752
BIOCHEMICAL SOCIETY TRANSACTIONS
Adenovirus Deoxyribonucleic Acid Synthesis in a Soluble Fraction Isolated from Infected Human Cells C. H. SHAW, D. M. REKOSH, W. C. RUSSELL and G. E. BLAIR Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, U.K.
Adenoviruses possess a linear double-stranded-DNA genome, of mol.wt. 23 x lo6 (Philipson et a/., 1975). In human cells, DNA synthesis is essentially virus-specific at about 17-18h after infection, by which time host-DNA synthesis is inhibited. DNA replication appears to proceed bidirectionally, synthesis beginning at or near the terminus of each DNA strand (Weingartner et al., 1976). Studies with temperaturesensitive mutants of adenovirus have suggested that at least one of the early nonstructural proteins is required for virus-DNA synthesis (Ensinger & Ginsberg, 1972). This is a single-stranded-DNA-binding protein of mo1,wt. 72000 that previous studies have shown to be phosphorylated (Russell & Blair, 1977). The aim of the present study was to establish a DNA-replication system in uitro and to examine the role of ‘early’ non-structural proteins in this process. HeLa cells were infected with 50 plaque-forming units of adenovirus type 5/cell and incubated at 37°C for 17h. The cells were labelled for 4min with [3H]thymidine and harvested immediately, or ‘chased’ for 60min with unlabelled medium. Nuclei were isolated from each culture, and a subnuclear fractionwas solubilized from these by treating with O.~M-(NH,),SO~as described by Wilhelm et al. (1976). The soluble fraction was then layered on to a 10-58 % (w/v) sucrose gradient and centrifuged at 285000g for 3 h. In the pulse-labelled cells, most of the trichloroacetic acid-precipitable radioactivity (i.e. labelled DNA) present in the nuclei was extracted into the soluble subnuclear fraction. This labelled DNA sedimented as a broad peak in the sucrose gradient at 50-100s. However, in the cells ‘chased’ with unlabelled medium, the labelled DNA sedimented mainly in a peak at about 40s. Both of these species are DNA-protein complexes that sediment faster than ‘naked’ adenovirus DNA (which sediments at 31 S). Removal of protein with sodium dodecyl sulphate and Pronase converted the 40s species into one that sedimented at 31s. Similar treatment of the 50-100s species revealed a heterogeneous size distribution of DNA molecules, suggestive of replicating adenovirus DNA. The 40s species may represent a more mature, stable form of replicated adenovirus DNA. One of the major proteins present in this soluble subnuclear fraction was shown by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis to be the single-strandedDNA-binding protein. Similar sucrose-gradient analysis showed that the DNAbinding protein, labelled for 30min in uiuo with [35S]methionine, was associated with the 50-100s peak, but not the 40s peak. When cells were ‘chased’ for 5h before harvesting, the DNA-binding protein was found in association with both peaks. In a 1 h period of labelling with [3sS]methionine, labelled DNA-binding protein appeared in association with both DNA peaks. However, when labelled for 1h with [32P]Pf, phosphorylated DNA-binding protein appeared only in the 50-100s peak. Newly synthesized phosphorylated DNA-binding protein apparently preferentially associates with replicating adenovirus DNA. The ability of the soluble subnuclear fraction to synthesize DNA was investigated. When the isolated fraction was incubated in the presence of Mg2+ ions, ATP and all four deoxynucleoside triphosphates, [3H]dTTP was incorporated into DNA without added template, primer or DNA polymerases. The endogenous DNA polymerase activity present in this fraction represents the major portion of endogenous activity in intact infected nuclei. A similar soluble fraction from uninfected cells did not incorporate labelled deoxynucleoside triphosphates, even though nuclei from uninfected cells exhibited significant incorporation. Immunoglobulin G purified from antiserum raised against the ‘early’ proteins of adenovirus type 5 (of which the single-strandedDNA-binding protein in the major antigen) inhibited DNA synthesis in uitro in the 1978
