Membrane transport of antisense oligonucleotides (ODN) is an inefficient process which requires special carriers for their intracellular delivery. We have developed a delivery system for AS-ODN and their phosphorothioate analogues (AS-PTO) directed against human immunodeficiency virus type 1 (HIV-1) tat mRNA for efficient transfection of HIV-1 target cells. Protamine was used to complex AS-ODN and AS-PTO to form nanoparticles with diameters of about 180 nm and surface charges in the range of −18 to +30 mV. Cellular uptake of these nanoparticles was significantly enhanced compared to naked oligonucleotides. A double labeling technique with fluorescently tagged protamine and AS-ODN was used to follow the intracellular fate of the nanoparticles. Protamine/AS-ODN nanoparticles showed release of the antisense compound leading to specific inhibition of tat mediated HIV-1 transactivation. In contrast, protamine/AS-PTO complexes were stable over 72 h, and failed to release AS-PTO. These results demonstrate that protamine/AS-ODN nanoparticles are useful for future therapeutical application to inhibit viral gene expression.

ABSTRACT Tat is among the required regulatory genes of human immunodeficiency virus type 1 (HIV-1). Tat functions both within infected cells as a transcription factor and as an extracellular factor that binds and alters bystander cells. Some functions of extracellular Tat can be neutralized by immune serum or monoclonal antibodies. In order to understand the antibody response to Tat, we are defining antibody epitopes and the effects of natural Tat sequence variation on antibody recognition. The dominant Tat epitope in macaque sera is within the first 15 amino acids of the protein amino terminus. Together with a subdominant response to amino acids 57 to 60, these two regions account for most of the macaque response to linear Tat epitopes and both regions are also sites for the binding of neutralizing antibodies. However, the dominant and subdominant epitope sequences differ among virus strains, and this natural variation can preclude antibody binding and Tat neutralization. We also examined serum samples from 31 HIV-positive individuals that contained Tat binding antibodies; 23 of the 31 sera recognized the amino terminus peptide. Similar to binding in macaques, human antibody binding to the amino terminus was affected by variations at positions 7 and 12, sequences that are distinct for clade B compared to other viral clades. Tat-neutralizing antibodies to the dominant amino terminus epitope are affected by HIV clade variation.

Human herpesvirus 8 or Kaposi's sarcoma-associated herpesvirus (HHV8/KSHV) is believed to be the most important etiopathological factor of Kaposi's sarcoma (KS) and some specific types of malignant lymphomas. The diagnostic and prognostic significance of serum viral load in endemic (African) areas is poorly understood. In AIDS-related KS (AKS) it has been shown that HIV-Tat may be of pathogenic importance and that immunoreactivity to Tat may have prognostic significance. Here we report on the quantitative analysis of HHV8 DNA in serum from Tanzanian patients with KS (n = 19), either AIDS-related (AKS) (n = 14) or endemic KS (EKS) (n = 5) and non-KS control individuals (n = 4). Fourteen AKS sera were also tested for HIV-tat antibodies by a direct ELISA assay. In AKS patients detectable (12 out of 14) serum HHV8 DNA levels showed a median of 1400 copies/ml as compared to a median of 200 copies/ml for EKS, but for one AKS case with an exceptionally high level (25,500 copies/ml). The serum HHV8 DNA levels were usually higher in males (n = 17; median 580 DNA copies/ml) as compared to females (n = 6; median 120 DNA copies/ml) and in early, patch stages (n = 8; median 2,750 copies/ml) as compared to late, nodular stages (n = 11; median 200 DNA copies/ml). Of fourteen sera from AKS patients, seven were positive for antibody against HIV-1 tat. Epitope analysis of the anti-tat antibody spectrum showed reactivity to various non-functional sites, but not towards the functional epitopes 46-60 (TAR-binding region).

