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• W1978670534 abstract "The efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apoprotein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe2+ chelator, 2,2′-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus, type 1. In a standard plaque assay, 50 μm BIP caused a 50% reduction in plaque-forming units with either virus. Strong inhibition was observed only when BIP was added within 3 h post-infection. This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inhibit the activity of vaccinia ribonucleotide reductase (RR), its synthesis, nor its stability indicating that BIP blocked the activation of the apoprotein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron-response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron-regulated protein found in the cytosol of mammalian cells. The efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apoprotein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe2+ chelator, 2,2′-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus, type 1. In a standard plaque assay, 50 μm BIP caused a 50% reduction in plaque-forming units with either virus. Strong inhibition was observed only when BIP was added within 3 h post-infection. This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inhibit the activity of vaccinia ribonucleotide reductase (RR), its synthesis, nor its stability indicating that BIP blocked the activation of the apoprotein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron-response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron-regulated protein found in the cytosol of mammalian cells. ribonucleotide reductase hydroxyurea 2,2′-bipyridyl vesicular stomatitis virus multiplicity of infection hours post-infection bathophenanthroline disulfonic acid cytosine arabinoside herpes simplex virus type 1 labile iron pool calcein salicylaldehyde isonicotinoyl hydrazone iron-response element desferrioxamine polyacrylamide gel electrophoresis Organisms have an ambiguous relationship with iron (1Hill H.A.O. Sarkar B. Biological Aspects of Metals and Metal-related Diseases.in: Raven Press, Ltd., New York1983: 15-21Google Scholar, 2Polla B.S. Biochem. Pharmacol. 1999; 57: 1345-1349Crossref PubMed Scopus (34) Google Scholar, 3Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 86: 1-85Crossref Scopus (4392) Google Scholar). Iron is essential to replication and growth, yet at the same time iron is cytotoxic. Iron is nutritionally essential due to its role as prosthetic group in a variety of enzymes and electron transfer proteins required for energy metabolism, for a variety of metabolic interconversions, and for the biosynthesis of the deoxyribonucleotides required for DNA synthesis and repair (4Aisen P. Wessling-Resnick M. Leibold E.A. Curr. Opin. Chem. Biol. 1999; 3: 200-206Crossref PubMed Scopus (405) Google Scholar). Cytotoxicity is due to the efficiency by which iron, as Fe2+, can support the production of oxygen radicals, particularly the hydroxyl radical, HO⋅ (3Halliwell B. Gutteridge J.M.C. Methods Enzymol. 1990; 86: 1-85Crossref Scopus (4392) Google Scholar). Consequently, cells and organisms tightly regulate the uptake, efflux, and compartmentalization of iron so as to modulate the amount of iron accumulated and to ensure that by the appropriate sequestration, the cytotoxic potential of the iron that is absorbed is appropriately suppressed (4Aisen P. Wessling-Resnick M. Leibold E.A. Curr. Opin. Chem. Biol. 1999; 3: 200-206Crossref PubMed Scopus (405) Google Scholar, 5Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation.in: International Thomson Publishing, New York1997: 157-216Google Scholar, 6Wessling-Resnick M. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 285-314Crossref PubMed Scopus (60) Google Scholar). Pathogens and their hosts share this ambiguous relationship with iron (7Weinberg E.D. Weinberg G.A. Curr. Opin. Infect. Dis. 1995; 8: 164-169Crossref Scopus (80) Google Scholar, 8Taramelli D. Brambilla S. Sala G. Bruccoleri A. Tognazioli C. Riviera-Uzielli L. Boelaert J.R. Infect. Immun. 