, at least nine different virus-specific polypeptides are synthesized. Two of these are noncapsid polypeptides, P5A and P6A, with respective molecular weights of 54,000 and 40,000. Pulse-chase experiments did not demonstrate a precursor-product relationship between any of these nine polypeptides. In
-P7 FIG. 5. Autoradiogram of the electrophoretic separation of the ‘%-labeled polypeptides in the particulate fractions of BTV-infected cells labeled with 2 $.X/ml “C-protein hydrolysate for respective periods of 20 min (A), 1 hr (B), and 4 hr (Cl starting 16 hr pi. at 31’. Particulate fractions were prepared and electrophoresed as described in Materials and Methods.
BTV-INFECTED
CELL
PROTEIN
SYNTHESIS
393
FRACTION NUMBER FIG. 6. Sucrose gradient sedimentation profiles of particulate fractions obtained from BTV-infected (0) and mock-infected (A) cell cultures. Cells were labeled for a 1-hr period as described in the legend to Fig. 5. Particulate fractions were prepared as described in Materials and Methods, layered onto preformed 10-3041, sucrose gradients in 0.15 M STE buffer, and centrifuged for 40 min at 34,600 rpm in a SW 50.1 rotor. Fractions were collected from the bottom of the tube and the radioactivity in 10 ~1 of each fraction determined. The position of 630 and 556 S markers are those of purified reovirus and BTV, respectively. Fractions lo-16 were pooled and centrifuged for 3 hr at 35,000 rpm.
A
B
-m -P2 -P3 ,
m
-P4 -P5 -P5A _ eP6
FIG. 7. Electrophoretic analysis of the polypeptides in the 400 S complex, isolated and concentrated as described in the legend to Fig. 6. After electrophoresis on a polyacrylamide slab gel, the gel was stained with Coomassie Brilliant Blue and photographed. Polypeptides in the complex (A) and in a BTV control (B) are shown.
ified (e.g., phosphorylated) form of one of the capsid polypeptides. It has been shown by Lamb and Choppin (1977) that phosphorylation can have a very significant effect on the migration of polypeptides in polyacrylamide gels. The only evidence of a possible relationship between P6A and one of the capsid polypeptides is that there are P6A antibodies in antiserum against purified BTV. Other explanations for this can be advanced, however, and the matter remains to be investigated. It is not known whether the BTV mRNA species are translated with equal frequency. This translation frequency is best calculated from densitometer tracings of the labeled virus polypeptides in the cytoplasmic extracts of infected cells in the late period of infection such as shown in Fig. 3. The actual calculation is as described in the legend to Table 2. The result depicted in Table 2 is the average of eight independent calculations using different cytoplasmic extracts. The molar ratio of corresponding mRNA species is as determined by Huismans and Verwoerd (1973) and the coding assignments are as proposed by Verwoerd et al. (1972) and Martin and Zweerink (1972) with polypeptides P5A and P6A assigned positions of best fit. In most cases, the ratio of translation to transcription frequency does not
394
HENDRIK
HUISMANS
TABLE 2 A COMPARISON OF THE MOLAR RATIO IN WHICH THE BTV POLYPEPTIDES AND THE BTV mRNA SPECIES ARE SYNTHESIZED IN INFECTED CELLS Molar ratio BTV polypeptides mRNA Relative molar amount ““;iFe;pMW X 10m3” Relative amount (?I#’ Relative molar (Wd amount’ molar ratio mRNA Pl P2 P3 P4 P5 P5A P6 P6A P7
140 110 101 82 61 54 42 40 29
2.9 f 10.5 f 8.7 f 8.5 f 12.3 f 25.0 k 6.3 k 18.0 f 7.0 *
0.4 1.5 1.3 0.8 1.4 2.5 0.7 1.5 0.9
1.1 5.2 4.7 5.6 10.9 25.2 a.2 24.4 14.6
3.8 5.3 4.2 8.0 8.1 18.2 12.8 16.5 12.0
0.29 0.98 1.12 0.70 1.34 1.38 0.64 1.47 1.22
a Molecular weight values of capsid polypeptides are from Verwoerd et al. (1972). ‘Calculated as described in Table 1 from densitometer tracings of the ?labeled polypeptides in the cytoplssmic extract of BTV-infected cells such as shown in Fig. 3, A and B. The value shown is the mean of eight different calculations using different cytoplasmic extracts labeled for a I-hr period with 2 @i/ml 14Cprotein hydrolysate. Also shown is the calculated SD. ’ Relative amount divided by corresponding molecular weight and normalized to a percentage. d From H&mans and Verwoerd (1973).
