©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of HIV Expression by Intracellular Calcium Pump Inhibition (*)

Béla Papp (§) , Randal A. Byrn

From the (1) Hematology-Oncology Research Laboratory, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have studied the role of intracellular calcium sequestration on human immunodeficiency virus (HIV) production by latently infected T-lymphocytic cells. Inhibition of the sarco-endoplasmic reticulum-type calcium transport ATPases by thapsigargin or cyclopiazonic acid induced activation of HIV production in the CEM-derived ACH-2 cells. An approximately 50% depletion of the thapsigargin-sensitive calcium pools as measured fluorimetrically of Indo-loaded cells fully activated virus production. Viral activation was manifest by increases in soluble viral core p24 production, increases in cellular immunofluorescent staining for viral antigens, and increased viral transcription as measured by HIV long terminal repeat-directed expression of the chloramphenicol acetyltransferase reporter gene. Virus induction could be blocked in a dose-dependent manner by the calcium channel blocker econazole. Virus production by the Jurkat-derived HIV-1-inducible J1.1 cells was not significantly stimulated by thapsigargin. These data indicate that intracellular calcium pool function is involved in the control of the transcription of proviral HIV in a cell type-specific manner within the T-lymphoid lineage and that ACH-2 cells represent a useful model for the study of calcium dependent activation of the transcription of proviral HIV.


INTRODUCTION

Intracellular calcium storage organelles play a key role in calcium signaling. These organelles sequester calcium ions from the cytoplasm through the action of the sarco-endoplasmic reticulum-type calcium transport ATPases (SERCA)() and release calcium into the cytoplasm via receptor operated calcium channels (see Refs. 1-5 and references therein). The depletion of intracellular calcium pools can be specifically achieved by inhibition of the SERCA-type calcium pumps using agents such as thapsigargin (Tg), cyclopiazonic acid, or di- tert-butyl benzohydroquinone (tBHQ, Refs. 6-13). These agents induce leakage of stored calcium into the cytosol, mimicking second messenger-induced calcium mobilization. The initial calcium release event sets in motion several distinct cellular signaling systems. Signals coupled to intracellular calcium pool depletion result in calcium influx into the cell (2, 14, 15, 16, 17, 18, 19, 20, 21) , and the changes in cytosolic calcium levels modulate calcium and calmodulin-regulated processes (2, 19, 20, 21, 22) . The use of SERCA inhibitors has already led to the observation that mobilization of intracellular calcium pools can generate signals that modulate cell activation and differentiation (3, 6, 23) , adaptation to altered metabolic conditions (24) , and expression of the c- fos, c- jun, and c- myb transcription factor genes (6, 25) .

Calcium homeostasis plays a key role in lymphocyte activation, and the resulting modulation of transcription factor activities could contribute to the regulation of HIV gene expression (26, 27, 28, 29, 30, 31, 32) . Recent studies of HIV pathogenesis have shown (33, 34, 35, 36, 37) that even during the asymptomatic phase of HIV disease, a significant fraction of lymphocytes found in lymph nodes carries proviral HIV, whereas only a small subfraction of these cells actively expresses virus at any given time. Thus the study of signaling systems involved in the regulation of HIV expression in latently infected cells may contribute to our understanding of the natural course of HIV-induced disease.

In the present work the effect of inhibition of the SERCA-type calcium pumps on the expression of proviral HIV in latently infected lymphocytic cells was studied. CEM-derived ACH-2 cells were treated with inhibitors of the SERCA-type calcium pumps and virus production was measured by supernatant viral core p24 antigen production and by immunofluorescent staining for HIV antigens. HIV LTR-driven transcription was studied using an LTR-CAT-transfected CEM-derived cell line. In parallel, intracellular calcium pool function was assessed by fluorimetry in Indo-loaded cells. Our results indicate that intracellular calcium pools associated with SERCA-type calcium pumps can exert an important control of the transcription of proviral HIV in latently infected cells.


MATERIALS AND METHODS

Chemicals

Cadmium chloride, caffeine, acetyl coenzyme-A, phorbol myristate acetate, ionomycin, fetal calf serum, hydrocortisone hemisuccinate, econazole, and Indo 1-AM were obtained from Sigma. TNF was obtained from Genzyme, Cambridge, MA. Thapsigargin, cyclopiazonic acid, and 2,5-di- tert-butyl-1,4-benzohydroquinone were obtained from Biomol Inc., Plymouth Meeting, PA.

