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J Biol Chem, Vol. 275, Issue 5, 3693-3698, February 4, 2000

Molecular Mechanism of Decreased Glutathione Content in Human Immunodeficiency Virus Type 1 Tat-transgenic Mice^*

Jinah Choi, Rui-Ming Liu^§, Ramendra K. Kundu^¶, Frank Sangiorgi^¶, Weicheng Wu, Robert Maxson^¶, and Henry Jay Forman^§**

From the  Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, California 90033, the ^§ Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, Birmingham, Alabama 35294, the ^¶ Department of Biochemistry and Molecular Biology, University of Southern California/Norris Hospital and Research Institute, Los Angeles, California 90089, and the  Department of Pathology, University of Southern California School of Medicine, Los Angeles, California 90089

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human immunodeficiency virus (HIV) progressively depletes GSH content in humans. Although the accumulated evidence suggests a role of decreased GSH in the pathogenesis of HIV, significant controversy remains concerning the mechanism of GSH depletion, especially in regard to envisioning appropriate therapeutic strategies to help compensate for such decreased antioxidant capacity. Tat, a transactivator encoded by HIV, is sufficient to cause GSH depletion in vitro and is implicated in AIDS-associated Kaposi's sarcoma and B cell lymphoma. In this study, we report a decrease in GSH biosynthesis with Tat, using HIV-1 Tat transgenic (Tat+) mice. A significant decline in the total intracellular GSH content in liver and erythrocytes of Tat+ mice was accompanied by decreased -glutamylcysteine synthetase regulatory subunit mRNA and protein content, which resulted in an increased sensitivity of -glutamylcysteine synthetase to feedback inhibition by GSH. Further study revealed a significant reduction in the activity of GSH synthetase in liver of Tat+ mice, which was linearly associated with their GSH content. Therefore, Tat appears to decrease GSH in vivo, at least partially, through modulation of GSH biosynthetic enzymes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human immunodeficiency virus (HIV)¹ infection is associated with a systemic decrease in the GSH content in humans (1, 2). Such biochemical alteration appears to be a direct consequence of retroviral infection rather than a late and indirect consequence of advanced disease state, is progressive in nature, and often occurs in the absence of any symptoms associated with AIDS (2-5). The importance of this decreased GSH is suggested by the fact that decreased GSH can enhance HIV expression via activation of NF-B (4, 6, 7) and contribute to diverse immune dysfunctions found in AIDS, including the loss of CD4+ T lymphocytes via apoptosis (8-11). In fact, such decline in the GSH content may play a role in the increased drug toxicity and oxidative damage, often found in HIV-infected individuals (12-15). Furthermore, increased GSH or other sulfhydryl compounds can inhibit viral replication (7, 16), HIV-1 reverse transcriptase activity (16, 17), and the late stage of viral expression (18). GSH content may even predict the survival of HIV-seropositive (HIV+) individuals (19, 20). Therefore, it is perhaps crucial to determine whether this decrease in GSH occurs through an accelerated loss of GSH or specific modulation of its synthesis, especially as it relates to potential therapeutic strategies to compensate for such decreased antioxidant capacity. However, despite over a decade of intensive research on this subject, the exact mechanism(s) causing decreased GSH content with HIV remains unclear.

Cells maintain their high GSH content in part by reducing GSSG back to GSH, preventing the loss of GSH as GSSG (12). Also, GSH is synthesized from its amino acid constituents via two consecutive reactions catalyzed by -glutamylcysteine synthetase (GCS; glutamate-cysteine ligase, EC 6.3.2.2) and GSH synthetase (GS; EC6.3.2.3). In multiorgan systems, GSH can be supplied from one organ to help supply necessary amino acids for intracellular synthesis of GSH in other organs (21, 22). Some cells may even take up GSH in its intact form (23). Therefore, HIV-associated reduction in the GSH content can result from decreased GSH synthesis or an increased rate of loss due to increased consumption, degradation, or transport/leakage of GSH or from a combination of these factors. It has been suggested that GSH consumption may increase in HIV infection as a result of increased oxidative stress (14, 15, 24-28). Nevertheless, it is not clear whether this can explain the systemic GSH depletion in HIV+ patients, since even severe oxidative stress cannot deplete systemic GSH content in vivo (29). This is especially true because GSH synthesis can increase in an adaptive response to oxidants and xenobiotics via multiple mechanisms (23, 30-32). In fact, even if the cells were losing a significant amount of GSH or GSSG to the outside of the cells, total GSH, or at least cysteine, would be expected to increase, rather than decrease, in the plasma. Therefore, increased oxidative stress may not be sufficient to explain the systemic GSH depletion in HIV infection.