the fact that it is the largest of the mRNA species and possibly translated less efficiently than the others. This result differs markedly from that obtained in reovirus-infected cells in which PolyRelative amount (%) of polypeptides at difcase more than a 30-fold difference between peptide ferent 2-hr intervals p.i. at 31”” the translation and the transcription ratios of some mRNA species have been reported E-10 12-14 16-18 20-22 26-28 (Zweerink and Joklik, 1970; Zweerink et al., 2.0 1.8 1.9 1.9 2.2 1.6 1971). 24.5 22.2 23.9 24.7 25.5 20.1 A question that remains is whether the a.2 7.0 a.2 9.2 9.2 12.5 ratio in which polypeptides are synthesized 4.4 4.1 4.8 3.7 3.3 2.0 changes during the course of infection. In 9.5 11.1 15.2 12.9 13.5 13.0 29.6 29.1 25.1 22.1 21.3 18.3 the early period of infection, this ratio can 24.6 20.9 25.4 25.0 32.5 only be calculated by immune precipitation. The result in Fig. 2 indicates that the a Relative amounts were calculated from densitomsame virus polypeptides are synthesized eter tracings of the results in Fig. 2B as described in the legend to Table 1. both early and late in the infection cycle. The relative amount also appears to remain deviate more than f0.4 from unity. This the same. This result was verified by quanwould indicate that these mRNA species tit&ion of the relative amounts from denare translated with much the same fre- sitometer tracings of the result in Fig. 2B. quency as that with which they are synthe- The results are shown in Table 3. There sized. The only exception is possibly Pl were only minor differences in relative which is translated with a lower frequency amounts and no significant changes during than that predicted from the relative abun- the course of infection. However, immune dance of mRNA species number 1. It is precipitation is not a satisfactory method doubtful, however, whether this is due to a for quantitating relative amounts, as was specific regulatory mechanism or merely to pointed out by Cross and Fields (1976); TABLE 3 RELATIVE AMOUNT OF LABELED POLYPEPTIDES IN THE IMMUNE PRECIPITATES OF SOLUBLE PROTEIN FRACTIONS OF BTV-INFECTED CELLS LABELED AT DIFFERENT INTERVALS POSTINFECTION AT 31”
BTV-INFECTED
CELL PROTEIN
therefore, one must evaluate these results with caution. The ratio in which the BTV polypeptides are synthesized is not identical to that in which they are found in the soluble fraction. Certain polypeptides are removed from the soluble fraction much more rapidly than others. For example, polypeptides P2 and P3 are synthesized at approximately the same rate (Table 2). They are also present in mature virions in almost the same ratio (Verwoerd et al., 1972). Nevertheless, pulse-chase experiments indicate that, whereas labeled P3 is rapidly removed from the soluble fraction, labeled P2 is chased into mature vii-ions very slowly. A much smaller, but reproducible, reduction in the relative amount of polypeptide P7 in the soluble pool was also observed (Table 1). This reduction in P3 and P7 could be related to the fact that both are major polypeptides of the virus core particle (Verwoerd et al., 1972). Whatever the explanation, it does appear as if the soluble protein pool size of P3 is much smaller than that of the other capsid polypeptides. Although polypeptide P5A is synthesized in a larger relative amount than any of the other virus polypeptides, only very small amounts (if any) are found in the soluble protein fraction. It is converted very rapidly into a high-molecular-weight complex of about 400 S. This complex contains P5A as probably the only protein component. The characteristics of this complex are the subject of a separate communication (Huismans and Els, 1979). It appears to be quite different from the noncapsid polypeptide complex described in reovirus-infected cells (Huismans and Joklik, 1976) and is composed of hollow tubular structures of indeterminate length. ACKNOWLEDGMENTS I would like to thank Mr. Peter Carter for excellent technical assistance, Mr. P. A. M. Wege for the provision of cell cultures and the Department of Agricultural Technical Services for the facilities provided. REFERENCES ANDERSON,M., CAWSTQN,T., and CHESEMAN,G. C. (1974). Molecular weight estimates of milk-fat-glob-