Cell Culture and Inhibitor Treatments

The T-lymphoblastoid CEM-SS cells, as well as the chronically HIV-1 infected ACH-2 and J1.1 cells (38, 39) , were obtained from the AIDS Research and Reference Reagent Program, National Institutes of Health and were grown in RPMI 1640 medium (Mediatech Inc., Washington, D. C.) supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine (Mediatech). The cells were harvested in the exponential phase of growth, then centrifuged at 1000 g for 10 min. Cell pellets were washed twice with 45 ml of complete medium by centrifugation, as above, and resuspended in complete medium at a density of 1 10cells/ml. Thapsigargin, cyclopiazonic acid, or tBHQ were added to the cells from stock solutions of 5 mM, 10 mM, and 0.5 M in MeSO, or ethanol, respectively, and the cells were further cultured in a humidified incubator at 37 °C and 5% CO. The concentration of the MeSO and ethanol diluents was less than 0.1% in the experiments, was included in appropriate control experiments, and did not interfere with the assays.

Cell counts and viabilities were determined using a hemocytometer and the trypan blue exclusion test and by an MTT conversion assay as described previously (40, 41) . In several experiments these two techniques were performed simultaneously on the same cells and gave closely comparable results.

Calcium Flux Measurements

Cells were harvested by centrifugation and resuspended in complete medium at a density of 2 10cells/ml and incubated with 1 µM Indo 1-AM for 30 min at 37 °C. The cells were then centrifuged, and the cell pellet was resuspended in 45 ml of complete medium and centrifuged as above. The cells were thereafter resuspended in complete medium at a density of 2 10cells/ml and stored at room temperature. 3-ml cell suspensions were mixed with 10 ml of Tyrode-HEPES solution (3) , centrifuged, and the cells were resuspended in 3 ml of Tyrode-HEPES. Cytoplasmic calcium concentrations were recorded as described previously (3) using a Perkin-Elmer LS-5B spectrofluorimeter equipped with a thermostatically controlled (37 °C) cuvette holder and stirring capability (C. N. Wood & Co, Longhorn, PA). Free cytoplasmic calcium concentration was calculated as described previously (3, 42) .

Data presented in this work represent the results of at least three independent experiments.

Virus Quantitation

HIV production was assessed by quantitating core p24 concentration in culture supernatants using the DuPont HIV-1 antigen capture ELISA system (DuPont), according to the manufacturer's instructions. Samples were assayed in duplicate. In this work we tested several lots of ACH-2 cells of different passage history and obtained essentially similar results. A slight variation in the basal level of core p24 production (ranging from 150 to 700 pg/10cells/day), however, was observed. Indirect immunofluorescence staining of methanol fixed cells was performed using human HIV-positive serum and anti-human IgG-fluorescein isothiocyanate conjugate (Tago, Burlingame, CA) as described previously (43) .

HIV-specific transcription was assessed in a CEM-derived cell line expressing the chloramphenicol acetyltransferase gene driven by the HIV-1 LTR. The pU3R-III CAT plasmid was modified by insertion of the hygromycin-B-phosphotransferase gene (44) . Stable introduction of this expression vector into CEM-SS cells was accomplished by electroporation (44) . Selection of stable transfectants was performed by culturing the cells for 5 weeks in the above medium supplemented with 400 units/ml hygromycin-B (Calbiochem). The resulting line, BWV-899, showed low basal levels of CAT expression. CAT assays following thapsigargin treatment of the cells were performed by standard methods (45) . Chloramphenicol acetylation was quantitated using an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


RESULTS

Concentration-dependent Stimulation of HIV Production by Calcium Pump Inhibitors

Thapsigargin (Fig. 1 A) as well as cyclopiazonic acid (Fig. 1 B), two structurally unrelated inhibitors of the SERCA-type calcium pumps, induced a concentration-dependent stimulation of virus production of ACH-2 cells. The effect occurred in the low nanomolar range for thapsigargin and in the low micromolar range for cyclopiazonic acid. The proliferation of the cells was also inhibited by these agents, as detected by MTT conversion. Similar results on cell growth have been observed previously with uninfected cells (46, 47, 48) . The magnitude of the stimulation of HIV production by the cells was comparable with that obtained using maximally effective TNF or phorbol myristate acetate treatment (Fig. 1, C and D).