Alternatively, GSH can be suppressed as a result of decreased synthesis. Helbling et al. (33) reported that GSH input into circulation is reduced in HIV+ individuals. In addition, a systemic decrease in total GSH content (2, 33) and decreased metabolic labeling of GSH in HIV+ individuals (34) all suggest reduced GSH biosynthesis in HIV infection. Indeed, decreased GSH content may allow development of oxidative stress rather than oxidative stress causing the decrease in GSH concentration. GSH synthesis may be compromised in HIV+ individuals because of the reduced availability of cysteine (1, 35, 36), a limiting substrate for GSH biosynthesis, and cysteine supplementation may restore the GSH content in these individuals (19, 34). However, it has been debated whether the restoration of cysteine content truly restores intracellular GSH content in HIV+ patients (33, 36), suggesting some defect in the utilization of cysteine for GSH biosynthesis. Therefore, the molecular basis of decreased GSH synthesis in HIV infection has been obscure.

HIV type 1 (HIV-1) Tat is a transactivator protein encoded by HIV that is essential for efficient viral replication (37). Tat is associated with AIDS-related dementia (38) as well as two of the most frequent cancers associated with AIDS: Kaposi's sarcoma (39) and B cell lymphoma (40). Tat has been shown to lower total and reduced GSH concentration in vitro in various cell lines and also the total sulfhydryl content in vivo in a transgenic animal model (27, 41, 42). In addition, Tat is sufficient to cause both the enhanced activation of NF-B and the increased susceptibility to apoptosis, observed with HIV (42-44). Tat also enhances drug toxicity in vivo (27). Therefore, Tat may be a viral agent that participates in many of the pathological changes that occur upon HIV infection. Consequently, determining the mechanism whereby Tat decreases GSH content may serve as an important step toward understanding how HIV suppresses the GSH content in humans.

The aim of the present investigation, therefore, was to test whether GSH synthesis is decreased in Tat-transgenic (Tat+) mice and, in the process, determine the mechanism underlying such reduced GSH synthesis with Tat. A significant decrease in the GSH content in liver and erythrocytes of Tat+ mice was associated with specific modulation of GCS and GS, the enzymes involved in GSH de novo synthesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tat+ Mice and Reagents-- C57Bl mice (Jackson Laboratory) had been previously engineered to express HIV-1 Tat constitutively and ubiquitously under the control of the chicken -actin promoter (40). All of the Tat+ mice used were homozygous for Tat and were derived from a single line. Food and water were provided ad lib. On the day of the experiment, 14-month-old Tat+ mice and age-matched control mice (NIA, National Institutes of Health, Bethesda, MD) were sacrificed in CO₂ chambers. Immediately, blood was drawn directly from the heart and collected in EDTA-containing test tubes (VWR Scientific, San Diego, CA).

All high performance liquid chromatography (HPLC) solvents were Baker Analyzed HPLC-grade reagents from VWR Scientific. All other chemicals were at least analytical grade and obtained from Sigma, unless indicated otherwise.

Intracellular GSH and Cysteine Measurements-- Total intracellular GSH ([GSH] + 2 × [GSSG]) and total cysteine ([cysteine] + 2 × [cystine]) concentrations were measured by HPLC according to the well established method of Fariss and Reed (45). Immediately after obtaining samples, both erythrocytes and liver samples were washed twice in 1× phosphate-buffered saline and acidified in 10% perchloric acid, 2 mM EDTA that contained the internal standard, -glutamylglutamic acid (Bachem, Torrance, CA). Liver samples were precipitated and sonicated twice in this solution to ensure complete GSH recovery. Samples were further processed and analyzed for their GSH and cysteine contents as described previously (46).

Northern Analysis of GCS mRNA Content-- Liver samples were homogenized in Trizol reagent (Life Technologies, Grand Island, NY), and total RNA was isolated, following the manufacturer's protocol. The amount of GCS light subunit (GCS-LS), GCS heavy subunit (GCS-HS), and glyceraldehyde 3-phosphate dehydrogenase mRNAs was measured by Northern analysis and quantitated with an InstantImager (Packard Instrument Co., Meriden, CT), as described previously (46).