SYNTHESIS
395
ule membrane protein-sodium sulphate complexes by electrophoresis in gradient acrylamide gels. Biothem. J. 139,653-669. CROSS, R. K., and FIELDS, B. N. (1976). Reovirusspecific polypeptides: Analysis using discontinuous gel electrophoresis. J. Viral. 19, 162-173. HOWELL,P. G., VERWOERD,D. W., and OELLERMANN, R. A. (1967). Plaque formation by bluetongue virus. Onderstepoort J. Vet. Res. 34, 317-332. HUISMANS, H., and ELS, H. J. (1979). Characterization of the tubules associated with the replication of three different orbiviruses. Virology, 92, 397-406. HUISMANS, H., and JOKLIR, W. K. (1976). Reoviruscoded polypeptides in infected cells: Isolation of two native monomeric polypeptides with affinity for single-stranded and double-stranded RNA, respectively. Virology 70, 411-424. HUISMANS, H., and VERWOERD, D. W. (1973). Control of transcription during the expression of the bluetongue virus genome. Virology 52,81-88. JOKLIK, W. K. (1974). Reproduction of Reoviridae. In “Comprehensive Virology 2” (H. Fraenkel-Conrat and R. R. Wagner, eds.), pp. 231-334. Plenum Press, New York. LAMB, R. A., and CHOPPIN, P. W. (1977). The synthesis of Sendai virus polypeptides in infected cells. 111. Phosphorylation of polypeptides. Virology 81, 382-397. MARTIN, S. A., and ZWEERINK, H. J. (1972). Isolation and characterization of two types of bluetongue virus particles. Virology 60,495~506. MORGAN, E. M., and ZWEERINK, H. J. (1974). Reovirus morphogenesis. Corelike particles in cells infected at 39” with wild-type reovirus and temperature-sensitive mutants of groups B and G. Virology 59, 556-569. MORGAN, E. M., and ZWEERINK, H. J. (1975). Characterization of transcriptase and replicase particles isolated from reovirus-infected cells. Virology 68, 455-466. PALMER, E. L., MARTIN, M. L., and MURPHY, F. A.
(1977). Morphology and stability of infantile gastroenteritis virus: Comparison with reovirus and bluetongue virus. J. Gen. Virol. 35,403-414. SHIPHAM, S. O., and DE LA REY, M. (1976). The isolation and preliminary genetic classification of temperature-sensitive mutants of bluetongue virus. Onderstepoort J. Vet. Res. 43, 189-192. STONE, K. R., SMITH, R. E., and JOKLIK, W. K. (1974). Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses. Virology 58, 86-109. VERWOERD,D. W. (1969). Purification and characterization of bluetongue virus. Virology 38, 203-212. VERWOERD,D. W., Louw, H., and OELLERMANN, R. A. (1970). Characterization of bluetongue virus ribonucleic acid. J. Virol5, 1-7.
396
HENDRIK
VERWOERD, D. W., Em, H. J., DE VILLIERS, E. M., and HUISMANS, H. (1972). Structure of the bluetongue virus capsid. J. Virol. 10, 733-794. VERWOEF~D,D. W., OELLERMANN, R. A., BROEKMAN, J., and WEISS, K. E. (1967).The serological relation-
ship of South African bovine enterovirus strains (Echo SA-I and II) and the growth characteristics iu ceU culture of the prototype strain (Echo SA-I). Onderstepoort J. Vet. Res. 34,41-52.
HUISMANS ZWEERINK, H. J., and JOKLIK, W. K. (1970). Studies
on the intracellular synthesis of reovirus-specified proteius. Virology 41,501-518. ZWEERINK, H. J., ITO, Y., and MATSUHISA, T. (1972).
Synthesis of reovirus double-stranded RNA within viriouhke particles. Virology 50,349-358. ZWEERINK, H. J., MCDOWELL, M. J. and JOKLIK, W.
K. (1971). EsaentiaI and nonessential noncapsid reovirus proteins. Virology 45,71fI-723.