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Influence of T4 superinfection on the formation of RNA bacteriophage coat protein
J. Mol. Biol. (1970)47, 599-603
Influence of T4 Superinfection on the Formation RNA Bacteriophage Coat Protein
of
Synthesis of RNA-phage coat protein is strongly inhibited following superinfection with phage T4; a sharp reduction in coat production is observed within several minutes after superinfection. In simultaneous mixed infection of cells of Escherichia coli with RNA phage and T-even phage (virulent DNA phage), parental RNA remains intact and is found in polysomes, but no RNA polymerase or replicative intermediates are found; in delayed T4 superinfection, infectious RNA is synthesized, but no progeny phage or defective particles are produced, and coat-protein antigen synthesis is strongly inhibited. We interpreted these results to mean that T4 blocks the messenger function of the RNA phage chromosome by inhibiting translation (Hattman & Hofschneider, 1967, 1968). Our earlier experiments on coat-protein antigen formation used a serological technique, with serum prepared against purified phage. Since it was not clear if such a preparation is also active against free virion subunits or nascent coat protein on ribosomes, we were cautious about stating that T4 blocks synthesis of the coat protein molecule per se. (However, a recent report indicates that the immune precipitation procedure also measures coat protein not contained in phage (Kaerner, 1969).) For this reason, we have examined formation of the coat protein directly, and the results of the observations are reported here. The rationale of the method utilizes several properties of RNA phage coat protein : (1) it is of low molecular weight (cu. 14,000) (Konigsberg, Weber, Notani & Zinder, 1966); (2) it lacks histidine (Konigsberg et al., 1966; Weber, Notani, Wikler & Konigsberg, 1966) ; (3) its synthesis at late infection times constitutes about 20 to 80% of total protein synthesis (Hattman & Hofschneider, 1967; Watanabe, Watanabe & August, 1968; Sugiyama & Stone, 1968). Therefore, coat formation can readily be analyzed by double-labeling of cells late in infection with [14C]histidine and [3H]phenylalanine and measuring 3H/14C ratios of protein fractions separated according to size. Preliminary control experiments verified that separation of f2 coat from E. coli proteins could be achieved by gel filtration on Sephadex GlOO (Fig. 1). In uninfected cells and T4aminfected cells, no substantial changein [3H]phenylalanine/[14C]histidine incorporation was observed, in contrast to f2-infected cells. Labeled coat-protein isolated from purified f2 phage was run separately as a marker and observed at the same position as the peak 3H/14C. It should be pointed out that we were able repeatedly to use the same column and thus compare profiles from experiment to experiment. Gel filtration profiles obtained from control f2-infected cells are compared to superinfected cells in Figures 2 and 3. In these experiments, T&m82 was added 40 minutes after primary infection with f2, and a 15minute labeling period commenced five minutes after T&m addition. It is clear that superinfection with T4am inhibits f2-coat protein synthesis. It should be noted that incorporation of label was three39
699
600
STANLEY
I
4 (a)
4
I
I
I
HATTMAN
I
~ Uninfected cells
(b) T4 am 82-Infected
cells
rf
25
30
35
40
45
15 20 25
Fraction no.
30
3.5 40
Fraction no.