Figure 1: Concentration-dependent activation by calcium pump inhibitors of HIV production of ACH-2 T-cells. 10cells/ml were incubated with various concentrations of thapsigargin ( A), cyclopiazonic acid ( B), TNF ( C), and phorbol myristate acetate ( D). The supernatant viral p24 antigen concentrations ( squares) and the viable cell densities ( circles) were determined on day 5 of culture using ELISA and MTT conversion, respectively.



In the cases above, growth arrest accompanied virus induction. Similar effects ( i.e. growth arrest and augmentation of HIV production) were obtained with tBHQ and the calcium ionophore ionomycin in the concentration ranges of 6.25-50 and 0.03-2 µM, respectively. On the other hand, hydrocortisone (0.156-10 µM), caffeine (0.15-5 mM), and cadmium ions (4-250 µM) blocked the proliferation of ACH-2 cells in a concentration-dependent manner without stimulating HIV production (results not shown).

Time Course of HIV Production

As shown on Fig. 2A, thapsigargin treatment of ACH-2 cells induced virus production within 48 h and sustained stimulation of virus production was observed during a 5-day period. This resulted in an approximately 70-fold enhancement of HIV p24 production in treated cultures, as compared with controls.


Figure 2: Time course of thapsigargin action on HIV production and growth of ACH-2 cells. A, the cells were exposed to 12.5 nM thapsigargin ( circles) or diluent ( squares) at initial cell densities of 10cells/ml, and supernatant core p24 levels were measured daily for 5 days. B, growth of ACH-2 cells in the absence ( squares) and in the presence ( circles) of 12.5 nM thapsigargin. Thapsigargin treatment resulted in growth arrest. C, cell growth and viability of control ( squares) and 12.5 nM thapsigargin-treated ( circles) ACH-2 cells, measured by MTT conversion. D, viability of control ( squares) and thapsigargin-treated ( circles) ACH-2 cells, as determined by trypan blue exclusion. E, inhibition of thapsigargin-induced HIV synthesis by econazole. Cells were exposed to various concentrations of econazole and treated with 2.7 nM thapsigargin ( squares). As a control, cells were exposed to econazole in the absence of thapsigargin ( triangles). Relative virus production is expressed as the ratio of supernatant p24 concentrations (pg/ml) and the MTT metabolizing activity (milli-optical density) of the cells at day 4 relative to control ( i.e. with thapsigargin without econazole).



Thapsigargin treatment of these cells resulted in an arrest of cell growth, as measured by cell counts (Fig. 2 B) and by MTT conversion (Fig. 2 C), without significant loss of viability (Fig. 2 D). When ACH-2 cells were treated with 2.7 nM thapsigargin in the presence of various concentrations of the calcium channel blocker econazole (Fig. 2 E) for 4 days, a dose-dependent inhibition of thapsigargin-induced HIV production could be obtained in the low micromolar econazole concentration range. Econazole alone did not modify viability or virus production of ACH-2 cells.

Immunofluorescence Analysis of HIV Expression

When HIV production by ACH-2 cells was evaluated by indirect immunofluorescence, less than 5% of the cells stained positive at base line (Fig. 3 A). After 12.5 nM thapsigargin treatment for 5 days, more than 95% of the cells became positive for HIV antigens (Fig. 3 B). TNF treatment (1000 units/ml, 5 days) served as a positive control in these studies (39) and fully activated HIV production by ACH-2 cells (Fig. 3 C).


Figure 3: Immunofluorescent staining of HIV antigen production of latently infected cells. A, nonstimulated ACH-2 cells; B, ACH-2 cells stimulated for 5 days with 12.5 nM thapsigargin. C, ACH-2 cells after 5 days treatment with 10units/ml TNF. D, chloramphenicol acetyltransferase activity of CEM-SS cells carrying an HIV LTR-CAT construct treated by thapsigargin for 4 days. CAT activity is stimulated by thapsigargin in a concentration-dependent manner in the nanomolar range.



CAT Assays

In order to study the effect of inhibition of SERCA-type calcium pumps on HIV transcription in a cellular context similar to ACH-2 cells, CEM-SS were transfected with a plasmid carrying an HIV-LTR CAT construct. As shown on Fig. 3D, 4 days of treatment with thapsigargin in the nanomolar range resulted in a concentration-dependent enhancement of CAT activity. This enhancement, up to 8-fold, indicates that inhibition of SERCA-type calcium pumps modulates the transcription of HIV-1 directed by the viral LTR.