2000; 68: 1724-1726Crossref PubMed Scopus (20) Google Scholar, 9Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1176) Google Scholar, 10Mabeza G.F. Loyevsky M. Gordeuk V.R. Weiss G. Pharmacol. Ther. 1999; 81: 53-75Crossref PubMed Scopus (100) Google Scholar, 11Howard D.H. Clin. Microbiol. Rev. 1999; 12: 394-404Crossref PubMed Google Scholar, 12Cornelissen C.N. Sparling P.F. Mol. Microbiol. 1994; 14: 843-850Crossref PubMed Scopus (202) Google Scholar, 13Cabantchik Z.I. Moody-Haupt S. Gordeuk V.R. FEMS Immun. Med. Microbiol. 1999; 26: 289-298Crossref PubMed Google Scholar). However, pathogens live within the context of the iron-restricted milieu that the host maintains as a key to the suppression of the cytotoxic potential of iron. Bacterial pathogens adapt to this iron-limited environment in several ways. One is the production of chelating agents that possess exceptionally large affinities for iron as either Fe2+ or Fe3+ (9Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1176) Google Scholar,11Howard D.H. Clin. Microbiol. Rev. 1999; 12: 394-404Crossref PubMed Google Scholar, 14Britigan B.E. Rasmussen G.T. Olakanmi O. Cox C.D. Infect. Immun. 2000; 68: 1271-1275Crossref PubMed Scopus (24) Google Scholar). Another strategy is to divert transferrin iron by producing transferrin receptors that compete with the host's (11Howard D.H. Clin. Microbiol. Rev. 1999; 12: 394-404Crossref PubMed Google Scholar, 12Cornelissen C.N. Sparling P.F. Mol. Microbiol. 1994; 14: 843-850Crossref PubMed Scopus (202) Google Scholar). The dependence on the host's supply of iron that pathogens exhibit makes them susceptible to the potential bacteriostatic effects of host iron limitation or chelation. A large body of evidence indicates that manipulation of host iron status does lead to a modulation of the proliferation and virulence of many bacteria and protozoa (10Mabeza G.F. Loyevsky M. Gordeuk V.R. Weiss G. Pharmacol. Ther. 1999; 81: 53-75Crossref PubMed Scopus (100) Google Scholar, 13Cabantchik Z.I. Moody-Haupt S. Gordeuk V.R. FEMS Immun. Med. Microbiol. 1999; 26: 289-298Crossref PubMed Google Scholar, 15Darnell G. Richardson D.R. Blood. 1999; 94: 781-792Crossref PubMed Google Scholar,16Richardson D.R. Can. J. Physiol. Pharmacol. 1997; 75: 1164-1180Crossref PubMed Scopus (67) Google Scholar). In contrast to such organisms, viruses have not evolved mechanisms for actively scavenging host iron. In part, this is no doubt due to the fact that viruses produce limited metabolic machinery except for that required for the replication of their genome. On the other hand, DNA viruses are directly dependent on iron for their proliferation as a result of the essential role that iron plays in the catalytic center of ribonucleotide reductase (RR)1 (17Chabes A., V., D. Larsson G. Liu A. Graslund A. Wijmenga S. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2474-2479Crossref PubMed Scopus (79) Google Scholar, 18Cooper C.E. Lynagh G.R. Hoyes K.P. Hider R.C. Cammack R. Porter J.B. J. Biol. Chem. 1996; 271: 20291-20299Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 19Lamarche N. Matton G. Massie B. Fontecave M. Atta M. Dumas F. Gaudreau P. Langelier Y. Biochem. J. 1996; 320: 129-135Crossref PubMed Scopus (17) Google Scholar). RR is encoded in most if not all large DNA viral genomes (e.g. pox and herpes viruses) and is produced early in infection to support the production of the dNTPs required for viral DNA synthesis (20Prichard M.N. Shipman Jr., C. Chemotherapy. 1995; 41: 384-395Crossref PubMed Scopus (11) Google Scholar, 21Jacobson J.G. Leib D.A. Goldstein D.J. Bogard C.L. Schaffer P.A. Weller S.K. Coen D.M. Virology. 1989; 173: 276-283Crossref PubMed Scopus (161) Google Scholar, 22Idowu A.D. Fraser-Smith E.B. Poffenberger K.L. Herman R.C. Antiviral Res. 1992; 17: 145-156Crossref PubMed Scopus (62) Google Scholar, 23Cameron J.M. McDougall I. Marsden H.S. Preston V.G. Ryan D.M. Subak-Sharpe J.H. J. Gen. Virol. 