AIDS-associated Kaposi's sarcoma (AKS) is particularly aggressive and it is one of the principal neoplasms in regions of Africa affected by both high endemic HHV8 and epidemic HIV infection. In this study, serum samples from 18 patients with Kaposi's sarcoma from Tanzania, mostly males (n = 15 vs 3), were subjected to analysis with respect to HHV8-DNA load and antibody spectrum against the HIV-1 tat protein. Of the 18 patients, 14 were HIV-1-positive. The median HHV8 virus load in the HIV-1-positive group was 2075 DNA copies/ml, compared to 450 copies/ml in the HIV-1-negative group. In the HIV-1-positive group, the males had a higher HHV8-DNA virus load as compared to females (median: 4600 vs 1400 genome copies per ml). Since tat can promote AKS development (4-6) by intercellular signalling pathways, and these signals can be abolished by anti-tat IgG (7-9), we have examined the anti-tat IgG spectrum in this study. It would be expected that the levels of serum HHV8-DNA are higher in KS patients who have low anti-tat IgG titer, or who are anti-tat IgG-negative. In the present study, seven out of fifteen AKS patients were positive for anti-tat IgG. Although, we have not seen a strict quantitative relationship between serum anti-tat IgG and HHV8-DNA levels, our data appear to suggest a correlation between the two parameters. In view of these observations and the published data, we suggest that cross-signalling pathways between the tat protein and HHV8-DNA are involved in the complexity of pathogenesis of Kaposi's sarcoma.

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.

In our laboratory, the gene transfer efficiency of some lipofection reagents (lipofectine, lipofectamine, DOTAP and Dosper) and histones H3 and H4 was compared to that of DEAE-Dextran (64). The histones H3 and H4 were found to have the highest transfection efficiency of all the agents tested. In the present study we have analyzed other parameters important for gene delivery by the histones H3 and H4. We transferred the HIV-1 tat gene to Jurkat cells and measured the transactivation of HIV-1-LTR by the transactivator protein, expressed in Jurkat cells. The expression of CAT as a reporter gene hybridized to LTR was a direct measure of transactivation potential. In order to investigate whether the transfection was only due to the positive ionic character of the histones H3 and H4 we tested other histones (H1 and H2A) and polylysine in our system. Under our experimental conditions, neither polylysine, nor the histones H1 and H2A were able to promote gene transfer in Jurkat cells. The inability of these reagents to promote gene transfer was not dependent on DNA condensation; in EMSA (Electrophoretic Mobility Shift Assay) all these reagents exhibited a strong retardation of DNA. In the presence of anti-histone-IgG the transfection potential of histones H3 and H4 was diminished in a concentration - dependent manner. To investigate whether the histone antibodies inhibited the condensation of DNA by histones we carried out gel retardation assays (EMSA) in the absence and in the presence of histone antibodies. Anti-histone-IgG had no effect on the retardation of histone-DNA complexes; on the contrary, retardation was increased. This observation has led us to postulate two models for the possible mechanism by which the histones H3 and H4 catalyze gene transfer in eucaryotic cells.

We analyzed patterns of antibody response to recombinant transactivator protein (human Immunodeficiency virus type 1 [HIV-1] tat) in serum samples from HIV-1-negative subjects (n = 60), HIV-1-infected asymptomatic patients (n = 20), HIV-1-infected patients with Kaposi's sarcoma (n = 25), and patients with Kaposi's sarcoma without HIV-1 infection. None of the healthy subjects possessed anti-tat immunoglobulin G (IgG) in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera had anti-tat IgG to all the functional domains of tat protein. Histochemical studies on lymph nodes from five asymptomatic HIV-1-infected patients showed that, in all cases, tat-positive cells were present within the germinal center at the stage of follicular fragmentation containing immunoblasts and small lymphocytes. Of the 25 HIV-1-infected patients with Kaposi's sarcoma, 4 were anti-tat IgG-positive; however, the epitope analysis revealed that IgG to functional domains of tat protein--in particular to transactivating response element (TAR)-binding site--were absent. All patients with Kaposi's sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG and a prevalence of different antibody profiles in different groups of patients indicated 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.