Western Analysis of GCS Proteins-- Mouse liver samples were sonicated briefly in 0.25 M sucrose, 1 mM EDTA, 20 mM Tris-HCl (pH 7.4) solution that contained a 2 µg/ml concentration each of leupeptin and aprotinin and 50 µg/ml phenylmethylsulfonyl fluoride. Erythrocytes were lysed by adding a 10× volume of distilled water. Following 20 min of centrifugation at 12,000 rpm and 1 h centrifugation at 100,000 rpm at 4 °C, supernatants were concentrated in Microcon-10 tubes (Millipore Corp., Bedford, MA). Then 10 µg of liver and 40 µg of erythrocyte cytosolic protein samples were denatured in 2% SDS, 6 M urea, and 300 mM dithiothreitol. Subsequently, GCS-HS and GCS-LS protein content was determined by Western analysis, using rabbit anti-rat GCS-HS and GCS-LS antibodies, as described previously (46).

GCS and GS Activity Assays-- GCS activity was determined in vitro, according to the method described by Yan and Huxtable (47) with slight modifications. After concentrating liver and erythrocyte proteins in Microcon-10 tubes as described under "Western Analysis of GCS Proteins," samples were washed further by repeatedly adding GCS lysis solution (0.1 M Tris-HCl, pH 8.2, 150 mM KCl, 20 mM MgCl₂, and 2 mM EDTA) to remove endogenous inhibitors, acceptors, and substrates. The reaction mixture consisted of GCS lysis solution that was supplemented with 25 mM sodium glutamate, 5 mM cysteine, 5 mM dithiothreitol, 10 mM ATP, and 0.04 mg/ml acivicin. Reactions were allowed to proceed for 30 min at 37°C upon adding protein at a final concentration of 0.1-1 mg/ml. Reactions were then terminated with 5% (w/v) 5-sulfosalicylic acid. After adjusting pH to 8-9 with 40 mM KOH/400 mM N-ethylmorpholine, samples were derivatized for 20 min in the dark with 3 mM monobromobimane (Calbiochem). Next, samples were acidified again with 5-sulfosalicylic acid and, after removing precipitated protein, were analyzed by HPLC, as described previously (46).

For the GS activity assay, glutamate was substituted with 30 mM glycine, and L-cysteine was substituted with 3 mM -glutamylcysteine (48). Dithiothreitol concentration was 6 mM. Protein was added to the reactions at a final concentration of 0-0.25 mg/ml.

Base-line levels of -glutamylcysteine and GSH were negligible, if present, in both GCS and GS activity assays; nevertheless, the base-line absorbance at the points where these compounds eluted was subtracted from each reaction. GCS and GS activities were both linear for at least 30 min at the protein concentrations used.

Statistical Analysis-- Data were expressed as means ± S.E. unless indicated otherwise and evaluated by Student's t test or one-way analysis of variance followed by the Tukey test. Pearson product moment correlation and linear regression analysis were used to identify statistically significant correlation or association between variables. For all analyses, p < 0.05 was considered statistically significant. The number of samples used in each experiment is indicated under "Results" or in the figure legends.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular GSH Content Is Decreased in Liver and Erythrocytes of Tat+ Mice Compared with Age-matched Controls-- HIV-1 Tat+ mice used in this investigation had been previously reported to develop B-cell lymphoma, but their livers did not display any histological abnormalities (40). However, total intracellular GSH content was significantly decreased to 57 ± 3% (Fig. 1A) and 80 ± 5% (Fig. 1B) of the age-matched control level in liver and erythrocytes of these mice, respectively. There was no significant difference in total GSH content between genders within each group (data not shown). A significant, positive linear correlation existed between liver and erythrocyte GSH content (r = 0.56; p < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Total intracellular GSH content is decreased in liver and erythrocytes from Tat+ mice compared with controls. Liver and red blood cell samples from age-matched control and Tat+ mice were processed immediately for their total intracellular GSH content. A, total liver GSH content ([GSH] + 2 × [GSSG]) was 49.1 ± 3.5 (n = 10) and 27.9 ± 1.6 (n = 6) in control and Tat+ mice, respectively. B, total GSH content in the red blood cells was 8.9 ± 0.4 (n = 10) in control mice and 7.2 ± 0.4 (n = 6) in Tat+ mice. Error bars represent S.D. *, statistically significant difference from control (p < 0.05).