1988; 69: 2607-2612Crossref PubMed Scopus (108) Google Scholar). This pattern is found in the host as well, because mammalian RR is cell cycle-regulated with strong induction of its synthesis in S phase concurrent with genome replication (24Eriksson S. Graslund A. Skog S. Thelander L. Tribukait B. J. Biol. Chem. 1984; 259: 11695-11700Abstract Full Text PDF PubMed Google Scholar). Eukaryotic and viral ribonucleotide reductase is a heterodimeric protein (17Chabes A., V., D. Larsson G. Liu A. Graslund A. Wijmenga S. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2474-2479Crossref PubMed Scopus (79) Google Scholar, 18Cooper C.E. Lynagh G.R. Hoyes K.P. Hider R.C. Cammack R. Porter J.B. J. Biol. Chem. 1996; 271: 20291-20299Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 19Lamarche N. Matton G. Massie B. Fontecave M. Atta M. Dumas F. Gaudreau P. Langelier Y. Biochem. J. 1996; 320: 129-135Crossref PubMed Scopus (17) Google Scholar, 25Nguyen H.-H.T. Ge J. Perlstein D.L. Stubbe J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12339-12344Crossref PubMed Scopus (42) Google Scholar). The vaccinia virus subunits are referred to as R1 and R2. R1, the large subunit (87 kDa), binds NTPs and is regulatory in nature (26Slabaugh M.B. Davis R.E. Roseman N.A. Mathews C.K. J. Biol. Chem. 1993; 268: 17803-17810Abstract Full Text PDF PubMed Google Scholar). R2, the small subunit (37 kDa), contains the active site of the enzyme that includes a diiron core and a catalytic tyrosyl radical (27Howell M.L. Sanders-Loehr J. Loehr T.M. Roseman N.A. Mathews C.K. Slabaugh M.B. J. Biol. Chem. 1992; 267: 1705-1711Abstract Full Text PDF PubMed Google Scholar). The genes encoding these two subunits are found on theHindIII F and I genomic fragments, respectively (26Slabaugh M.B. Davis R.E. Roseman N.A. Mathews C.K. J. Biol. Chem. 1993; 268: 17803-17810Abstract Full Text PDF PubMed Google Scholar, 27Howell M.L. Sanders-Loehr J. Loehr T.M. Roseman N.A. Mathews C.K. Slabaugh M.B. J. Biol. Chem. 1992; 267: 1705-1711Abstract Full Text PDF PubMed Google Scholar). Both are early genes in that they are temporally expressed prior to DNA replication in the vaccinia infectious cycle (28Howell M.L. Roseman N.A. Slabaugh M.B. Mathews C.K. J. Biol. Chem. 1993; 268: 7155-7162Abstract Full Text PDF PubMed Google Scholar). That a DNA virus requires the pool of dNTPs provided by RR in support of its replicative cycle is indicated by the effect of hydroxyurea (HU). HU inactivates ribonucleotide reductases by quenching the catalytic tyrosyl radical. HU blocks vaccinia replication in cultured cells (29Slabaugh M.B. Mathews C.K. J. Virol. 1986; 60: 506-514Crossref PubMed Google Scholar). The vaccinia infection, and the essential role of RR in it, provides a useful biologic system in which to explore the mechanism by which iron-dependent enzymes obtain the metal that is critical to their activity. In effect, infection introduces in a regulated fashion a gene that encodes an iron-dependent enzyme whose biologic function (support of DNA synthesis and viral replication) and enzyme activity (NDP/NTP reduction) can be readily assayed. Analysis of the iron acquisition pathway employed by the viral RR will permit us to identify the iron pool(s) and proteins involved. This provides us with a useful model system that will permit the analysis of how iron is trafficked to iron apoproteins, and perhaps, how modulation of this trafficking could specifically impact on the replication of cell pathogens. In this study, we have used the membrane-permeant, Fe2+chelator, 2,2′-bipyridyl (BIP), as a probe of whether and how cellular iron is required for a productive infection by vaccinia virus in cultured cells. BIP has been established to interact with the “labile” iron pool within eukaryotic cells (30Breuer W. Epsztejn S. Cabantchik Z.I. J. Biol. Chem. 1995; 270: 24209-24215Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 31Epsztejn S. Kakhlon O. Glickstein H. Breuer W. Cabantchik I. Anal. Biochem. 1997; 248: 31-40Crossref PubMed Scopus (322) Google Scholar, 32Konijn A.M. Glickstein H. Vaisman B. Meyron-Holtz E.G. Slotki I.N. Cabantchik Z.I. Blood. 