15 20
25
30 3.5
40
Fraction no. Fm. 1. Sephadex GlOO gel filtration patterns of proteins synthesized in uninfected cells (a), T4am82-infected cells (b) and ft-infected cells (c). E. co& strain K38 was grown to the appropriate optical density and the incorporation period begun. Labeling was with [3H]phenylalanine (0.5 PC/ml.) and [l%]histidine (0.1 pc/ml.). (a) at o.n.,,,=0.40, a IO-min incorporation period was started in the uninfected cells; (b) at period was 0.D .540=0.37, cells were infected with T4am82, and 5 min later a 20-min incorporation 0.26, cells were infected with f2; and 45 min later a l&mm incorporation started; (0) at o.D.~~~= period was started. At 60 min post-infection, the 0.D.540 had increased to 0.50. Incorporation was terminated by a 3-min chase with 100 pg cold amino wcids/ml., followed by pouring over crushed ice and centrifugation. Phenol extraction and dialysis were as described by Vifmela, Algranati t Ochoa (1967) except for the following modification: the final dialysis was with 0.01 M-potassium phosphate (pH 7.2) + 1 o/0 2-mercaptoethanol + 0.5% Sarkosyl. This buffer was used for the Sephadex gel filtration as well. Samples of 0.4 to O-6 ml. were applied to a GlOO Sephadex column (bed volume = 30 ml.); in (b) and (c), 2%drop fractions were collected (0.6 ml.), and in (a) 20.drop fractions were collected. The fractions were precipitated with cold trichloroaoetic acid and filtered onto Whatman GF/A glass fiber disks. Counting was in a Packard 3375 sointillation counter set for double-isotope counting; 8H overlap in 14C channel was O.O20/0, and 14C overlap in sH channel was 11% for unquenched standards. The raw data are plotted with no corrections for efficiency of overlap (on filters, the W overlap in 3H is actually higher than with the ideal standards). Recoveries through extraction and dialysis were 80 to 90% for both isotopes, and all counts applied to the column were recovered. The results depicted above were obtained in three independent experiments,
LETTERS
TO
THE
EDITOR
601
Fraction no. FIG. 2. Sephadex GlOO gel filtration patterns of proteins synthesized in f2-control infection and T4am82 superinfection. Cells were grown as described in Fig. 1 and infected with f2 at o.D.,,,=0*28. At 40 mm postinfection, T4am82 was added to one portion of the culture. 5 min later, the pulse was started for a 15-mm period and terminated; procedures were as described in Fig. 1. Recovery of 3H and l*C was 80% for the control and 90% for the superinfection. 0.4 ml. portions for the f2-control infection (a) and 0.5 ml. for the superinfection (b) were applied in separate column runs; 25-drop fractions were collected.
Fraction no. FIG. 3. Sephadex GlOO gel filtration patterns of proteins and T4arn82 superinfection. Conditions were similar to those described in Fig. 2, except of [3H]phenylalanine. Recovery was 90% for the control and of the f2-control infection (a) and 0.6 ml. of the superinfection runs; 25drop fractions were collected.
synthesized
in f2-oontrol
infection
that [3H]alsnine was used in place 100% for the superinfection. 0.4 ml. (b) were applied in separate column
to fivefold lower in the superinfected cultures compared to the control infections; this was observed previously with [35S]sulfate incorporation (Hattman & Hofschneider, 1967). This is probably due to both continued growth (increase of O.D.,,,) and higher rate of protein synthesis in the control infection. The nature of the proteins observed by the Sephadex assay was confirmed by polyaorylamide gel electrophoresis (data not shown). Briefly, in all cases the major peak of labeled proteins excluded on GIOO Sephadex was separated into many smaller peaks (the 3H/14C ratio remaining constant). The included labeled proteins yielded a large major peak; however, an elevated 3H/14C ratio was found only with the control infection, and this material co-electrophoresed with purified marker protein.
602
STANLEY
HATTMAN
In the preceding experiments, the incorporation of labeled amino acids was during a 15-minute period, corresponding to 5 to 15 minutes post T&m-superinfection. Infectious RNA production is known to proceed in delayed superinfections such as described here, although there is a depression in synthesis (Hattman & Hofschneider, 196’7; Yarosh & Levinthal, 1967). Therefore, it is not excluded that an apparent direct block in coat production might be attributed to reduced progeny RNA formation; it is known, in fact, that the level of coat production is influenced by the level of progeny RNA (Oeschger & Nathans, 1966). Therefore, an attempt was made to minimize such a possibility by confining the pulse period to only three minutes, and started at two minutes after T4am superinfection. The results of two such independent experiments are depicted in Figure 4. In these experiments, there was only a twofold reduction in total label incorporation into superinfected cultures compared to control infections. It can be seen that the elution profiles in the superinfected cuItures are different from those observed for longer pulse periods (Figs 2 and 3) ; I have not investigated this point further. Similar patterns were obtained with [3H]phenylalanine and [3H]alanine. The results clearly show the
Fraction
Fraction
no.
no,
Fm. 4. Sephadex Cl00 gel f?ltr&ion patterns of proteins synthesized in f2-control infection and T4am82 superinfeotion. Cells infected with f2 for 42 min were superinfected with T4am82.2 min later, a pulse was started for & 3-min period; incorporation was halted by a 2-min ohase with cold amino acids. In (a) and (b) labeling w&s with [3H]phenylalanine (2.5 pc/ml.) and [Wlhistidine (1.0 pc/ml.); in (c) and (d) labeling w&s with [*H]altmine (0.5 &ml) and [14C]histidine (0.1 &ml.).