575th MEETING, GLASGOW
753
soluble fraction from infected cells, whereas preimmune immunoglobulin G did not. These results are consistent with there being an important role for the DNA-binding protein in adenovirus-DNA replication as suggested by Van der Vliet et al. (1977). Further studies are required to characterize the products of DNA synthesis in vitro, and the host-cell enzymes and virus proteins involved in this process. Ensinger, M. J. & Ginsberg, M. S. (1972) J. Virol. 10, 328-339 Philipson, L., Pettersson, U. & Lindberg, U. (1975) Molecular Biology of Adenouiruses, pp. 11-16, Springer-Verlag, Vienna and New York Russell, W. C. & Blair, G. E. (1977) J. Gen. Virol. 34, 19-35 Weingartner. B., Winnacker, E. L., Tolun, A. & Pettersson, U. (1976) Cell 9, 259-268 Wilhelm, J., Brison, O . , Kedinger, C. & Chambon, P. (1976) J. Virol. 19, 61-81 Van der Vliet, P. C., Zandberg, J. & Jansz, H. S. (1977) Virology 80, 98-110
The Effect of Oxidised Nicotinamide-Adenine Dinucleotide on Ribonucleic Acid Synthesis in Isolated Nuclei from Baby-Hamster Kidney Cells (BHK-21/C13) HENRY M. FURNEAUX and COLIN K. PEARSON Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 1 AS, Scotland, U.K.
Several studies on mammalian cell metabolism have established that there is a relationship between the intracellular concentration of NAD+ and the growth state of the cell (Wintzerith et al., 1961; Ferris & Clark, 1971; Schwartz et al., 1974). This relationship led to the idea, originally proposed by Morton (1958), that NAD+ may control cellular growth. The way in which this could occur is not readily understandable in terms of the classic role of NAD+ as a cofactor in biological redox reactions. There are, however, reactions in which NAD+ participates not as a cofactor but as a substrate. In particular, it is now appreciated that the ADP-ribose moiety of NAD+ may be covalently bound to proteins (Sugimura, 1973; Hilz & Stone, 1976;-Shall et al., 1977). In mammalian cells the enzyme that catalyses this reaction (ADP-ribosylation) is localized in the nucleus, the site of NAD+ biosynthesis. The mammalian enzyme [poly(ADP-ribose) polymerase] can also catalyse the addition of further ADP-ribose units (by a 2’-1’ glycosidic linkage), forming the homopolymer poly(ADP-ribose). Poly(ADP-ribose) polymerase modifies all classes of nuclear protein in uitro (Rickwood et al., 1977). Thus it seems unlikely that this modification reaction will have a unique role in cellular metabolism. It does, however, suggest that this modification reaction may be the link between the intracellular NAD+ concentration and the growth state of the cell. If we accept this hypothesis, then it seems likely that ADP-ribosylation of nuclear proteins will be involved in the three major cellular growth processes, i.e. DNA, RNA and protein synthesis. In the case of DNA and RNA synthesis it is possible to test this involvement directly. Isolated nuclei from mammalian cells are capable of synthesizing DNA and RNA in vitro (Kidwell & Mueller, 1969; Zylber &Penman, 1971). A possible experimental approach therefore would be to examine the effect of NAD+ on DNA and RNA synthesis in isolated nuclei. The effect of NAD+ on DNA synthesis in isolated nuclei has been extensively studied (Burzio & Koide, 1970; Roberts et al., 1973; Burzio et al., 1975; Suhadolnik et al., 1977). On the other hand there are few reports dealing specifically with the involvement of ADP-ribosylation in RNA synthesis. Miiller & Zahn (1976) reported that the activity of the endogenous RNA polymerase in isolated nuclei from quail oviduct is inhibited by the addition of NAD+. They also provided evidence for a covalent linkage between
Vol. 6
BIOCHEMICAL SOCIETY TRANSACTIONS
Adenovirus Deoxyribonucleic Acid Synthesis in a Soluble Fraction Isolated from Infected Human Cells C. H. SHAW, D. M. REKOSH, W. C. RUSSELL and G. E. BLAIR Division of Virology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, U.K.