1999; 94: 2128-2134Crossref PubMed Google Scholar). This pool accounts for ∼20% of the newly arrived iron in a cell (30Breuer W. Epsztejn S. Cabantchik Z.I. J. Biol. Chem. 1995; 270: 24209-24215Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) and appears to be the pool that is sensed by regulatory factors such as iron-response element-binding protein (IREPB/IRP1, cytosolic aconitase) (5Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions in Gene Regulation.in: International Thomson Publishing, New York1997: 157-216Google Scholar, 33Hanson E.S. Leibold E.A. J. Biol. Chem. 1998; 273: 7588-7593Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 34Picard V. Epsztejn S. Santambrogio P. Cabantchik Z.I. Beaumont C. J. Biol. Chem. 1998; 273: 15382-15386Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and, in the yeast Saccharomyces cerevisiae, the iron-regulated transcription factor, Aft1p (35Hassett R.F. Romeo A.M. Kosman D.K. J. Biol. Chem. 1998; 273: 7628-7636Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 36Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (287) Google Scholar). We show here that this pool appears also to support a productive viral infection by providing the iron required for the activation of the vaccinia RR. Vaccinia virus, strain WR, herpes simplex virus type 1, strain KOS, and the African green monkey kidney cell line BSC40 were used for this study. Monolayer cultures were grown Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% calf serum (HyClone, Logan, UT). Viral stocks were prepared from 48-h infected monolayers; these were titered in a standard plaque assay (37Condit R.C. Motyczka A. Virology. 1981; 113: 224-241Crossref PubMed Scopus (15) Google Scholar). This same assay was used to determine viral yield in both vaccinia and herpesvirus infections. A plaque assay was used also to demonstrate that BIP did not inhibit a vesicular stomatitis virus (VSV) infection. VSV is an RNA virus. The labeling procedure of Niles et al. (38Niles E.G. Lee-Chen G.-J. Shuman S. Moss B. Broyles S.S. Virology. 1989; 172: 513-522Crossref PubMed Scopus (62) Google Scholar) was used to follow protein synthesis in vaccinia-infected cells. Confluent monolayers of BSC40 were inoculated with vaccinia virus at a multiplicity of infection (m.o.i.) of 20 for 30 min. The medium was aspirated and replaced with virus-free medium. At varying times after infection, the medium was removed; the cells were washed once with prewarmed phosphate-buffered saline, and the cells were pulse-labeled for 15 min with 0.8 ml of prewarmed phosphate-buffered saline containing 100 μCi/ml [35S]methionine (>1175 Ci/mmol, PerkinElmer Life Sciences). The label was removed, and the cells were moved into 1.0 ml of SDS-electrophoresis sample buffer (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). The labeled proteins were fractionated on a 10% SDS-PAGE gel and were subsequently visualized in the gel by autoradiography. The protocol described by Condit and Motyczka was used (37Condit R.C. Motyczka A. Virology. 1981; 113: 224-241Crossref PubMed Scopus (15) Google Scholar). Duplicate 60-mm dishes of BSC40 monolayers were infected at an m.o.i. of 10. At varying times post-infection, the cells were pulse-labeled for 15 min with 15 μCi of [3H]thymidine (>80 μCi/mmol, PerkinElmer Life Sciences). Cells were scraped from the dishes into 1 ml of water and mixed with 10% trichloroacetic acid. The precipitates were collected and washed on glass fiber filters and counted in a Beckman scintillation spectrophotometer. Cell extracts were prepared from control cells, and cells were treated with BIP (100 μm) for 3 h and used in an RNA gel shift assay for IRP1 (40Leibold E.A. Munro H.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2171-2175Crossref PubMed Scopus (552) Google Scholar) using as probe a 92-base ribooligonucleotide containing the IRE from the human ferritin L chain mRNA (41Walden W.E. Daniels-McQueen S. Brown P.H. Gaffield L. Russell D.A. Bielser D. Bailey L.C. Thach R.