John P. Bader

THE ROLE OF DEOXYRIBONUCLEIC ACID IN THE SYNTHESIS OF ROUS SARCOMA VIRUS.

Abstract The role of deoxyribonucleic acid (DNA) in the growth of Rous sarcoma virus (RSV) was examined utilizing actinomycin and 5-bromodeoxyuridine (BUDR). Actinomycin D (3 μg/ml) suppressed synthesis of RSV in chick embryo cells within 4 hours after addition and prior to any obvious change in the microscopic morphology of the cells. Growth of vesicular stomatitis virus, an RNA-containing virus, was not inhibited by actinomycin. BUDR also inhibits growth of RSV. Cells pretreated with BUDR could support the growth of RSV if BUDR was removed, but exposure of pretreated cells to additional BUDR inhibited virus growth tenfold. Cultures treated with BUDR in which no cellular growth was detectable and DNA synthesis was minimal could still support the growth of RSV. Inhibition of RSV growth by BUDR was reversible by thymidine. These results indicate that the growth of RSV requires the synthesis of a new DNA, and the possibility arises that the RSV particle may contain a DNA moiety.

The requirement for DNA synthesis in the growth of rous sarcoma and rous-associated viruses

Abstract Antagonists of DNA inhibited the growth of Rous sarcoma virus (RSV) and Rous-associated virus (RAV). Actinomycin D and mitomycin D inhibited the growth of both viruses and demonstrated a general involvement of DNA in virus synthesis. A specific requirement for DNA synthesis in virus growth was demonstrated using 5-iododeoxyuridine and cytosine arabinoside. Inhibition of virus growth by 5-iododeoxyuridine was reduced by the presence of thymidine in the medium, and deoxycytidine prevented inhibition by cytosine arabinoside. The requirement for DNA synthesis in the growth of RSV was transitory, which suggests that a viral DNA “primer” is synthesized in infected cells during the growth cycle of RSV.

Thiazolobenzimidazole: Biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

Biological and biochemical anti-HIV activity of the benzothiadiazine class of nonnucleoside reverse transcriptase inhibitors

A series of benzothiadiazine derivatives were screened against the human immunodeficiency virus (HIV) and certain structure-activity relationships were defined for anti-HIV activity in this chemical class. The selected representative NSC 287474 was a highly potent inhibitor of HIV-induced cell killing and HIV replication in a variety of human cell lines, as well as in fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2, and also against both nevirapine- and pyridinone-resistant strains (N119 and A17) of HIV-1, which are cross-resistant to several structurally diverse nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase, but not HIV-2 reverse transcriptase. Combination of NSC 287474 with AZT synergistically inhibited HIV-1-induced cell killing in vitro. The compound did not inhibit the replication of the Rauscher murine leukemia retrovirus or the simian immunodeficiency virus. The benzothiadiazine class of compounds represents a new active anti-HIV-1 chemotype within the diverse group of nonnucleoside reverse transcriptase inhibitors.

3-Deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase, inhibits reproduction of rous sarcoma virus and transformation of chick embryo cells

Abstract 3-Deazaadenosine, a known potent inhibitor of adenosylhomocysteine hydrolase, exerts an inhibitory effect on the reproduction of Rous sarcoma virus (RSV-BH) and on the malignant transformation of chick embryo cells by this virus. The inhibitory effect is reversible. The increased capacity for glucose uptake, a biochemical characteristic of chick embryo cells transformed by RSV-BH, is inhibited by 3-deazaadenosine, and the ability of the infected cells to grow in suspension is blocked. After prolonged exposure to 3-deazaadenosine, the morphological phenotype characteristic of transformed cells largely disappeared, and transformed cells resembled noninfected cells. Deazaadenosine inhibits the reproduction of Sindbis virus, Newcastle disease virus, and vesicular stomatitis virus to a lesser degree than RSV-BH. Deazaadenosine, 0.1 m M , has no effect on DNA or protein synthesis in cells, and only a slight effect on RNA synthesis. No incorporation of 3-deaza[ 14 C]adenosine into cellular nucleic acids was found. Deazaadenosine produces an increase in the intracellular level of adenosylhomocysteine, with the concomitant appearance of a relatively large amount of 3-deazaadenosylhomocysteine; the ratio of intracellular adenosylmethionine to adenosylhomocysteine, or (adenosylhomocysteine + 3-deazaadenosylhomocysteine) is decreased from 150 to 19, and 1.4 respectively. It is postulated that 3-deazaadenosine inhibits virus activities by its ability to inhibit adenosylhomocysteine hydrolase, resulting in an inhibition of methylation reaction(s) required for virus growth and replication.

The uptake of nucleosides by cells in culture. I. Inhibition by heterologous nucleosides.