We expressed tat protein from HIV-1 coding AA 1-67 (strain HIV-Bru) in the inducible vector pTrc 99A in E. coli. The tat coding region was cloned in the ATG site of the expression vector. A sequence coding for 15 AA was added at the 3'region as a molecular marker. After sonification, the tat protein was routinely detected in Western blots using the Mab developed by us. Following precipitation and centrifugation the resulting pellets were dissolved and purified by three different methods: 1. immunoaffinity-chromatography using Affi-gel HZ coupled with a Mab recognizing the N-terminal sequence of HIV-1-tat; 2. ion exchange chromatography using DEAE-52 cellulose, and 3. isoelectric focusing in free solution. The resulting protein extracts obtained from the three purification protocols were checked in ELISA with the antibody. The peak fraction from all the procedures showed tat activity. No cross reaction in the presence of sera from uninfected persons was observed. The results showed that the purification of tat protein using monoclonal antibodies leads to highly purified preparations.

D-Penicillamine, a structural analog of cysteine, has the ability to chelate metal ions and reacts with cysteine. We have shown earlier that D-Penicillamin is a potential inhibitor of tat-mediated transactivation of HIV-1-LTR (14) and possesses anti-HIV-1 activity (23). Following this approach, we evaluated the anti-tat and anti-HIV-1 activity of several sulfhydryl compounds with chelating properties. The tested compounds: N-(2-Mercapto-propionyl)-glycin (MPG), 2,3-Dimercapto-propanol (DMP) and 2,3-Dimercapto-propane-sulfonic acid (DMPS) exhibited an inhibitory effect on the tat-mediated transactivation in Jurkat cells, as well as in U937 cells. The highest inhibitory response was shown by DMP leading to about 50% inhibition of transactivation in Jurkat cels and an 80% inhibition in U937 cells. On the contrary, DMPS (30 micrograms/ml) had no inhibitory effect in U937 cells, but did exhibit a 50% inhibition of transactivation in Jurkat cells at 30 micrograms/ml. The antiviral activity of DMP and DMPS was evaluated in H9 cells. In the concentration range which is effective for antiviral effect, both the compounds were highly cytotoxic. Mercapto-propionyl-glycin, although a weak inhibitor of transactivation, was able to inhibit synctia formation to more than 90% and inhibit the viral antigene expression to about 70%. The concentration of MPG needed to achieve this antiviral effect was very high, but it had no cytotoxicity at this concentration. We suggest that a search for compounds using this approach may be useful in developing potential inhibitors of tat-mediated transactivation.

Analysis of protein secretion was performed for a macrophage-like cell line, which was established from the peritoneal cells of NMRI mice treated with 17 alpha-ethinylestradiol. The protein secretion pattern was investigated by computerized analysis of high resolution two-dimensional gel electrophoresis (2-D PAGE) and compared to that of control macrophages, intraperitoneally activated by bacterial lipopolysaccharide. The transformed cells encode a number of low molecular weight proteins (10-20 kDa), which were not observed in control cells under identical experimental conditions. In conclusion the transformation of peritoneal macrophages by 17 alpha-ethinylestradiol leads to an upregulation of polypeptides, in particular of low molecular weight proteins. A high similarity between the induced low molecular weight protein secretion by macrophages of 17 alpha-ethinylestradiol-treated and that of 2,3,7,8-tetrabromodibenzo-p-dioxin-treated mice was found.

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.

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 Kaposi's sarcoma (n = 25) and of people with Kaposi's 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 Kaposi's 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 Kaposi's 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.