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9503-9507Crossref PubMed Scopus (81) Google Scholar, 42Swenson G.R. Patino M.M. Beck M.M. Gaffield L. Walden W.E. Biol. Met. 1991; 4: 48-55Crossref PubMed Scopus (19) Google Scholar). The extracts were prepared by detergent lysis (0.5% Nonidet P-40) in 10 mm Hepes (pH 7.5) containing 10 mm KCl, 1 mm dithiothreitol, 1 mmphenylmethanesulfonyl fluoride, and RNasin (40 units, Promega, Madison, WI). The riboprobe was transcribed from plasmid pTZ18RM1 (see Ref. 42Swenson G.R. Patino M.M. Beck M.M. Gaffield L. Walden W.E. Biol. Met. 1991; 4: 48-55Crossref PubMed Scopus (19) Google Scholar, kindly supplied by Dr. William Walden) with T7 RNA polymerase, labeled with [α-32P]UTP following the standard procedure (Technical Manual TM016, Promega Corp., Madison, WI), and purified on a 10% polyacrylamide gel containing 8 m urea. The binding reactions (40Leibold E.A. Munro H.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2171-2175Crossref PubMed Scopus (552) Google Scholar, 42Swenson G.R. Patino M.M. Beck M.M. Gaffield L. Walden W.E. Biol. Met. 1991; 4: 48-55Crossref PubMed Scopus (19) Google Scholar) were resolved on a 6% polyacrylamide gel; the gel was dried and analyzed using a Bio-Rad PhosphorImager and Molecular Analyst software. The labeling and immunoprecipitation protocols were as described by Howell et al. (28Howell M.L. Roseman N.A. Slabaugh M.B. Mathews C.K. J. Biol. Chem. 1993; 268: 7155-7162Abstract Full Text PDF PubMed Google Scholar). Duplicate 60-mm dishes of BSC40 monolayers were infected at an m.o.i. of 10. At varying times post-infection, the cells were pulse-labeled for 1 h with 80 μCi [35S]methionine, harvested, and washed. A cell extract was prepared in a lysis buffer containing 50 mm Tris-HCl (pH 6.8), 1% SDS, 0.008% bromphenol blue, and 7.5% glycerol. Extract was incubated with polyclonal antibody to either the R1 or R2 subunit of vaccinia virus (kindly supplied by Dr. Christopher Mathews), and the immunocomplexes were isolated by adsorption to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech). After washing, the pellet was resuspended in 50 μl of Laemmli buffer and boiled, and 25 μl was applied to a 10% SDS-PAGE gel. The labeled R1 and R2 subunits were visualized by autoradiography. The assay described by Slabaugh et al. (43Slabaugh M.B. Johnson T.L. Mathews C.K. J. Virol. 1984; 52: 507-514Crossref PubMed Google Scholar) was followed. Monolayers of BSC40 cells (100-mm dishes) were infected at an m.o.i. of 10. At various times post-infection, the medium was removed, and the dishes were placed on ice. All further manipulations were performed at 4 °C. Following washing in a 25 mm Hepes buffer containing 10 mm dithiothreitol, cell extracts were prepared in a hypotonic Hepes lysis buffer with the assistance of a Dounce homogenizer. Aliquots (20 μl) of these extracts were used in a ribonucleotide reductase assay mixture containing 25 μm(∼200 cpm/pmol) [3H]cytidine 5′-diphosphate (>20 Ci/mmol, Amersham Pharmacia Biotech). After 30 min the reaction mixtures were quenched by the addition of 10 m perchloric acid. The mixture was clarified by centrifugation, and 40 μl of the supernatant was transferred to a fresh tube that was tightly capped. Following boiling to convert all nucleotides to monophosphates and clarification of this mixture by centrifugation, a marker solution containing 20 mm each of CMP, dCMP, and dUMP was added, and the total nucleotides were fractionated by chromatography on plastic-backed cellulose thin layer plates. dUMP is a secondary product as a result of the dCMP deaminase activity present in BSC40 cell extracts. The markers permitted visualization of the reaction products under UV light; the corresponding regions of the plates were removed and counted in a Beckman scintillation spectrophotometer. Confluent monolayers of BSC40 cells were inoculated with vaccinia virus at an m.o.i. of 10, either in the absence of 2,2′-bipyridyl (control) or in the presence of BIP from 10 to 100 μm. Initial tests demonstrated that at 100 μm BIP did not markedly inhibit the growth of these cells to confluency and did not cause significant changes in cell morphology or survival in confluent