LETTERS
TO
THE
EDITOR
603
near elimination of recognizable f2 coat production; in Bgure 4(c), there may have been a small amount of coat production. These results support the view that inhibition of f2 coat synthesis by T4am is mediated by direct translation inhibition, and that this process is expressed within the first few minutes after T4am superinfection. It should be pointed out that premature chain termination does not seem to be the mode of translation inhibition. If 20%-length coat molecules were produced, then an increased 3H/14C ratio would still have been observed. This follows from the location of the four phenylalanine residues (out of 129), which are at positions 4, 7, 25 and 95 with respect to the N-terminal amino acid. However, production of shorter or acid-soluble peptides is not ruled out. Recently, I have been examining in vitro protein synthesis programmed with f2 RNA (or poly U) in extracts derived from normal and T4am-infected cells. I have been unable to demonstrate any significant difference between the ability of normal and T4am extracts to support polypeptide synthesis. In addition, double-labeling and gel electrophoresis analysis indicate that normal f2 coat is made in T4am-extract. The excellent technical assistance of Mrs Judith Hambley is gratefully acknowledged, This investigation was supported by a Public Health Service research grant no. AI 08738-01 from the National Institute of Allergy and Infectious Disease. Department of Biology University of Rochester Rochester, New York 14627, U.S.A.
STANLEY
HATTUN
Received 22 August 1969 REFERENCES P. H. (1967). J. Mol. Biol. 29, 173. P. H. (1968). J. Mol. BioZ. 37, 513.
Hattman, S. & Hofschneider, Hattman, S. & Hofschneider, Kaerner, H. C. (1969). J. Mol. Biol. 42, 259. Konigsberg, M., Weher, K., Notani, G. & Zinder, N. (1966). J. BioZ. Chem. 14, 2579. Oeschger, M. P. & Nt thans, D. (1966). J. Mol. BioZ. 22, 235. Sugiyama, T. & Stone, H. O., Jr (1968). J. Mol. BioZ. 36, 91. Viiiunla, E., Algranati, I. D. & Ochoa, S. (1967). Europ. J. Biochem. 1, 3. Watanabe, M., Watanabe, H. 8z August, J. T. (1968). J. Mol. BioZ. 33, 11. Weher, K., Notani, G., Wikler, M. & Konigsberg, W. (1966). J. Mol. BioZ. 20, 423. Yarosh, E. & Levinthal, C. (1967). J. Mol. BioZ. 30, 329.
Influence of T4 Superinfection on the Formation RNA Bacteriophage Coat Protein
of
Synthesis of RNA-phage coat protein is strongly inhibited following superinfection with phage T4; a sharp reduction in coat production is observed within several minutes after superinfection. In simultaneous mixed infection of cells of Escherichia coli with RNA phage and T-even phage (virulent DNA phage), parental RNA remains intact and is found in polysomes, but no RNA polymerase or replicative intermediates are found; in delayed T4 superinfection, infectious RNA is synthesized, but no progeny phage or defective particles are produced, and coat-protein antigen synthesis is strongly inhibited. We interpreted these results to mean that T4 blocks the messenger function of the RNA phage chromosome by inhibiting translation (Hattman & Hofschneider, 1967, 1968). Our earlier experiments on coat-protein antigen formation used a serological technique, with serum prepared against purified phage. Since it was not clear if such a preparation is also active against free virion subunits or nascent coat protein on ribosomes, we were cautious about stating that T4 blocks synthesis of the coat protein molecule per se. (However, a recent report indicates that the immune precipitation procedure also measures coat protein not contained in phage (Kaerner, 1969).) For this reason, we have examined formation of the coat protein directly, and the results of the observations are reported here. The rationale of the method utilizes several properties of RNA phage coat protein : (1) it is of low molecular weight (cu. 14,000) (Konigsberg, Weber, Notani & Zinder, 1966); (2) it lacks histidine (Konigsberg et al., 1966; Weber, Notani, Wikler & Konigsberg, 1966) ; (3) its synthesis at late infection times constitutes about 20 to 80% of total protein synthesis (Hattman & Hofschneider, 1967; Watanabe, Watanabe & August, 1968; Sugiyama & Stone, 1968). Therefore, coat formation can readily be analyzed by double-labeling of cells late in infection with [14C]histidine and [3H]phenylalanine and measuring 3H/14C ratios of protein fractions separated according to size. Preliminary control experiments verified that separation of f2 coat from E. coli proteins could be achieved by gel filtration on Sephadex GlOO (Fig. 1). In uninfected cells and T4aminfected cells, no substantial changein [3H]phenylalanine/[14C]histidine incorporation was observed, in contrast to f2-infected cells. Labeled coat-protein isolated from purified f2 phage was run separately as a marker and observed at the same position as the peak 3H/14C. It should be pointed out that we were able repeatedly to use the same column and thus compare profiles from experiment to experiment. Gel filtration profiles obtained from control f2-infected cells are compared to superinfected cells in Figures 2 and 3. In these experiments, T&m82 was added 40 minutes after primary infection with f2, and a 15minute labeling period commenced five minutes after T&m addition. It is clear that superinfection with T4am inhibits f2-coat protein synthesis. It should be noted that incorporation of label was three39
699
600
STANLEY
I
4 (a)
4
I
I
I
HATTMAN
I
~ Uninfected cells
(b) T4 am 82-Infected
cells
rf
25
30
35
40
45
15 20 25
Fraction no.