Adenoviruses possess a linear double-stranded-DNA genome, of mol.wt. 23 x lo6 (Philipson et a/., 1975). In human cells, DNA synthesis is essentially virus-specific at about 17-18h after infection, by which time host-DNA synthesis is inhibited. DNA replication appears to proceed bidirectionally, synthesis beginning at or near the terminus of each DNA strand (Weingartner et al., 1976). Studies with temperaturesensitive mutants of adenovirus have suggested that at least one of the early nonstructural proteins is required for virus-DNA synthesis (Ensinger & Ginsberg, 1972). This is a single-stranded-DNA-binding protein of mo1,wt. 72000 that previous studies have shown to be phosphorylated (Russell & Blair, 1977). The aim of the present study was to establish a DNA-replication system in uitro and to examine the role of ‘early’ non-structural proteins in this process. HeLa cells were infected with 50 plaque-forming units of adenovirus type 5/cell and incubated at 37°C for 17h. The cells were labelled for 4min with [3H]thymidine and harvested immediately, or ‘chased’ for 60min with unlabelled medium. Nuclei were isolated from each culture, and a subnuclear fractionwas solubilized from these by treating with O.~M-(NH,),SO~as described by Wilhelm et al. (1976). The soluble fraction was then layered on to a 10-58 % (w/v) sucrose gradient and centrifuged at 285000g for 3 h. In the pulse-labelled cells, most of the trichloroacetic acid-precipitable radioactivity (i.e. labelled DNA) present in the nuclei was extracted into the soluble subnuclear fraction. This labelled DNA sedimented as a broad peak in the sucrose gradient at 50-100s. However, in the cells ‘chased’ with unlabelled medium, the labelled DNA sedimented mainly in a peak at about 40s. Both of these species are DNA-protein complexes that sediment faster than ‘naked’ adenovirus DNA (which sediments at 31 S). Removal of protein with sodium dodecyl sulphate and Pronase converted the 40s species into one that sedimented at 31s. Similar treatment of the 50-100s species revealed a heterogeneous size distribution of DNA molecules, suggestive of replicating adenovirus DNA. The 40s species may represent a more mature, stable form of replicated adenovirus DNA. One of the major proteins present in this soluble subnuclear fraction was shown by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis to be the single-strandedDNA-binding protein. Similar sucrose-gradient analysis showed that the DNAbinding protein, labelled for 30min in uiuo with [35S]methionine, was associated with the 50-100s peak, but not the 40s peak. When cells were ‘chased’ for 5h before harvesting, the DNA-binding protein was found in association with both peaks. In a 1 h period of labelling with [3sS]methionine, labelled DNA-binding protein appeared in association with both DNA peaks. However, when labelled for 1h with [32P]Pf, phosphorylated DNA-binding protein appeared only in the 50-100s peak. Newly synthesized phosphorylated DNA-binding protein apparently preferentially associates with replicating adenovirus DNA. The ability of the soluble subnuclear fraction to synthesize DNA was investigated. When the isolated fraction was incubated in the presence of Mg2+ ions, ATP and all four deoxynucleoside triphosphates, [3H]dTTP was incorporated into DNA without added template, primer or DNA polymerases. The endogenous DNA polymerase activity present in this fraction represents the major portion of endogenous activity in intact infected nuclei. A similar soluble fraction from uninfected cells did not incorporate labelled deoxynucleoside triphosphates, even though nuclei from uninfected cells exhibited significant incorporation. Immunoglobulin G purified from antiserum raised against the ‘early’ proteins of adenovirus type 5 (of which the single-strandedDNA-binding protein in the major antigen) inhibited DNA synthesis in uitro in the 1978