cultures (data not shown). Plates were titered for the number of viable virus produced after 48 h of infection (Fig. 1). The data show that BIP caused a 3–4-log decrease in viral yield with the sharpest decrease occurring between 40 and 80 μm. Early viral gene expression precedes DNA replication. Both are required for intermediate and late gene expression. A survey of protein production during a vaccinia infection serves as a temporal measure of the progression of the infectious cycle. To determine where in this cycle the BIP-dependent inhibition of virus formation occurred, cells were pulse-labeled with [35S]methionine at different times, i.e. hours post-infection (hpi). Radiolabeled proteins were separated by SDS-PAGE and observed by autoradiography (Fig. 2). Analysis of protein synthesis in the control infected cells (no BIP) demonstrated the onset of viral early protein production (examples designated by E in Fig.2) within 3 hpi followed by the shut-off of synthesis of these and host proteins at 6 hpi. The loss of host and early virus gene expression precedes onset of the viral late protein synthesis that is dependent on the initiation of viral DNA replication. The effect of BIP (100 μm in this experiment) was striking. BIP did not inhibit the production of viral early proteins. However, it delayed the shut-off of viral early and host protein synthesis and prevented the accumulation of late viral proteins. This phenotype would be consistent with the inhibition by BIP of viral DNA replication. Viral DNA replication was assessed directly by measuring the rate of [3H]thymidine incorporation into DNA at different times post-infection. A typical time course of labeling is shown in Fig.3. Although the non-infected control cells (closed triangles) exhibited a constant level of [3H]thymidine incorporation (average value over the 9-h experiment, 12,650 cpm), the vaccinia-infected cells showed a 10-fold increase in incorporation at 3–6 hpi that then declined to control levels at 9 hpi (open circles). BIP inhibited this virus-dependent burst of DNA synthesis in a concentration-dependent manner that closely paralleled the concentration-dependent effect on viral yield seen in Fig.1. Thus, 40 μm BIP inhibited [3H]thymidine incorporation by <10% (open triangles), whereas 60 μm BIP inhibited >90% (closed circles) with little further inhibition at 100 μm BIP (labeling not different from uninfected control cells, closed triangles). This result also was fully consistent with the inhibition by BIP of late protein synthesis (Fig. 2) since expression of late genes, but not early ones, requires concurrent DNA synthesis. In summary, both labeling experiments indicate that BIP inhibits viral DNA synthesis and that this results in a block to further progression of the infectious cycle including expression of viral intermediate and late genes. This block was reversible as indicated by the additional results shown in Fig. 3. At 6 hpi, the BIP (100 μm) was removed from a set of cultures, and DNA synthesis was measured in these now BIP-free cells. Within 1 h, DNA synthesis began and followed a time course similar to the virus-infected cell controls (cf. open squares to open circles). This recovery of virus-dependent DNA synthesis led to a productive infection since the viral yield from these cultures was equivalent to the yield from cultures that had never been treated with BIP (2.8 versus 2.5 × 109 plaque-forming units, respectively). Viral infected cells from which the BIP had not been removed did not exhibit this DNA synthesis pattern at any time over the 12-h experiment (closed squares) and exhibited the 3-log decrease in viral yield shown in Fig. 1. BIP appears to block virus production by inhibiting viral DNA replication. Thus, BIP appears to work early in the infectious cycle. If the BIP effect was restricted to an early step in infection, then at some time point post-infection, BIP would no longer be able to inhibit. This inference was tested by measuring both incorporation of [3H]thymidine at 6 hpi and viral yield at 48 hpi in cells that