Abstract Millimolar levels of nucleosides inhibited the uptake of trace levels of other, labeled nucleosides into the RNA and DNA of chicken, rat and mouse cells in culture. The incorporation of label into cold acid-soluble and insoluble fractions was invariably inhibited to the same extent. Every ribo- and deoxyribonucleoside tested inhibited the uptake of each of the other nucleosides. The effect was immediate and rapidly reversible. The synthesis of RNA was shown not to be inhibited, since the incorporation of 32P into RNA was unaffected by excess nucleosides. In contrast, the incorporation of 32P into DNA was inhibited by thymidine, which is known to block the formation of deoxycytidine triphosphate. The active inhibitory compound probably is the nucleoside itself rather than a phosphorylated derivative. Cells deficient in thymidine kinase activity were as responsive to the inhibitory action of thymidine as were cells containing the kinase. Also, the extracted nucleoside kinases were generally unaffected by the various nucleosides (and their derivatives) found inhibitory in whole cells. The results suggest enzyme-like transport systems for nucleosides susceptible to inhibition by heterologous nucleosides. The use of radioactive nucleosides in the study of the metabolic effect of millimolar levels of other nucleosides may be complicated by the block occurring in their assimilation into the intracellular pool.

Studies on the relationship between glycolysis and (Na++K+)-ATPase in cultured cells

In several tissues a coupling between glycolysis and (Na+ + K+)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na+ + K+)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na+ + K+)-ATPase was estimated by the initial rate of ouabain-sensitive K+ influx after K+ reintroduction to K+-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K+-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na+ due to other transport processes related to glycolysis, such as Na+-H+ exchange, Na+-glucose cotransport, and K+-H+ exchange were ruled out as mediators of this effect on (Na+ + K+)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na+ + K+)-ATPase to these cultured cells.

Metabolic requirements for infection by Rous sarcoma virus: I. The transient requirement for DNA synthesis

Abstract The replication of Rous sarcoma virus (RSV) was demonstrated to require DNA synthesis only during the early stages of the infectious cycle. A specific inhibitor of DNA synthesis, d -arabinosyl cytosine (cytosine arabinoside), prevented virus growth if added within 8 hours after initial exposure of cells to RSV, but virus growth was unaffected by later addition of the antibiotic. The addition of puromycin or 2,6-diaminopurine riboside later than 8 hours after RSV prevented further increase of infectious virus, demonstrating that RNA and protein synthesis are continuously required for growth of RSV. Cells maintained on a serum-deficient medium lost the ability to synthesize DNA, while RNA and protein synthesis continued at a high rate. Cells infected after serum depletion yielded little virus if maintained on serum-deficient medium, but cells infected prior to serum depletion produced large quantities of virus for as long as 39 days after serum depletion. These experiments support the interpretation of antimetabolite data and demonstrate that the DNA required for virus replication is not a “pool” of DNA synthesized early in infection and incorporated later into virions. A requirement for protein synthesis during the early phase of RSV-infection was examined using puromycin and d -arabinosyl cytosine. The DNA-synthetic phase proceeded in the presence of puromycin, demonstrating that preexisting cellular enzymes are sufficient to establish the required DNA.

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

Abstract The synthesis of a new analog of adenosylhomocysteine (AdoHcy), 5′-deoxy-5′-(isobutylthio)-3-deazaadenosine, is described. The compound is a non-competitive inhibitor of AdoHcy hydrolase, with a K i of 0.4 mM, as compared to a K i of 3 μM for 3-deazaadenosine, a competitive inhibitor of the enzyme. 5′-Deoxy-5′-(isobutylthio)-3-deazaadenosine is not hydrolyzed by AdoHcy hydrolase. 5′-Deoxy-5′-(isobutylthio)-3-deazaadenosine when tested has a selective antiviral activity against Rous sarcoma virus in chick embryo cells, and against Gross murine leukemia virus in mouse embryo cells. Possible mechanisms of this anti-viral activity are discussed.

Inhibitors of glycosylation reverse retroviral interference

Abstract Cells which are productively infected with retroviruses are extremely resistant to superinfection with the homologous virus. We found that in certain cases, this resistance is largely or entirely eliminated by overnight treatment of the cells with either of two inhibitors of glycosylation (2-deoxyglucose or tunicamycin). This loss of viral interference was accompanied by a loss of gp70 from the cell surface, as demonstrated by surface labeling with 125 I and radioimmunoprecipitation. In other cell-virus combinations, these inhibitors gave much smaller reductions in viral interference and in the level of gp70 at the cell surface. The most likely explanation for these phenomena is that inhibition of glycosylation of the env precursor polyprotein prevents its subsequent processing into gp70 and transport to the cell surface; to the extent that the cellular pool of gp70 is depleted during an overnight treatment, cellular receptors become available for interaction with superinfecting virus particles.