Intervirology II: 182-187 (1979)
Poly(Adenosine Diphosphate Ribose) Synthesis during Herpes Simplex Virus Infection Werner E. G. Müller, Dietrich Falke. Rudolf K. Zahn and Josef Arendes1 Institut für Physiologische Chemie und Institut für Medizinische Mikrobiologie, Universität Mainz, Mainz
Key Words. Herpes simplex virus • Poly(adenosine diphosphate ribose) synthesis ■ Herpes simplex virus DNA synthesis • Poly(adenosine diphosphate ribose) polymerase Summary. Immediately after infection of baby hamster kidney cells with herpes simplex virus (HSV), cellular DNA synthesis was blocked, while extensive HSV DNA synthesis began. These dramatic alterations of the control mechanisms for these two DNA synthesizing systems were not accompanied by a change in the poly(adenosine diphosphate ribose) polymerase activity.
1 We gratefully acknowledge loans from the 'Aka demie der Wissenschaften und Literatur", Mainz (FRG). Address inquiries to: Prof. Dr. W. E.G. Müller, In stitut für Physiologische Chemie, Abteilung Ange wandte Molekularbiologie. Duesbergweg, D-6500 Mainz (FRG)
Received: April 22; revised: June 8, 1978
suggested that the synthesis of the polyanion poly(A DP-Rib) may be related to chromosome condensation in mitosis [5], It was the aim of the present study to deter mine whether poly(ADP-Rib) synthesis plays a regulatory function in the DNA synthesis of herpes simplex virus(HSV)-infected cells. This biological model seemed to be suitable for a clarification of whether poly(ADP-Rib) syn thesis is correlated with DNA formation, be cause after HSV infection dramatic alterations in the control mechanisms of the two DNA synthesizing systems occur.
Materials and Methods Compounds The following materials were obtained: [3H]dThd (spec.act. 5 Ci/mmol) and nicotinamide-[U-MC]ade
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:05:08 AM
Polyfadenosine diphosphate ribose) [poly (ADP-Rib)] was first discovered by Chambon el at. [1] as an in vitro reaction product. Re cently, it was shown that poly(ADP-Rib) also occurs in vivo [2, 3], The data about the phys iological role of poly(ADP-Rib) so far avail able do not allow definite conclusions. Some in vitro studies seem to indicate that poly(ADPRib) formation is involved in DNA and/or RNA synthesis [4]; in other reports it has been
183
Poly(ADP-Rib) Synthesis and HSV Infection
nine dinucleotide (spec.act. 260 mCi/mmol) from the Radiochemical Centre (Amersham, England); N A D ', phosphodiesterase I, DNase I, RNasc A, micrococcal nuclease and alkaline phosphatase from Boehringer (Mannheim, FRG); and lysine-rich histone from calf thymus (type III) from Sigma Chemical Co. (St. Louis, Mo.). Cells Baby hamster kidney (BHK) 21 cells were grown in monolayer cultures as described previously [6], For all experiments, cells were used 32 h after seeding; they are considered to be in the stationary phase of growth at this time [6],
The purified nuclei were suspended in 6 vol o f Tris-HCl buffer [50 mM Tris-HCI, 1 mM EDTA, 5 mM NaF, 30% (v/v) glycerol, pH 8.0] containing 1 M NaCI and were homogenized by three strokes in a Dounce homogenizer with the tight-fitting pestle. After stirring for 60 min, the preparation was centrifuged in an SW50 rotor o f the Spinco L2-65 for 60 min at 50,000 rpm and subsequently dialyzed (15 h, 2 ) against Tris-HCl (without NaCI). The dialysate was centrifuged again in an SW50 rotor for 60 min at 50,000 rpm. This frac tion subsequently was subjected to DEAE-Sephadex chromatography. Soluble poly(ADP-Rib) polymerase was obtained by a stepwise gradient [II].