were treated with BIP (100 μm) at various times post-infection. In Fig. 4 the DNA synthesis in these cells was compared with that in uninfected cells (mock) and in control infected cells (none, no BIP added). The data demonstrate that to inhibit strongly DNA synthesis at 6 hpi, BIP must be added by 3 hpi (stippled bars). A similar result was observed in regards to the inhibition of viral yield (Fig. 5, closed circles). The results were completely consistent with the inference above that BIP acted early in infection and that the mechanism of BIP inhibition of virus production was linked to the BIP inhibition of DNA synthesis.Figure 5Bypyridyl, hydroxyurea, and Ara-C inhibit viral replication at different times post-infection; a membrane-impermeant Fe2+ chelator, BPS , does not inhibit. BIP (100 μm, closed circles), hydroxyurea (10 mm, open triangles), and Ara-C (100 μg/ml, open squares) were added to vaccinia-infected monolayers of BSC40 cells at the times post-infection indicated in the figure. The viral yield in these infected cells was then determined at 48 hpi by a standard plaque assay. The inhibition of vaccinia virus infection by the membrane-impermeant Fe2+ chelator, BPS (100 μm), was also tested. This reagent was without inhibitory effect (open circles). The data in the figure are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To probe further the mechanism of the BIP inhibition, the temporal effect of other inhibitors on viral DNA synthesis and yield was determined. In particular, HU inhibits active RR by reducing the ferric iron core. This results in the loss of the tyrosyl radical essential to the catalytic activity of this enzyme (27Howell M.L. Sanders-Loehr J. Loehr T.M. Roseman N.A. Mathews C.K. Slabaugh M.B. J. Biol. Chem. 1992; 267: 1705-1711Abstract Full Text PDF PubMed Google Scholar). RR is an early protein produced by vaccinia and other large DNA viruses and is required for a productive infection (20Prichard M.N. Shipman Jr., C. Chemotherapy. 1995; 41: 384-395Crossref PubMed Scopus (11) Google Scholar, 26Slabaugh M.B. Davis R.E. Roseman N.A. Mathews C.K. J. Biol. Chem. 1993; 268: 17803-17810Abstract Full Text PDF PubMed Google Scholar, 27Howell M.L. Sanders-Loehr J. Loehr T.M. Roseman N.A. Mathews C.K. Slabaugh M.B. J. Biol. Chem. 1992; 267: 1705-1711Abstract Full Text PDF PubMed Google Scholar). Alternatively, we employed cytosine arabinoside (Ara-C), which directly inhibits DNA polymerization. The temporal sensitivity of viral-dependent DNA synthesis to these two inhibitors is shown in Fig. 4. The corresponding inhibition of viral yield is shown in Fig. 5. Unlike BIP, HU inhibited DNA synthesis by 70% even if added at 5 hpi (Fig. 4, shaded bars), whereas Ara-C retained full inhibition when added at this time (solid bars). A similar pattern was observed in regard to viral yield (Fig. 5). HU (open triangles) and Ara-C (open squares) were equally effective in inhibiting viral yield when added up to 2 hpi. However, differences were observed when these inhibitors were added at later times. In contrast to BIP, HU was fully effective when added up to 3 hpi, while in contrast to both of the other compounds Ara-C was fully effective even when added 4 hpi. The premise underlying our original prediction that BIP would inhibit vaccinia infection was that this lipophilic, membrane-permeant Fe2+ chelator would readily diffuse into cells and sequester iron within the cells that otherwise would be available in support of viral replication. The data show clearly that BIP does inhibit viral replication. A membrane-impermeant iron chelator was used" @default.
• W1978670534 created "2016-06-24" @default.
• W1978670534 creator A5010922704 @default.
• W1978670534 creator A5023724784 @default.
• W1978670534 creator A5026280856 @default.
• W1978670534 creator A5077919054 @default.
• W1978670534 date "2001-06-01" @default.
• W1978670534 modified "2023-09-27" @default.
• W1978670534 title "Intracellular Chelation of Iron by Bipyridyl Inhibits DNA Virus Replication" @default.
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