Calcium Flux Determinations

In order to correlate the extent of intracellular calcium pump inhibition with the degree of stimulation of HIV production, ACH-2 cells were exposed to thapsigargin and studied fluorimetrically using Indo-1. The inhibitor concentration of 12.5 nM used in these experiments induced a complete activation of HIV production (see Fig. 1). Although thapsigargin treatment of ACH-2 cells induced an acute rise in cytoplasmic calcium levels, the resting cytoplasmic calcium concentration of the cells returned to that of nontreated cells after an overnight incubation in the presence of the inhibitor (Fig. 4 A). Similar results have been reported previously for uninfected lymphoid cells (46, 48, 49) .


Figure 4: Calcium flux measurements in control ( Tg) and previously thapsigargin-treated ( +Tg) ACH-2 cells. A, at day 1 following 12.5 nM thapsigargin treatment the cells were treated with a second, 4 µM dose of thapsigargin in the absence of extracellular calcium. After the completion of the calcium release from internal pools, 0.5 mM calcium was added to the medium, and calcium influx was observed. B, thapsigargin-treated cells were loaded by Indo in calcium-free medium, and by consecutive additions of calcium and EGTA, the cells were placed in 0.5 mM calcium-containing and calcium-free medium. The thapsigargin-treated cells repeatedly exhibited an initial rise in the cytoplasmic calcium concentration when placed in calcium containing medium.



The addition of a second, higher dose of thapsigargin (4 µM) revealed that the internal calcium pools were partially depleted, as the second thapsigargin treatment resulted in an approximate 50% diminished calcium mobilization, as compared with untreated cells (Fig. 4 A). The initial thapsigargin treatment resulted in transient rises of the cytosolic calcium concentration when the cells were placed in calcium containing medium (Fig. 4 B), a phenomenon not seen in control cells. Similar results were obtained after 5 days of continuous thapsigargin treatment (results not shown). A partial depletion of the thapsigargin-sensitive calcium pools was therefore sufficient to fully activate HIV production.


DISCUSSION

As shown in this report, the inhibition of intracellular calcium sequestration by thapsigargin, cyclopiazonic acid, or tBHQ, three structurally unrelated inhibitors of the SERCA-type calcium pumps, fully activated HIV production by the latently infected ACH-2 T-cell line. Over a 5-day period of treatment more than 95% of the cells became positive for HIV antigen production as opposed to less than 5% prior to treatment. This stimulation occurred in the range of inhibitor concentrations previously shown to inhibit the SERCA calcium pumps in situ in membrane preparations made from uninfected cells (50) . Thapsigargin induction of HIV synthesis could be blocked by the calcium channel blocker econazole. This indicates that calcium influx into the cells through econazole-sensitive calcium channels, present in lymphoid cells (51, 52) , opened by the depletion of thapsigargin-sensitive calcium pools is involved in virus induction in ACH-2 cells.

Intracellular calcium pump inhibitor treatment induced the synthesis and release of soluble viral antigen into the culture supernatant and increased virus antigen expression within the cell, as measured by p24 ELISA and immunofluorescent staining, respectively, indicating de novo virus synthesis. On the transcriptional level, assuming that thapsigargin treatment did not modify CAT mRNA or protein turnover, it can be concluded that thapsigargin treatment of the CEM-SS-derived BWV-899 cells increased the transcription of the chloramphenicol acetyltransferase reporter gene directed by an HIV LTR. A 50% depletion of the thapsigargin-mobilizable calcium pool was sufficient for the maximal stimulation of HIV production. Such a treatment, similar to that seen on uninfected cells (3) , resulted in an enhanced calcium influx across plasma membrane calcium channels opened as a consequence of pool depletion.

