Ilhan Demirhan

D-Penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein

D‐Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV‐1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co‐transfected with plasmids containing HIV‐LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D‐penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration‐dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 μg/ml of D‐penicillamine. Earlier experiments have shown that D‐penicillamine at 40 μg/ml can completely inhibit HIV‐1 (HTLV‐III B) replication in H9 cells [(1986) Drug Res. 36, 184–186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV‐1 replication by D‐penicillamine.

Detection of distinct patterns of anti-tat antibodies in HIV-infected individuals with or without Kaposi's sarcoma.

Patterns of antibody response to recombinant transactivator protein (HIV-1 tat) in serum samples from HIV-1-negative persons (n = 60), HIV-1-infected asymptomatic persons (n = 20), HIV-1-infected people with Kaposis sarcoma (n = 25) and of people with Kaposis sarcoma without HIV-1 infection have been analyzed. None of the healthy people had anti-tat IgG in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera have anti-tat IgG to all the functional domains of tat protein. Four of the 25 HIV-1-infected patients with Kaposis sarcoma were anti-tat IgG-positive; however, epitope analysis revealed that IgG to functional domains of tat protein, in particular to TAR-binding site, were absent. All patients with Kaposis sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG, and prevalence of different antibody profiles in different groups of patients suggest the pathophysiologic role of tat protein. Thus, a passive immunization with anti-tat IgG could be a useful strategy to influence the pathophysiologic state of the disease.

Intercellular traffic of human immunodeficiency virus type 1 transactivator protein defined by monoclonal antibodies

Monoclonal antibodies (mAbs) directed against the amino‐terminal region (N‐terminal sequence 2–19) of transactivator protein (tat) of HIV‐1 have been shown to inhibit intercellular transactivation mediated by the extracellular tat protein. The intracellular transactivation was not significantly affected by anti‐tat mAbs. The specificity of anti‐tat mAbs in abolishing the transactivating potential of extracellular tat is documented by studies with mAbs to HIV‐1 reverse transcriptase, or to a human mammary cancer protein. None of these antibodies showed any inhibitory effect on intercellular transactivation. Specific interaction of anti‐tat IgG with tat protein expressed in Jurkat cells is further supported by experiments on immunoblotting. Extracellular tat is responsible for signals which induce a variety of biological responses in HIV‐infected cells, as well as in uninfected cells. The fact that anti‐tat mAbs can abolish the intercellular traffic of tat protein offers a unique strategy in the development of vaccines against AIDS.

Docking studies reveal a selective binding of D-penicillamine to the transactivator protein of human immunodeficiency virus type 1

DOCK and Affinity studies were carried out to study the binding of D‐ and L‐penicillamine to the transactivator protein (tat) of human immunodeficiency virus type 1 (HIV‐1). These studies reveal a selective binding of D‐penicillamine to the cysteine‐rich region covering amino acid residues 20–38 of the tat protein. A careful analysis of the components of the binding energy of the D‐ and L‐isomers reveals that the D‐isomer has a more favorable van der Waals interaction resulting from an optimal placement of the dimethylthiomethyl side chain in the binding site. This observation matches the experimental data that D‐penicillamine is a more potent inhibitor of tat‐mediated transactivation than the L‐isomer. The docking and experimental data offer an interesting approach to design structural molecules with potential application to block signal functions of the tat protein in HIV‐1 pathogenesis.

Inhibitors of DNA polymerases: their selectivity and mode of action.

The first DNA polymerase in mammalian cells was discovered by Bollum (1958), but only in the last years the existence of multiple forms of DNA polymerases was recognized in these cells. In the recent years, there has been a great interest in the purification and characterization of different eukaryotic DNA polymerases, and to study the functional role(s) of each of these enzymes. The field of DNA polymerases gained an unexpected importance as in 1970, a novel DNA polymerase was discovered as a constituent of retrovirus (Temin and Mizutani, 1970; Baltimore 1970), now known as reverse transcriptase (RT). In addition, several new DNA-polymerizing activities are induced in eukaryotic cells infected with DNA viruses. Another unique enzyme which also exhibits a deoxyribonucleotide polymerizing activity is the terminal deoxyribonucleotidyl transferase (TdT). This enzyme, though a cellular enzyme, is unique in the sense that, so far, only thymus tissue has been shown to contain it. In the strictest sense TdT is not a DNA polymerase as it does not require a template to direct the DNA synthesis; nevertheless, it does catalyze the incorporation of deoxyribonucleotides into DNA and thus we consider it here as one of the DNA synthetic enzymes. Like RT the TdT also merits considerable attention as a biochemical marker’for certain types of leukemias, as will be discussed by Dr. Sarin and Dr. Mertelsmann is this proceeding.