30
3.5 40
Fraction no.
15 20
25
30 3.5
40
Fraction no. Fm. 1. Sephadex GlOO gel filtration patterns of proteins synthesized in uninfected cells (a), T4am82-infected cells (b) and ft-infected cells (c). E. co& strain K38 was grown to the appropriate optical density and the incorporation period begun. Labeling was with [3H]phenylalanine (0.5 PC/ml.) and [l%]histidine (0.1 pc/ml.). (a) at o.n.,,,=0.40, a IO-min incorporation period was started in the uninfected cells; (b) at period was 0.D .540=0.37, cells were infected with T4am82, and 5 min later a 20-min incorporation 0.26, cells were infected with f2; and 45 min later a l&mm incorporation started; (0) at o.D.~~~= period was started. At 60 min post-infection, the 0.D.540 had increased to 0.50. Incorporation was terminated by a 3-min chase with 100 pg cold amino wcids/ml., followed by pouring over crushed ice and centrifugation. Phenol extraction and dialysis were as described by Vifmela, Algranati t Ochoa (1967) except for the following modification: the final dialysis was with 0.01 M-potassium phosphate (pH 7.2) + 1 o/0 2-mercaptoethanol + 0.5% Sarkosyl. This buffer was used for the Sephadex gel filtration as well. Samples of 0.4 to O-6 ml. were applied to a GlOO Sephadex column (bed volume = 30 ml.); in (b) and (c), 2%drop fractions were collected (0.6 ml.), and in (a) 20.drop fractions were collected. The fractions were precipitated with cold trichloroaoetic acid and filtered onto Whatman GF/A glass fiber disks. Counting was in a Packard 3375 sointillation counter set for double-isotope counting; 8H overlap in 14C channel was O.O20/0, and 14C overlap in sH channel was 11% for unquenched standards. The raw data are plotted with no corrections for efficiency of overlap (on filters, the W overlap in 3H is actually higher than with the ideal standards). Recoveries through extraction and dialysis were 80 to 90% for both isotopes, and all counts applied to the column were recovered. The results depicted above were obtained in three independent experiments,
LETTERS
TO
THE
EDITOR
601
Fraction no. FIG. 2. Sephadex GlOO gel filtration patterns of proteins synthesized in f2-control infection and T4am82 superinfection. Cells were grown as described in Fig. 1 and infected with f2 at o.D.,,,=0*28. At 40 mm postinfection, T4am82 was added to one portion of the culture. 5 min later, the pulse was started for a 15-mm period and terminated; procedures were as described in Fig. 1. Recovery of 3H and l*C was 80% for the control and 90% for the superinfection. 0.4 ml. portions for the f2-control infection (a) and 0.5 ml. for the superinfection (b) were applied in separate column runs; 25-drop fractions were collected.
Fraction no. FIG. 3. Sephadex GlOO gel filtration patterns of proteins and T4arn82 superinfection. Conditions were similar to those described in Fig. 2, except of [3H]phenylalanine. Recovery was 90% for the control and of the f2-control infection (a) and 0.6 ml. of the superinfection runs; 25drop fractions were collected.
synthesized
in f2-oontrol
infection
that [3H]alsnine was used in place 100% for the superinfection. 0.4 ml. (b) were applied in separate column
to fivefold lower in the superinfected cultures compared to the control infections; this was observed previously with [35S]sulfate incorporation (Hattman & Hofschneider, 1967). This is probably due to both continued growth (increase of O.D.,,,) and higher rate of protein synthesis in the control infection. The nature of the proteins observed by the Sephadex assay was confirmed by polyaorylamide gel electrophoresis (data not shown). Briefly, in all cases the major peak of labeled proteins excluded on GIOO Sephadex was separated into many smaller peaks (the 3H/14C ratio remaining constant). The included labeled proteins yielded a large major peak; however, an elevated 3H/14C ratio was found only with the control infection, and this material co-electrophoresed with purified marker protein.