575th MEETING, GLASGOW
753
soluble fraction from infected cells, whereas preimmune immunoglobulin G did not. These results are consistent with there being an important role for the DNA-binding protein in adenovirus-DNA replication as suggested by Van der Vliet et al. (1977). Further studies are required to characterize the products of DNA synthesis in vitro, and the host-cell enzymes and virus proteins involved in this process. Ensinger, M. J. & Ginsberg, M. S. (1972) J. Virol. 10, 328-339 Philipson, L., Pettersson, U. & Lindberg, U. (1975) Molecular Biology of Adenouiruses, pp. 11-16, Springer-Verlag, Vienna and New York Russell, W. C. & Blair, G. E. (1977) J. Gen. Virol. 34, 19-35 Weingartner. B., Winnacker, E. L., Tolun, A. & Pettersson, U. (1976) Cell 9, 259-268 Wilhelm, J., Brison, O . , Kedinger, C. & Chambon, P. (1976) J. Virol. 19, 61-81 Van der Vliet, P. C., Zandberg, J. & Jansz, H. S. (1977) Virology 80, 98-110
The Effect of Oxidised Nicotinamide-Adenine Dinucleotide on Ribonucleic Acid Synthesis in Isolated Nuclei from Baby-Hamster Kidney Cells (BHK-21/C13) HENRY M. FURNEAUX and COLIN K. PEARSON Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 1 AS, Scotland, U.K.
Several studies on mammalian cell metabolism have established that there is a relationship between the intracellular concentration of NAD+ and the growth state of the cell (Wintzerith et al., 1961; Ferris & Clark, 1971; Schwartz et al., 1974). This relationship led to the idea, originally proposed by Morton (1958), that NAD+ may control cellular growth. The way in which this could occur is not readily understandable in terms of the classic role of NAD+ as a cofactor in biological redox reactions. There are, however, reactions in which NAD+ participates not as a cofactor but as a substrate. In particular, it is now appreciated that the ADP-ribose moiety of NAD+ may be covalently bound to proteins (Sugimura, 1973; Hilz & Stone, 1976;-Shall et al., 1977). In mammalian cells the enzyme that catalyses this reaction (ADP-ribosylation) is localized in the nucleus, the site of NAD+ biosynthesis. The mammalian enzyme [poly(ADP-ribose) polymerase] can also catalyse the addition of further ADP-ribose units (by a 2’-1’ glycosidic linkage), forming the homopolymer poly(ADP-ribose). Poly(ADP-ribose) polymerase modifies all classes of nuclear protein in uitro (Rickwood et al., 1977). Thus it seems unlikely that this modification reaction will have a unique role in cellular metabolism. It does, however, suggest that this modification reaction may be the link between the intracellular NAD+ concentration and the growth state of the cell. If we accept this hypothesis, then it seems likely that ADP-ribosylation of nuclear proteins will be involved in the three major cellular growth processes, i.e. DNA, RNA and protein synthesis. In the case of DNA and RNA synthesis it is possible to test this involvement directly. Isolated nuclei from mammalian cells are capable of synthesizing DNA and RNA in vitro (Kidwell & Mueller, 1969; Zylber &Penman, 1971). A possible experimental approach therefore would be to examine the effect of NAD+ on DNA and RNA synthesis in isolated nuclei. The effect of NAD+ on DNA synthesis in isolated nuclei has been extensively studied (Burzio & Koide, 1970; Roberts et al., 1973; Burzio et al., 1975; Suhadolnik et al., 1977). On the other hand there are few reports dealing specifically with the involvement of ADP-ribosylation in RNA synthesis. Miiller & Zahn (1976) reported that the activity of the endogenous RNA polymerase in isolated nuclei from quail oviduct is inhibited by the addition of NAD+. They also provided evidence for a covalent linkage between
Vol. 6
