- Email: [email protected]
Cytotoxicity Resulting from Addition of HIV-1 Nef N-Terminal Peptides to Yeast and Bacterial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
232, 707–711 (1997)
RC976349
Cytotoxicity Resulting from Addition of HIV-1 Nef N-Terminal Peptides to Yeast and Bacterial Cells Ian G. Macreadie,*,1 Melinda G. Lowe,* Cyril C. Curtain,* Dean Hewish,† and A. A. Azad* *Biomolecular Research Institute, Parkville, Victoria; and †Division of Biomolecular Engineering, Commonwealth Scientific and Industrial Research Organisation, Parkville, Victoria, Australia
Received February 7, 1997
The Nef protein of human immunodeficiency type 1 (HIV-1) has been implicated in diverse intracellular functions; however, extracellular functions have been less studied. Nef and the N-terminus of Nef possess membrane-perturbing and fusogenic activities in artificial membranes that also cause cytotoxicity to human cells, including lymphocytes. The present study investigates the toxicity of HIV-1 Nef peptides employing yeast and bacterial cells. The N-terminal portion of Nef was found to cause cell killing in Escherichia coli and in a variety of yeast cells. This activity was enhanced by myristylation of the Nef N-terminus, a modification that did not lead to toxicity in a control peptide. Cell death in yeast was due to permeabilization of the cell membrane as determined by the propidium iodide uptake of peptide-treated cells. Extracellular Nef, or its breakdown products, may have effects similar to the Nef peptides described here and could be responsible, at least in part, for the death of cells in lymphoid tissues during AIDS. Assays using yeast or bacteria are convenient, inexpensive, and robust and should be useful in further analysis and screening of inhibitors of this activity associated with HIV-1 Nef. q 1997 Academic Press
The genomes of all primate lentiviruses contain nef, a highly conserved gene that overlaps the 3* LTR. Recent evidence suggests that the nef gene product, a myristylated protein of 205 aa (Fig. 1A), is involved in AIDS pathogenesis since mutations in HIV-1 nef leading to the loss of Nef correlate with no progression to patho1 To whom correspondence should be addressed at Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia. Fax: (61-3) 9342 4301. E-mail: [email protected]. Abbreviations: aa, amino acids; AIDS, aquired immunodeficiency syndrome; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; LTR, long terminal repeat; myr, myristylated; PBS, phosphate buffered saline; PI, propidium iodide; SIV, simian immunodeficiency virus.
genesis, even though HIV-1 infection persists (1,2). Genetically engineered point and deletion mutations in molecularly cloned SIVmacnef also implicate Nef in AIDS pathogenesis in rhesus monkeys (3). Numerous studies have shown that Nef is implicated in a diverse range of activities within the cell. However, there has been little consideration of an extracellular mode of action, even though there is some evidence that Nef exists in an extracellular form. Antibodies to Nef appear early after HIV-1 infection (4-13), and the C-terminus of Nef has been observed on the surface of HIV-1 infected cells (14). Further, in a vaccinia expression system Nef was reported to be ‘secreted’ from cells into the medium (15), in a yeast expression system Nef was released from the cells under conditions of stress (16), and in a baculovirus expression system it was found to be present on the cell surface (17). In this study the properties of Nef in its extracellular form have been considered by employing peptides that correspond to native Nef N-terminus. Previously, it was noted that the N-terminus of Nef bore a similarity with bee venom melittin (see Fig. 1B) and that Nef, particularly Nef N-terminal peptides, could cause perturbation and fusion of artificial membranes (18). Subsequently these N-terminal peptides were shown to exhibit in vitro killing of human lymphocytes and red blood cells (20). In this study we determined that Nef N-terminal peptides could also kill yeast and bacterial cells, in a manner that is consistent with the killing observed in human cells. The microbial cell assays provide an additional means for the analysis of Nef function and for the convenient screening of inhibitors of such an activity. MATERIALS AND METHODS Strains and Growth. The yeast strains reported in this study were Saccharomyces cerevisiae stain DY150 (MATa ura3-52 leu23,112 trp1-1 ade2-1 his3-11 can1-100), Candida glabrata strain L5 (leu), Candida albicans clinical isolate JRW#5, Kluyveromyces lactis stain MW98-8c (MATa uraA arg lys) and Schizosaccharomyces
707
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pombe (h0ade ura leu1-32). Strains were grown in YEPD (1% yeast extract, 2% peptone, 2% glucose). Escherichia coli strain MC1061 (F0araD139 D(ara-leu)7696 / galE15 galK16 D(lac)X74 rpsL (Strr) hsdR2 (r0 KmK) mcrA mcrB1) was also employed for toxicity studies and plated onto 2 x YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl). Peptide synthesis. Peptides were synthesized on an Applied Biosystems 430A Peptide Synthesiser as previously described (18). Peptides were analysed for purity by aa composition, HPLC, mass spectrometry and/or sequence analysis. Peptides are myrNef2-22 myr, GGKWSKSSVIGWPAVRERMRR; Nef2-22 , GGKWSKSSVIGWPAVRERMRR; and myrNef31-50 , myrGAVSRDLEKHGAITSSNTAA. Peptide treatments. Peptides were purified to homogeneity by HPLC and then dissolved in water. Aliquots of peptide solutions were added to cells suspended in a final volume of 100 ml water, and after 1 h. or other specified time the mixture was plated onto the appropriate solidified medium. Plates were incubated for one to three days, depending on the strain, and the numbers of viable colonies were determined. Microscopy of yeast. PI uptake of treated cells was examined by microscopy immediately after the addition of PI to 1 mg/ml to cells in suspension. Fluorescence microscopy utilised an Olympus BH2RFCA microscope equipped with an excitation cube/filter B for PI fluorescence. Flow cytometry analysis. Cells were analysed by forward angle and 907 light scatter as well as PI fluorescence using a Coulter EPICS Elite flow cytometer. PI was added to 25 mg/ml and dye penetration was measured by the presence of fluorescence emission at 520 nm. Gating was adjusted such that the population defined as ‘‘PI stained’’ comprised 5% of the control cell population.