The samples were also examined for their intracellular cysteine content. No significant change in the total cysteine content could be detected in liver (0.36 ± 0.07 and 0.23 ± 0.04 nmol/mg protein in control and Tat+ mice, respectively, p > 0.05) or erythrocytes (0.13 ± 0.02 and 0.12 ± 0.05 nmol/mg protein in control and Tat+ mice, respectively, p > 0.05) of control (n = 10) versus Tat+ mice (n = 6) with the statistical tests employed. There was no significant correlation between liver and erythrocyte total cysteine content (r = 0.38; p > 0.05). Furthermore, comparing the data with those in Fig. 1, no significant correlation was found between total GSH and cysteine concentrations in either liver (r = 0.31, p > 0.05) or erythrocytes (r = 0.31, p > 0.05).

GCS-LS mRNA Content Is Decreased in Liver from Tat+ Mice-- To test whether Tat modulated GCS, the first enzyme in GSH de novo synthesis, we determined the level of GCS heavy (catalytic, GCS-HS) and light (regulatory) subunit mRNAs in livers from control and Tat+ mice. Northern analysis detected one GCS-HS and two GCS-LS mRNA species in mouse liver, as in rat liver (Fig. 2A). GCS-HS mRNA content was not different between two groups of mice. There was also no significant change in the higher molecular weight GCS-LS mRNA band. However, the lower molecular weight GCS-LS mRNA content was significantly decreased to 56 ± 4% of the control level in Tat+ mice (Fig. 2B).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   GCS-LS mRNA content is decreased in liver samples from Tat+ mice compared with controls. A, total RNA isolated from control (n = 3) and Tat+ mice (n = 4) livers were analyzed by Northern analysis for GCS mRNA content. B, data are expressed as ratio of GCS to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA contents. The image was quantitated using InstantImager. *, statistically significant difference from control (p < 0.05).

GCS-LS Protein Content Is Decreased in Liver and Erythrocytes of Tat+ Mice-- Next, we examined whether the GCS-LS protein content was likewise reduced in Tat+ mice relative to controls by Western analysis. GCS-HS and -LS proteins were identified, based on the size of rat kidney GCS-HS and -LS proteins (data not shown). GCS-LS protein content was significantly decreased to 41 ± 16% of the control level in livers from Tat+ mice (Fig. 3A), consistent with the decrease in the lower molecular weight GCS-LS mRNA band (Fig. 2). Erythrocytes from the same mice exhibited a similar decline in the GCS-LS protein content, to 48 ± 20% of the control level (Fig. 3B). There was no statistically significant decline in GCS-HS protein content.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   GCS-LS protein content is decreased in liver and erythrocytes from Tat+ mice compared with controls. Liver (A) and erythrocytes (B) from control (n = 9) and Tat+ mice (n = 5) were tested for their GCS protein content. Ten µg of liver and 40 µg of erythrocyte cytosolic protein was analyzed by Western analysis, using rabbit anti-rat GCS-HS and GCS-LS antibodies. Black bar, control mice; gray bar, Tat+ mice. *, statistically significant difference from control (p < 0.05).

Decreased GCS-LS Protein Content Was Accompanied by Increased Sensitivity of GCS to Inhibition by GSH-- GCS-HS exhibits all of the catalytic activity of the holoenzyme and is feedback-inhibited by GSH (49). GCS-LS, on the other hand, is believed to decrease the K_m of GCS-HS for glutamate and increase its K_i for GSH (50). Consistent with the lack of any change in the GCS-HS protein content, GCS activity was not decreased in liver or erythrocytes of Tat+ mice measured with saturating substrate concentration (5.2 ± 0.4 in control versus 6.2 ± 0.4 nmol/mg/min in Tat+ mouse liver; 0.81 ± .05 in control versus 0.81 ± 0.03 nmol/mg/min in Tat+ mouse erythrocytes).