602
STANLEY
HATTMAN
In the preceding experiments, the incorporation of labeled amino acids was during a 15-minute period, corresponding to 5 to 15 minutes post T&m-superinfection. Infectious RNA production is known to proceed in delayed superinfections such as described here, although there is a depression in synthesis (Hattman & Hofschneider, 196’7; Yarosh & Levinthal, 1967). Therefore, it is not excluded that an apparent direct block in coat production might be attributed to reduced progeny RNA formation; it is known, in fact, that the level of coat production is influenced by the level of progeny RNA (Oeschger & Nathans, 1966). Therefore, an attempt was made to minimize such a possibility by confining the pulse period to only three minutes, and started at two minutes after T4am superinfection. The results of two such independent experiments are depicted in Figure 4. In these experiments, there was only a twofold reduction in total label incorporation into superinfected cultures compared to control infections. It can be seen that the elution profiles in the superinfected cuItures are different from those observed for longer pulse periods (Figs 2 and 3) ; I have not investigated this point further. Similar patterns were obtained with [3H]phenylalanine and [3H]alanine. The results clearly show the
Fraction
Fraction
no.
no,
Fm. 4. Sephadex Cl00 gel f?ltr&ion patterns of proteins synthesized in f2-control infection and T4am82 superinfeotion. Cells infected with f2 for 42 min were superinfected with T4am82.2 min later, a pulse was started for & 3-min period; incorporation was halted by a 2-min ohase with cold amino acids. In (a) and (b) labeling w&s with [3H]phenylalanine (2.5 pc/ml.) and [Wlhistidine (1.0 pc/ml.); in (c) and (d) labeling w&s with [*H]altmine (0.5 &ml) and [14C]histidine (0.1 &ml.).
LETTERS
TO
THE
EDITOR
603
near elimination of recognizable f2 coat production; in Bgure 4(c), there may have been a small amount of coat production. These results support the view that inhibition of f2 coat synthesis by T4am is mediated by direct translation inhibition, and that this process is expressed within the first few minutes after T4am superinfection. It should be pointed out that premature chain termination does not seem to be the mode of translation inhibition. If 20%-length coat molecules were produced, then an increased 3H/14C ratio would still have been observed. This follows from the location of the four phenylalanine residues (out of 129), which are at positions 4, 7, 25 and 95 with respect to the N-terminal amino acid. However, production of shorter or acid-soluble peptides is not ruled out. Recently, I have been examining in vitro protein synthesis programmed with f2 RNA (or poly U) in extracts derived from normal and T4am-infected cells. I have been unable to demonstrate any significant difference between the ability of normal and T4am extracts to support polypeptide synthesis. In addition, double-labeling and gel electrophoresis analysis indicate that normal f2 coat is made in T4am-extract. The excellent technical assistance of Mrs Judith Hambley is gratefully acknowledged, This investigation was supported by a Public Health Service research grant no. AI 08738-01 from the National Institute of Allergy and Infectious Disease. Department of Biology University of Rochester Rochester, New York 14627, U.S.A.
STANLEY
HATTUN
Received 22 August 1969 REFERENCES P. H. (1967). J. Mol. Biol. 29, 173. P. H. (1968). J. Mol. BioZ. 37, 513.
Hattman, S. & Hofschneider, Hattman, S. & Hofschneider, Kaerner, H. C. (1969). J. Mol. Biol. 42, 259. Konigsberg, M., Weher, K., Notani, G. & Zinder, N. (1966). J. BioZ. Chem. 14, 2579. Oeschger, M. P. & Nt thans, D. (1966). J. Mol. BioZ. 22, 235. Sugiyama, T. & Stone, H. O., Jr (1968). J. Mol. BioZ. 36, 91. Viiiunla, E., Algranati, I. D. & Ochoa, S. (1967). Europ. J. Biochem. 1, 3. Watanabe, M., Watanabe, H. 8z August, J. T. (1968). J. Mol. BioZ. 33, 11. Weher, K., Notani, G., Wikler, M. & Konigsberg, W. (1966). J. Mol. BioZ. 20, 423. Yarosh, E. & Levinthal, C. (1967). J. Mol. BioZ. 30, 329.