Enzyme Assays The chromatin-bound poly(ADP-Rib) polymerase was assayed in a reaction mixture containing (in a total volume of 70 p.1) 100 mM Tris-HCl, pH 8.5, 6 mM MgCl2 , 60 mM KCI, 4 mM dithiothreitol, 50 ¡¿M [,-,C]NAD+ (2 mCi/mmol), and 20 p.1 o f the enzyme sample (purified nuclei) to be assayed. The mixture was incubated for 10 min at 25 . Acid-insoluble radioactiv ity was determined as described [12]. Incorporation ofdThd The extractable poly(ADP-Rib) polymerase assay For the incorporation o f [3H]dThd into DNA, mixture (50 ¡xl) contained the following components: 2.5 ¡xCi [3H]dThd was added to 10 ml culture fluid (one 60 mM Tris-HCl, pH 8.0, 15 mM MgCle, 50 mM KCI, Petri dish) and incubated for 30 min. The acid-insoluble I mM EDTA, 4 mM dithiothreitol, 50 u M [NC]NADmaterial o f the cell pellet was determined as described (2 mCi/mmol), 1.7 ¡xg/ml native herring DNA, 0.4 [7] . For the experiments to determine the onset o f HSV |xg/m! lysine-rich histone, and the enzyme sample to be DNA synthesis analytically (by CsCI gradient centri assayed (30 ¡zl). The mixture was incubated at 25 for fugation), HSV-infectcd cells from 10 Petri dishes were 10 min. Acid-insoluble radioactivity was determined used. Each Petri dish was incubated in the presence of as described [12], 2.5 [zCi [3H]dThd (in 10 ml medium) for 60 min. DNA was isolated subsequently. Other Procedures DNA was analyzed according to Burton [13]. RNA Isopycnic Gradient Centrifugation was determined as described [14]. For protein determi DNA from cells from 10 Petri dishes was isolated nations the method o f Lowry et al. [15] was used. [8] and subsequently purified by CsCI density gradient Herring sperm DNA, isolated according to Xah/t centrifugation [9]. et al. [16], was a gift o f H. Mack (lllertissen, FRG). Viruses HSV (type 1) strain Lennette was used. The cells (4 x I08 cells/Petri dish) were infected at 20 PFU/cell. Time zero was considered to be at the end of the ad sorption period (1 h). The infected cells were incubated at 37° for 0-11 h and then harvested.
Results Pattern o f Synthesis o f Cellular and Viral DNA The incorporation rate of [3H]dThd into DNA in the intact ceil system was drastically
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:05:08 AM
Enzyme Preparations Chromatin-bound poly(ADP-Rib) polymerase was isolated as follows. Nuclei were isolated according to McGuire and O' M alley[\0\. The packed, purified nuclei were suspended in 10 mM Tris-HCl, pH 8.0, and 250 mM sucrose. This preparation was characterized by a DNA; RNA ratio of 4.28 and a DNA:protein ratio of 0.35. Extractable poly(ADP-Rib) polymerase was iso lated as follows. Nuclei were isolated from cells [10].
184
M üIler/Falke/Zahn/A rendes
£
Poly(Adenosine Diphosphate Ribose) Synthesis during Herpes Simplex Virus Infection Werner E. G. Müller, Dietrich Falke. Rudolf K. Zahn and Josef Arendes1 Institut für Physiologische Chemie und Institut für Medizinische Mikrobiologie, Universität Mainz, Mainz
Key Words. Herpes simplex virus • Poly(adenosine diphosphate ribose) synthesis ■ Herpes simplex virus DNA synthesis • Poly(adenosine diphosphate ribose) polymerase Summary. Immediately after infection of baby hamster kidney cells with herpes simplex virus (HSV), cellular DNA synthesis was blocked, while extensive HSV DNA synthesis began. These dramatic alterations of the control mechanisms for these two DNA synthesizing systems were not accompanied by a change in the poly(adenosine diphosphate ribose) polymerase activity.
1 We gratefully acknowledge loans from the 'Aka demie der Wissenschaften und Literatur", Mainz (FRG). Address inquiries to: Prof. Dr. W. E.G. Müller, In stitut für Physiologische Chemie, Abteilung Ange wandte Molekularbiologie. Duesbergweg, D-6500 Mainz (FRG)
Received: April 22; revised: June 8, 1978
suggested that the synthesis of the polyanion poly(A DP-Rib) may be related to chromosome condensation in mitosis [5], It was the aim of the present study to deter mine whether poly(ADP-Rib) synthesis plays a regulatory function in the DNA synthesis of herpes simplex virus(HSV)-infected cells. This biological model seemed to be suitable for a clarification of whether poly(ADP-Rib) syn thesis is correlated with DNA formation, be cause after HSV infection dramatic alterations in the control mechanisms of the two DNA synthesizing systems occur.