To determine the effect of decreased GCS-LS protein content in Tat+ mice, we examined the effect of GSH on GCS activity in liver and erythrocytes from the two groups of mice. GCS activity from control and Tat mouse liver and erythrocytes were measured at low (0.5 mM) and high (25 mM) glutamate concentrations in the absence and presence of 10 mM GSH, and the ratios of the GCS activity in the absence and presence of GSH at low and high glutamate within the same sample were compared between the Tat and control groups. The results suggest that the major difference between the GCS activity in control and Tat is that the latter is inhibited to a greater extent by GSH (Fig. 4). This greater GSH feedback inhibition would be expected to contribute to a lowering of the GSH steady state that was observed.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Sensitivity of GCS to inhibition by GSH is increased in liver and erythrocytes from Tat+ mice compared with controls. The ratio of the differences in GCS activity at low (0.5 mM) and high (25 mM) glutamate concentrations in the absence and presence of GSH was determined in control (n = 8 for liver; n = 10 for erythrocytes) and Tat+ mice (n = 5 for liver and erythrocytes). The sensitivity of GCS to inhibition by GSH was calculated as follows: sensitivity = (1/vol  1/voh)/(1/(1/(sol  soh))/((1/vil  1/vih)/1/(1/sil  1/sih)), where v represents GCS activity; s represents glutamate concentration; and the subscripts o, i, l, and h represent uninhibited ( GSH), inhibited (+ GSH), low glutamate and high glutamate, respectively. In the experiments here, sol = sil and sol = sil and soh = sih; therefore, sensitivity = (1/vol  1/voh)/(1/vil  1/vih). *, statistically significant difference from control (p < 0.05).

GS Activity Is Also Decreased in Livers from Tat+ Mice-- We also examined the possibility of a change in the activity of GS, the next enzyme in GSH biosynthesis. GS activity was significantly decreased to 73 ± 4% of the control level in Tat+ mouse liver (Fig. 5) but not in the erythrocytes (0.53 ± 0.03 in control versus 0.54 ± 0.01 nmol/mg/min in Tat+ mice, p > 0.05). Interestingly, GS activity was linearly associated with total intracellular GSH content of liver (r = 0.72; p < 0.01; r² = 0.51).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   GS activity is decreased in Tat+ mouse liver. Liver samples from control (n = 9) and Tat+ mice (n = 5) were analyzed for their GS activity by HPLC. *, statistically significant difference from control (p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acute infection of C57Bl mice with murine AIDS virus can diminish their liver GSH content 2 weeks postinfection (51). In this report, we show that HIV-1 Tat expression is sufficient to decrease GSH content in the same strain of mice and, furthermore, present a potential mechanism for this decreased GSH content: a specific modulation of GCS and GS. Specifically, down-regulation of GCS-LS in Tat+ mice was associated with an increased sensitivity of GCS to inhibition by GSH. No change in cysteine concentration could be demonstrated with the tests employed, perhaps due to the limitations in the instrument and the sample size. Regardless of whether cysteine content is altered in Tat+ mice, GCS is believed to be the rate-limiting enzyme in GSH biosynthesis (52), and therefore, such a decline in GCS-LS protein content is likely to result in a decreased rate of GSH biosynthesis. This, together with the down-regulation of manganese superoxide dismutase by Tat, may allow the observed reduction in GSH content. It is presently unknown whether the down-regulation of GCS-LS by Tat is sufficient to cause GSH depletion.

The role of GS modulation in decreased GSH concentration is less obvious. Although the high correlation between GS activity and GSH content (r = 0.72; p < 0.05) suggests a possible role of GS in determining GSH status, at least in mouse liver, GS is generally not believed to govern the rate of GSH synthesis. It is perplexing, therefore, how GS activity could be related to the GSH status. However, similar association between GS activity and GSH content has been reported by others (53, 54). In addition, while GCS is usually regarded as the rate-limiting enzyme in GSH synthesis (52), increasing evidence also suggests that GS activity can be regulated (55-62). Obviously, if GS activity falls significantly, it could become the rate-limiting step. -Glutamylcysteine, produced by GCS, has been suggested to be channeled efficiently to GS (63). It is known that physical interaction or close spatial arrangement of proteins that participate in a given signal transduction or biochemical pathway enables an efficient relay of signals to effector molecules as well as the channeling of intermediates between interacting metabolic enzymes (64). Thus, it is not at all surprising that the enzymes in GSH biosynthesis may be regulated coordinately and perhaps even interdependently, through protein-protein interaction. Nevertheless, the observed decline in the GCS-LS protein content may be sufficient to explain the decreased GSH content in Tat+ mice, and therefore, the physiological significance of ~30% decline in the GS activity in Tat+ mice remains unclear at the moment.