Thapsigargin action appeared to be specific to ACH-2 T-cells. Virus production of the Jurkat-derived latently infected T-cell line J1.1 did not change significantly following exposure to this drug, even though growth arrest was observed. This is in agreement with other reported observations, indicating that HIV production by J1.1 cells is controlled by distinct mechanisms from those acting in ACH-2 cells (53) . Uninduced ACH-2 cells contain significantly higher levels of the viral rev protein than J1.1 cells (53) . As this protein is involved in the transition from latent to productive infection, it can be hypothesized that intracellular calcium pool depletion by SERCA inhibitors sets in motion-activating mechanisms that require a certain level of preexisting rev. Moreover, HIV synthesis is regulated by a complex set of cellular transcription factors (26, 27, 28, 29, 30, 31, 32) . Differences in the expression of transcription factors that are activated upon calcium pool depletion may also account for the different responsiveness of ACH-2 and J1.1 cells. Thapsigargin may be useful in future studies to elucidate the differences between the HIV control mechanisms of these two model T-cell types.

The mobilization of calcium from intracellular storage pools is a key component of lymphocyte activation. Since the activation of latently HIV-infected lymphocytes leads to virus production and cytopathy, the study of the calcium-dependent signaling systems involved in the regulation of HIV expression may contribute to our understanding of the natural course of HIV-induced disease.

SERCA inhibitors offer a unique tool to study the mechanisms by which intracellular calcium pools and calcium entry from the extracellular medium interact in regulating HIV expression, as, unlike the action of calcium ionophores, the selective depletion of intracellular calcium pools activates the cells' own calcium entry mechanisms. In this work this process was exemplified by the inhibition of thapsigargin-induced HIV synthesis by the imidazole-type calcium channel blocker econazole. Therefore the data and the new experimental model presented in this paper may be useful in elucidating the effects of calcium homeostasis on the virus production of latently infected cells.

HIV is known to induce several perturbations of the calcium-dependent cellular signal transduction system in the infected cell. Elevated resting cytoplasmic calcium concentrations and increased inositol trisphosphate levels have been observed (54, 55) . Such changes may activate calcium release from intracellular pools (1, 2, 22, 56, 57, 58, 59, 60, 61, 62, 63) and, as shown in our work, could thereby stimulate virus production. The activation of calcium signaling by HIV therefore may represent a positive feedback mechanism in the regulation of virus replication and may represent a new target for the pharmacological modulation of HIV expression.


FOOTNOTES

*
This work was supported in part by the Agence Nationale de Recherches sur le SIDA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to the memory of Lugosi Antalné.

§
To whom correspondence should be addressed: U 348 INSERM, Hôpital Lariboisière 8, rue Guy Patin 75010 Paris, France. Fax: 33-1-49-95-85-79.

The abbreviations used are: SERCA, sarco-endoplasmic reticulum-type calcium transport ATPase; HIV, human immunodeficiency virus; LTR, long terminal repeat; Tg, thapsigargin; tBHQ, 2,5-di- tert-butyl-1,4-benzohydroquinone; TNF, tumor necrosis factor-; CAT, chloramphenicol acetyltransferase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay.


ACKNOWLEDGEMENTS

We are indebted to Drs. J. E. Groopman, D. Zhang, J. Enouf, . Enyedi, T. Kovcs, and B. Sarkadi for helpful discussions. ACH-2 and J1.1 cells were obtained through the AIDS Research and Reference Program (Division of AIDS, NIAID, National Institutes of Health) contributed by Dr. T. Folks. The excellent technical assistance of H. Chhay and C. Falcione is greatly appreciated.