Materials and Methods Compounds The following materials were obtained: [3H]dThd (spec.act. 5 Ci/mmol) and nicotinamide-[U-MC]ade
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:05:08 AM
Polyfadenosine diphosphate ribose) [poly (ADP-Rib)] was first discovered by Chambon el at. [1] as an in vitro reaction product. Re cently, it was shown that poly(ADP-Rib) also occurs in vivo [2, 3], The data about the phys iological role of poly(ADP-Rib) so far avail able do not allow definite conclusions. Some in vitro studies seem to indicate that poly(ADPRib) formation is involved in DNA and/or RNA synthesis [4]; in other reports it has been
183
Poly(ADP-Rib) Synthesis and HSV Infection
nine dinucleotide (spec.act. 260 mCi/mmol) from the Radiochemical Centre (Amersham, England); N A D ', phosphodiesterase I, DNase I, RNasc A, micrococcal nuclease and alkaline phosphatase from Boehringer (Mannheim, FRG); and lysine-rich histone from calf thymus (type III) from Sigma Chemical Co. (St. Louis, Mo.). Cells Baby hamster kidney (BHK) 21 cells were grown in monolayer cultures as described previously [6], For all experiments, cells were used 32 h after seeding; they are considered to be in the stationary phase of growth at this time [6],
The purified nuclei were suspended in 6 vol o f Tris-HCl buffer [50 mM Tris-HCI, 1 mM EDTA, 5 mM NaF, 30% (v/v) glycerol, pH 8.0] containing 1 M NaCI and were homogenized by three strokes in a Dounce homogenizer with the tight-fitting pestle. After stirring for 60 min, the preparation was centrifuged in an SW50 rotor o f the Spinco L2-65 for 60 min at 50,000 rpm and subsequently dialyzed (15 h, 2 ) against Tris-HCl (without NaCI). The dialysate was centrifuged again in an SW50 rotor for 60 min at 50,000 rpm. This frac tion subsequently was subjected to DEAE-Sephadex chromatography. Soluble poly(ADP-Rib) polymerase was obtained by a stepwise gradient [II].
Enzyme Assays The chromatin-bound poly(ADP-Rib) polymerase was assayed in a reaction mixture containing (in a total volume of 70 p.1) 100 mM Tris-HCl, pH 8.5, 6 mM MgCl2 , 60 mM KCI, 4 mM dithiothreitol, 50 ¡¿M [,-,C]NAD+ (2 mCi/mmol), and 20 p.1 o f the enzyme sample (purified nuclei) to be assayed. The mixture was incubated for 10 min at 25 . Acid-insoluble radioactiv ity was determined as described [12]. Incorporation ofdThd The extractable poly(ADP-Rib) polymerase assay For the incorporation o f [3H]dThd into DNA, mixture (50 ¡xl) contained the following components: 2.5 ¡xCi [3H]dThd was added to 10 ml culture fluid (one 60 mM Tris-HCl, pH 8.0, 15 mM MgCle, 50 mM KCI, Petri dish) and incubated for 30 min. The acid-insoluble I mM EDTA, 4 mM dithiothreitol, 50 u M [NC]NADmaterial o f the cell pellet was determined as described (2 mCi/mmol), 1.7 ¡xg/ml native herring DNA, 0.4 [7] . For the experiments to determine the onset o f HSV |xg/m! lysine-rich histone, and the enzyme sample to be DNA synthesis analytically (by CsCI gradient centri assayed (30 ¡zl). The mixture was incubated at 25 for fugation), HSV-infectcd cells from 10 Petri dishes were 10 min. Acid-insoluble radioactivity was determined used. Each Petri dish was incubated in the presence of as described [12], 2.5 [zCi [3H]dThd (in 10 ml medium) for 60 min. DNA was isolated subsequently. Other Procedures DNA was analyzed according to Burton [13]. RNA Isopycnic Gradient Centrifugation was determined as described [14]. For protein determi DNA from cells from 10 Petri dishes was isolated nations the method o f Lowry et al. [15] was used. [8] and subsequently purified by CsCI density gradient Herring sperm DNA, isolated according to Xah/t centrifugation [9]. et al. [16], was a gift o f H. Mack (lllertissen, FRG). Viruses HSV (type 1) strain Lennette was used. The cells (4 x I08 cells/Petri dish) were infected at 20 PFU/cell. Time zero was considered to be at the end of the ad sorption period (1 h). The infected cells were incubated at 37° for 0-11 h and then harvested.
Results Pattern o f Synthesis o f Cellular and Viral DNA The incorporation rate of [3H]dThd into DNA in the intact ceil system was drastically
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 4:05:08 AM
Enzyme Preparations Chromatin-bound poly(ADP-Rib) polymerase was isolated as follows. Nuclei were isolated according to McGuire and O' M alley[\0\. The packed, purified nuclei were suspended in 10 mM Tris-HCl, pH 8.0, and 250 mM sucrose. This preparation was characterized by a DNA; RNA ratio of 4.28 and a DNA:protein ratio of 0.35. Extractable poly(ADP-Rib) polymerase was iso lated as follows. Nuclei were isolated from cells [10].
184
M üIler/Falke/Zahn/A rendes
£