We studied the gene transfer efficiency of lipofection reagents in comparison to DEAE-Dextran. DOTAP, Dosper, and Lipofectin have lower transfection efficiency; Lipofectamine has a 2.5-fold better efficiency compared with DEAE-Dextran. We report a novel and highly efficient DNA transfer system based on the DNA-binding proteins histone 3 and histone 4. We have transferred the HIV-1 tat gene and measured the transactivation of HIV-1 LTR by the transactivator protein, expressed in Jurkat cells. The HIV-1 LTR was linked to the CAT gene as a reporter. Compared to DEAE-Dextran-mediated transfection, histone-mediated transfection resulted in a sevenfold higher expression of the CAT gene. The maximum transfection efficiency mediated by histones is dependent on the relative concentration (DNA:histone ratio) and the incubation time. In a gel-retardation assay, an optimal complex formation was observed under the same conditions that allowed the highest transfection efficiency. This ability of histones to increase the delivery and transgenic expression of foreign DNA in eukaryotic cells is not simply due to the positive ionic character of the histone proteins. Polylysine, histone H1, and histone H2A were unable to mediate gene transfection in our system. Monoclonal antibodies that recognize antigenic determinant present on all five histone proteins (anti-histone, pan) were able to neutralize the transfection-enhancing potential of histone 3 and histone 4. However, anti-histone IgG enhanced the retardation of mobility of histone-DNA complexes. The results of this study allow us to conclude that histones H3 and H4 can catalyze gene transfer and gene expression in eukaryotic cells without any requirement for additional constituents. For this reason, we have termed the new gene-delivery system as histonefection.

The protein secretion patterns in a macrophage-like cell line (CBrD), established from the peritoneal cells of NMRI mice treated with the dioxin analog 2,3,7,8-tetrabromodibenzo-p-dioxin (TBrDD), were analyzed by high resolution two-dimensional gel electrophoresis (2-D PAGE), and compared to the pattern of proteins secreted by control macrophages which were intraperitoneally activated by bacterial lipopolysaccharide. The most striking alterations were observed in the low molecular range. The transformed cells encode a number of low molecular mass proteins (10-20 kDa) which were not detected in control cells under identical experimental conditions. The protein pattern with respect to isoelectric point, molecular weight, optical density (OD) and area of the spot (in mm2) has been depicted by computer analysis in relation to a standardized spot outline and the spot's background (in OD). It is concluded that the transformation of murine peritoneal macrophages by TBrDD leads to an upregulation of proteins, in particular of low-molecular-weight proteins.

The reverse transcriptase - RNA dependent DNA polymerase of Bovine Leukemia Virus (BLV) was isolated and characterized. The enzyme has a molecular weight of about 80kd and the isoelectric point is 7.6. The enzyme prefers magnesium, as a divalent cation using synthetic homopolymeric template primer poly (C) oligo (dG). Monoclonal antibodies directed against reverse transcriptase of human immunodeficiency virus type I (HIV-I) did not crossreact with the isolated polymerase.

We have used an in vitro approach to study the efficiency of antisense oligonucleotides in inhibiting LTR-(HIV-1)-directed CAT expression catalyzed by tat protein, the functional protein of the transactivator gene. We selected the target sequence localized near the 5' end of the tat mRNA. The following conclusions can be drawn from the data presented here: a) Antisense oligonucleotides modified by conjugation of cholesterol at the 3' end have a severalfold higher inhibitory response, b) inhibitory response is dependent on the mode of introducing oligonucleotides, and c) the inhibition by antisense oligonucleotides is sequence specific and directed towards the targeted region. This approach could be useful for targeting functional regions of regulatory gene products and designing gene-targeted inhibitors of virus replication.

We have established a cell line from peritoneal macrophages of mice treated intraperitoneally with N-methyl-N-nitrosourea. The present communication describes the identification of an RNA-dependent DNA-polymerase activity in a particulate fraction from supernatants of the cell culture. This activity is similar to retroviral Reverse Transcriptase (RT) based on its template specificity and ionic preference. The proof of the retrovirus-like nature of RT was obtained by ultracentrifugation of the pelleted proteins secreted in the medium on a sucrose gradient. The main RT activity was obtained in fractions of 1.14-1.16 g/ml densities, which are comparable to those of type C retroviruses. The presented data support constitutive expression of the retrovirus gene in a chemically transformed cell line.