REFERENCES
  1. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205 [CrossRef][Medline] [Order article via Infotrieve]
  2. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  3. Sarkadi, B., Tordai, A., Homolya, L., Scharff, O., and Grdos, G. (1991) J. Membr. Biol. 123, 9-21 [Medline] [Order article via Infotrieve]
  4. MacLennan, D. H., Toyofuku, T., and Lytton, J. (1992) Ann. N. Y. Acad. Sci. 671, 1-10 [Medline] [Order article via Infotrieve]
  5. Grover, A. K., and Khan, I. (1992) Cell Calcium 13, 9-17 [CrossRef][Medline] [Order article via Infotrieve]
  6. Schaefer, A., Magocsi, M., Stocker, U., Kosa, F., and Marquardt, H. (1994) J. Biol. Chem. 269, 8786-8791 [Abstract/Free Full Text]
  7. Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336 [CrossRef][Medline] [Order article via Infotrieve]
  8. Thastrup, O., Foder, B., and Scharff, O. (1987) Biochem. Biophys. Res. Commun. 142, 654-660 [Medline] [Order article via Infotrieve]
  9. Thastrup, O., Linnebjerg, H., Bjerrum, P. J., Knudsen, J. B., and Christensen, S. B. (1987) Biochim. Biophys. Acta 927, 65-73 [CrossRef][Medline] [Order article via Infotrieve]
  10. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Bjerrum, P. J., and Hanley, M. R. (1989) Agents Action 27, 17-23 [Medline] [Order article via Infotrieve]
  11. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., and Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466-2470 [Abstract]
  12. Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W., Jr. (1989) J. Biol. Chem. 264, 12266-12271 [Abstract/Free Full Text]
  13. Kijima, Y., Ogunbunmi, E., and Fleischer, S. (1991) J. Biol. Chem. 266, 22912-22918 [Abstract/Free Full Text]
  14. Mason, M. J., Garcia-Rodriguez, C., and Grinstein, S. (1991) J. Biol. Chem. 266, 20856-20862 [Abstract/Free Full Text]
  15. Mason, M. J., Mahaut-Smith, M. P., and Grinstein, S. (1991) J. Biol. Chem. 266, 10872-10879 [Abstract/Free Full Text]
  16. Zweifach, A., and Lewis, R. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6295-6299 [Abstract]
  17. Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814 [CrossRef][Medline] [Order article via Infotrieve]
  18. Parekh, A. B., Terlau, H., and Stuhmer, W. (1993) Nature 364, 814-818 [CrossRef][Medline] [Order article via Infotrieve]
  19. Gardner, P. (1989) Cell 59, 15-20 [Medline] [Order article via Infotrieve]
  20. Grinstein, S., and Klip, A. (1989) Bull. N. Y. Acad. Sci. 65, 69-79
  21. Haverstick, D. M., and Gray, L. S. (1993) Mol. Biol. Cell 4, 173-184 [Abstract]
  22. Donnadieu, E., Bismuth, G., and Trautmann, A. (1992) J. Biol. Chem. 267, 25864-25872 [Abstract/Free Full Text]
  23. Tao, J., and Haynes, D. H. (1992) J. Biol. Chem. 267, 24972-24982 [Abstract/Free Full Text]
  24. Li, W. W., Alexandre, S., Cao, X., and Lee, A. S. (1993) J. Biol. Chem. 268, 12003-12009 [Abstract/Free Full Text]
  25. Schontal, A., Sugarman, J., Heller Brown, J., Hanley, M. R., and Feramisco, J. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7096-7100 [Abstract]
  26. Haseltine, W. A. (1991) FASEB J. 5, 2349-2360 [Abstract/Free Full Text]
  27. Stein, B., Baldwin, A. S., Jr., Ballard, D. W., Greene, W. C., Angel, P., and Herrlich, P. (1993) EMBO J. 12, 3879-3891 [Abstract]
  28. Molitor, J. A., Walker, W. H., Doerre, S., Ballard, D. W., and Greene, W. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 10028-10032 [Abstract]
  29. Nabel, G., and Baltimore, D. (1987) Nature 326, 711-713 [CrossRef][Medline] [Order article via Infotrieve]
  30. Perkins, N. D., Edwards, N. L., Duckett, C. S., Agranoff, A. B., Schmid, R. M., and Nabel, G. J. (1993) EMBO J. 12, 3551-3558 [Abstract]
  31. Pomerantz, R. J., Trono, D., Feinberg, M. B., and Baltimore, D. (1990) Cell 61, 1271-1276 [Medline] [Order article via Infotrieve]
  32. Negulescu, P. A., Shastri, N., and Cahalan, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2873-2877 [Abstract]
  33. Graziosi, C., Pantaleo, G., Demarest, J. F., Cohen, O. J., Vaccarezza, M., Butini, L., Montroni, M., and Fauci, A. (1993) AIDS 7, Suppl. 2, S53-S58