Our findings on Tat+ mice apparently lead to the next question, which is whether the modulation of GSH biosynthetic enzymes also occurs in HIV infection. A similar decline in GSH content has been found in the liver and erythrocytes of HIV+ individuals (34, 65). Hepatic GSH depletion is particularly interesting, as liver serves as a reservoir for HIV, and various cells found in liver, such as Kupffer cells, lymphocytes, sinusoidal endothelial cells, and even the hepatocytes themselves, can be infected by the virus (66). Thus, Tat may directly decrease GSH synthesis in the liver of HIV+ patients. This may explain the systemic decrease in the GSH content found in these individuals, because liver is the organ primarily responsible for GSH in circulation (21, 22). In addition, although HIV is not found ubiquitously in infected individuals, Tat is secreted by HIV-infected cells and also taken up rapidly by various cells (37). Thus, any cell that Tat enters can potentially suffer from decreased GSH biosynthesis. Moreover, Tat may affect GSH biosynthesis through an indirect mechanism, such as by production of diverse cytokines, which may also exert a systemic effect on GSH synthesis. Therefore, Tat is a potential viral agent that causes the systemic GSH depletion observed in humans infected with HIV. Whether GSH synthetic enzymes are indeed suppressed in HIV+ individuals in a Tat-dependent manner should be tested in the future.

It is uncertain whether a partial depletion in the GSH content will have significant effects on metabolism, and the answer is likely to depend on the GSH concentration and metabolic demands of each tissue. However, any effect of decreased GSH concentration will certainly be amplified in situations of increased oxidative burden. Due to the primary and secondary infections, HIV+ individuals tend to have activated respiratory burst of the immune cells, elevated level of pro-oxidative cytokine (24, 25), and increased intake of pharmaceutical compounds that either cause oxidative damage (e.g. zidovudine (27)) or require GSH for detoxification (13). These, along with a decrease in overall antioxidant capacity (14, 28), will undoubtedly create a higher demand for GSH and, in turn, result in an altered redox poise of cells. This may worsen various aspects of viral pathogenesis such as enhanced viral replication, potentiation of apoptotic cell death (44), and increased susceptibility to oxidative stress and drug toxicity. A 10-40% decrease in GSH content can completely inhibit lymphocyte activation in vitro (8). Indeed, the very fact that Tat modulates GSH biosynthesis in addition to suppressing the activity of manganese superoxide dismutase (27, 41, 42) may testify to the importance of redox regulation in HIV pathogenesis. Specifically, these pro-oxidative effects of Tat may enhance its NF-B-dependent transactivating potential (6, 7, 42, 43), suggesting that the effect of Tat on the GSH status is a specific and self-promoting event in HIV pathology. It should be noted, however, that this study was aimed at discovering the mechanism by which Tat decreases GSH content. The role that the decrease in GSH plays in HIV pathology was not addressed in the present investigation.

Finally, it is interesting that the symptoms of GS/GCS deficiencies and AIDS overlap. Both are associated with neurological complications (24, 67), decreased cysteine content (35, 36, 68), sensitivity to certain drugs (13, 67), abnormal leukocyte function (24, 67), and anemia (24, 67). It will be interesting to determine the possible role of GSH deficiency in these AIDS-associated symptoms as well as B-cell lymphoma and Kaposi's sarcoma, the two most frequent AIDS-associated cancers that are both associated with Tat (39, 40). Indeed, the wide spectrum of various aspects of HIV pathogenesis that may be related to changes in the GSH status is striking. The possible therapeutic value of strategies for restoring GSH in HIV infection remains a debatable issue. Nonetheless, the results here suggest potential avenues for further exploration.

	ACKNOWLEDGEMENT

We acknowledge Dr. Beth Schomer and Lin Gao for advice on Western analysis of proteins.

	FOOTNOTES

^* This work was supported by the Dolores Zohrab Liebmann Fellowship and National Institutes of Health Grant ES05511.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 1665 University Blvd., Ryals 317, Birmingham, AL 35294-0022. Tel.: 205-975-8949; Fax: 205-975-6341; E-mail: hforman@uab.edu.

	ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; HIV+, HIV-seropositive; GCS, -glutamylcysteine synthetase; GCS-HS, -glutamylcysteine synthetase heavy (catalytic) subunit; GCS-LS, -glutamylcysteine synthetase light (regulatory) subunit; GS, glutathione synthetase; HPLC, high performance liquid chromatography; Tat+, Tat-transgenic.

	REFERENCES

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