Yael Teitz

Inhibition of human immunodeficiency virus by N-methylisatin-β4′:4′-diethylthiosemicarbazone and N-allylisatin-β-4′:4′-diallythiosemicarbazone

Abstract N -methylisatin- β 4′:4′-diethylthiosemicarbazone(M-IBDET) and N -allylisatin- β -4′:4′-diallylthiosemicarbazone(A-IBDAT) inhibit the production of Human Immunodeficiency virus (HIV). Virus inhibition was related to the thiosemicarbazone derivative (TSCD) concentrations and time of treatment. Inhibition of HIV production was confirmed by various parameters of virus assay employing reverse transcriptase activity, plaque forming units (PFU) and levels of viral structural proteins. Effective antiviral TSCD concentrations ranged from 0.17 μM to 2.04 μM for M-IBDET, and from 1.45 μM to 17.4 μM for A-IBDAT. Treatment of the chronic HIV-infected cells for 48 h with 0.34 μM M-IBDET or 2.9 μM A-IBDAT caused about 50% inhibition in as virus yield ED 50 as assayed by the PFU method. Almost 2 logs of virus infectivity (PFU) was suppressed after 48 h of treatment with 17.4 μM A-IBDAT. Therapeutic index values of 20 and 30 were found for M-IBDET and A-IBDAT, respectively. A significant selective inhibition of HIV structural protein synthesis was shown by both M-IBDET and A-IBDAT.

Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.

Growth rate determines activity of porphobilinogen deaminase both in nonmalignant and malignant cell lines

PBGD activity and growth rate were determined in cultures of rat embryo fibroblasts, nontransformed and MLV/MS transformed fibroblastic cell lines; NIH-3T3 cells, and in a mouse lymphosarcoma cell line [L-929]. The two parameters examined correlate positively (P less than 0.001). The results of this investigation would seem to indicate clearly that porphobilinogen deaminase activity is related to growth. However, these experiments do not rule out the possibility that malignant transformation per se also causes changes in porphobilinogen deaminase activity.

Inhibition of Feline Immunodeficiency Virus Production and HIV Tat Activity by Thiosemicarbazone Derivatives

Two thiosemicarbazone derivatives (TSCD), N-methylisatin-β-4′:4′-diethylthiosemicarbazone (M-IBDET) and N-allylisatin-β-4′:4-diallylthiosemicarbazone (A-IBDAT), were tested for their anti-feline immunodeficiency virus (FIV) activity in FL4/FIV cells. This cell line consists of feline T-lymphocytes chronically infected with FIV. FIV production in FL4/FIV cells was inhibited by M-IBDET and A-IBDAT. Virus inhibition was proportional to drug concentrations and time of treatment. The effective antiviral drug concentrations ranged from 0.06 to 0.64 μm for M-IBDET and from 0.45 to 8.7 μm for A-IBDAT. Tests performed to determine the therapeutic index (TI) value for each drug indicated TI values of 10 and 20 for M-IBDET and A-IBDAT, respectively. Continuous treatment of the cells with low doses of the drugs, given at constant time intervals, for a whole month succeeded in suppressing the chronic infection. Experiments directed towards understanding the mode of inhibition of FIV by TSCD indicated that A-IBDAT is involved in repression of the synthesis of the p24 structural protein of FIV. Examination of the possibility that the TSCD are also involved in suppression of the activity of HIVs tat regulatory protein showed a clear dose-responsive inhibition of HIVs tat-mediated transactivation by M-IBDET and A-IBDAT.

Accelerated heme synthesis and degradation in transformed fibroblasts

Various parameters of the heme biosynthetic pathway were studied in two cell lines, one nontransformed and the other malignantly transformed (MLV/MS), both replicating at the same rate. Using the above system enabled us to distinguish between phenomena characteristic of the malignant transformation per se and those due to accelerated growth rate. Heme synthesis and degradation as well as the activities of ALAS, ALAD, PBGD, and FC were found to be increased in the transformed cells. However, the concentration of intracellular heme was markedly reduced from 30.4 +/- 4.4 pmole/mg protein in nontransformed cells to 10.5 +/- 2.6 pmole/mg protein in transformed cells. These observations show that malignant transformation leads to changes in heme metabolism unrelated to growth rate in this cell line.

Glycoprotein Enrichment in Moloney Leukemia Virus Structural Proteins Released from Interferon-Treated Cells

Interferon treatment of Moloney-leukemia-virus-infected cells (3T3/MLV) leads to the formation of virus particles enriched with viral structural glycoproteins, in addition to the inhibition of virus production. A preferential inhibitory effect on incorporation of RNA and proteins rather than glycoproteins was found in the released virus particles from interferon-treated cells. Enrichment in 70,000- and 45,000-dalton glycoprotein (gP-70, gP-45) in these particles was further demonstrated by polyacrylamide analysis of viral proteins pulse-labeled with [3H]-leucine. Viral glycoproteins released as soluble antigens were also determined. A 40% reduction was found in gP-70 and gP-45 released from interferon-treated cells. Radioimmunoprecipitation of pulse-chase-labeled cellular viral proteins showed no effect of interferon on the formation of viral structural 30,000-, 15,000- to 12,000-dalton proteins, and gP-70 and gP-45 from their respective precursors. The uncoordinate effect of interferon inhibition on viral 30,000-dalton protein and gP-70 is discussed.

RNA Directed DNA Polymerase in C-Type Particles from Normal Rat Thymus Cultures and Moloney Leukemia Virus

For the last few years we have been studying the metabolic process involved in the morphogenesis of the Moloney leukemia virus (MLV) and, particularly, in identifying and characterizing the enzymic system responsible for the synthesis of the RNA moiety (the genetic material) of this murine leukemia virus. Hopefully, such metabolic studies will throw light on how oncogenic viruses transform the cells they infect; however, it should be emphasized that different viral genes may be involved in viral reproduction in contrast to viral transformation.

Transplantation of Mouse Genes into Rat Cells Chronically Infected by Moloney Murine Leukemia Virus (M0-MuLV)

Three rat cell lines (RE, ES and RT21) chronically infected with Moloney Murine Leukemia Virus (M0-MuLV) became sensitive to infection with EMC virus and to the antiviral effect of mouse interferon. These two properties are specific to mouse cells only. EMC virus multiplies in M0-MuLV infected rat cells to a titer of 108 pfu and destroys the infected cells. Mouse interferon induces an antiviral state in these cells when challenged with EMC or VSV viruses, and causes inhibition of 97 per cent of M0-MuLV release. These properties are observed only in rat cells chronically infected with M0-LV obtained from mouse, but not in those infected with Moloney Leukemia Virus obtained from rat (M0-RLV).