  34. Embertson, J., Zupancic, M., Ribas, J. L., Burke, A., Racz, P., Tenner-Racz, K., and Haase, A. T. (1993) Nature 362, 359-362 [CrossRef][Medline] [Order article via Infotrieve]
  35. Embertson, J., Zupancic, M., Beneke, J., Till, M., Wolinsky, S., Ribas, J. L., Burke, A., and Haase, A. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 357-361 [Abstract]
  36. Pantaleo, G., Graziosi, C., Demarest, J. F., Butini, L., Montroni, M., Fox, C. H., Orenstein, J. M., Kotler, D. P., and Fauci, A. S. (1993) Nature 362, 355-358 [CrossRef][Medline] [Order article via Infotrieve]
  37. Fauci, A. S. (1993) Science 262, 1011-1018 [Medline] [Order article via Infotrieve]
  38. Perez, V. L., Rowe, T., Justement, J. S., Butera, S. T., June, C. H., and Folks, T. M. (1991) J. Immunol. 147, 3145-3148 [Abstract/Free Full Text]
  39. Folks, T. M., Clouse, K. A., Justement, J., Rabson, A., Duh, E., Kehrl, J. H., and Fauci, A. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2365-2368 [Abstract]
  40. Pauwels, R., Balzarini, J., Baba, M., Snoeck, R., Schols, D., Herdewijn, P., Desmyter, J., and De Clerk, E. (1988) J. Virol. Methods 20, 309-321 [CrossRef][Medline] [Order article via Infotrieve]
  41. Schwartz, O., Henin, Y., Marechal, V., and Montagnier, L. (1988) AIDS Res. Hum. Retroviruses 4, 441-448 [Medline] [Order article via Infotrieve]
  42. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450 [Abstract]
  43. Zeira, M., Byrn, R. A., and Groopman, J. E. (1990) AIDS Res. Hum. Retroviruses 6, 629-639 [Medline] [Order article via Infotrieve]
  44. Papp, B., Zhang, D., Groopman, J. E., and Byrn, R. A. (1994) AIDS Res. Hum. Retroviruses 10, 775-780 [Medline] [Order article via Infotrieve]
  45. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Ed., Vol. 3, pp. 16.59-16.65, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  46. Ghosh, T. K., Bian, J., Short, A. D., Rybak, S. L., and Gill, D. L. (1991) J. Biol. Chem. 266, 24690-24697 [Abstract/Free Full Text]
  47. Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4986-4990 [Abstract]
  48. Waldron, R. T., Short, A. D., Meadows, J. J., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 11927-11933 [Abstract/Free Full Text]
  49. Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981 [Abstract/Free Full Text]
  50. Papp, B., Enyedi, A., Kovcs, T., Sarkadi, B., Wuytack, F., Thastrup, O., Grdos, G., Bredoux, R., Lévy-Toledano, S., and Enouf, J. (1991) J. Biol. Chem. 266, 14593-14596 [Abstract/Free Full Text]
  51. Mason, M. J., Mayer, B., and Hymel, L. J. (1993) Am. J. Physiol. 264, C654-C662
  52. Nordstrom, T., Nevanlinna, H. A., and Andersson, L. C. (1992) Exp. Cell Res. 202, 487-494 [Medline] [Order article via Infotrieve]
  53. Butera, S. T., Roberts, B. D., Lam, L., Hodge, T., and Folks, T. M. (1994) J. Virol. 68, 2726-2730 [Abstract]
  54. Nye, K. E., and Pinching, A. J. (1990) AIDS 4, 41-45 [Medline] [Order article via Infotrieve]
  55. Nye, K. E., Knox, K. A., and Pinching, A. J. (1991) AIDS 5, 413-417 [Medline] [Order article via Infotrieve]
  56. Ino, M. (1990) J. Gen. Physiol. 95, 1103-1122 [Abstract]
  57. Bezprozvanny, I., Watras, J., and Ehrlich, B. E. (1991) Nature 351, 751-754 [CrossRef][Medline] [Order article via Infotrieve]
  58. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Science 252, 443-446 [Medline] [Order article via Infotrieve]
  59. Parys, J. B., Sernett, S. W., DeLisle, S. D., Snyder, P. M., Welsh, M. J., and Campbell, K. P. (1992) J. Biol. Chem. 267, 18776-18782 [Abstract/Free Full Text]
  60. Iino, M., and Endo, M. (1992) Nature 360, 76-78 [CrossRef][Medline] [Order article via Infotrieve]
  61. Zhang, B.-X., Zhao, H., and Muallem, S. (1993) J. Biol. Chem. 268, 10997-11001 [Abstract/Free Full Text]
  62. Combettes, L., and Champeil, P. (1994) Science 265, 813 [Medline] [Order article via Infotrieve]
  63. Finch, E. A., and Goldin, S. M. (1994) Science 265, 813-815 [Medline] [Order article via Infotrieve]

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