RESULTS The N-terminal portion of Nef kills yeast cells. Because of the convenience of a microbial model system, we investigated a peptide, myrNef2-22 , corresponding to the first 21 aa of the native Nef protein, for its effects on yeast cells. The myrNef2-22 peptide was added to cells suspended in water, held for 60 min. and then plated onto rich growth media (YEPD) to determine
FIG. 1. Nef and its comparison to mellittin. (A) Structure of the 206 aa Nef primary translation product from pNL4-3 (19) that is subsequently processed to remove the N-terminal methionine and myristylate the new N-terminus. The sequences underlined correspond to the chemically-synthesized parts of Nef that have been used in this study. (B) Comparison (modified from 18) of melittin (24 of the 26 residues) and the myrNef2-22 used in this study. Identical residues are boxed.
FIG. 2. Effect of myrNef2-22 on yeast colony formation. Yeast cells were suspended to a density of Ç104 cells/ml in water and myrNef2-22 was added to a final concentration of 10 mM. After 60 min, equal aliquots of untreated and treated cells were spread onto solidified YEPD and plates were examined after incubation at 287C. The photograph shows the colonies formed after 1 day for C. albicans and C. glabrata, 2 days for K. lactis and S. cerevisiae, and 3 days for Sz. pombe without peptide treatment. With peptide treatment all plates were identical—no colonies formed.
colony-forming ability. Four species of budding yeast, Candida glabrata, Candida albicans, Kluyveromyces lactis, and Saccharomyces cerevisiae, as well as the fission yeast, Schizosaccharomyces pombe, were tested. All yeast species responded similarly to the peptide treatments. Figure 2 shows that treatment with a 10 mM concentration of myrNef2-22 caused the loss of colony formation of the entire cell population. Although further experiments were performed on the above strains and numerous other strains, results below are restricted to those involving the S. cerevisiae strain, DY150, since cell killing was observed as a general phenomenon in all yeast strains tested. Effects of Nef myristylation on activity. The importance of N-terminal myristylation of the Nef peptide was examined using two additional peptides. myrNef31-50 is a peptide that contains an internal portion of Nef that was unnaturally myristylated at its N-terminus. Addition of 1 mM myrNef31-50 peptide to cells caused no loss of colony formation (Fig. 3A), showing that myristylation per se is not a cause of peptide toxicity. The second peptide, Nef2-22 , is the non-myristylated version of myrNef2-22 . As shown in Figure 3A, treatment with a 1 mM amount of Nef2-22 resulted in 80% loss of colony formation, while the same concentration of myrNef2-22 caused total loss of colony formation. To compare the myrNef2-22 and Nef2-22 in more quantitative terms, dose response studies were performed. In a 30 min. interval the lowest myrNef2-22 concentration for complete cell killing was about 1 mM (Fig. 3B) while the Nef2-22 peptide required about three times that level to cause a similar effect. Concentrations of myrNef2-22
708
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
branes are permeabilized. Following peptide treatment cells were examined for staining with PI. By fluorescence microscopy, cells treated with myrNef2-22 were stained with PI within 30 min. of the peptide addition while untreated cells did not stain (data not shown). PI staining is useful to distinguish unstained viable cells or cells with an intact membrane from those that are dead or have a compromised membrane (21). Quantitation of the peptide-induced permeabilization was investigated by flow cytometry (Fig. 4). Essentially all of the cells treated with myrNef2-22 were stained with PI after a 30 min. treatment. In contrast Nef2-22 caused about a third of cells to be permeabilized, while the treatment with the control peptide, myrNef31-50 , produced about 5% PI-staining, similar to the untreated cells. These data indicate that the Nef N-terminal peptides disrupt the plasma membrane and other cell membranes leading to cell death.
FIG. 3. Effect of myristylation of Nef peptides on yeast cell killing. (A) S. cerevisiae cells were treated with a 1 mM concentration of the Nef peptides indicated on the figure, according to the procedures described in the legend to Fig. 2. (B) Dose responses for treatments of 30 min with myrNef2-22 and Nef2-22 .
down to 50 nM were partially effective but below this concentration there was no effect. Thus the myristylated end group either adds stability or enhances the activity of the Nef2-22 peptide. It is worthy of comment that in these studies petite mutants (naturally arising mutants of S. cerevisae that delete parts of their mitochondrial genome to become respiratory deficient) had a subtle preferential survival over respiration competent cells. This observation was clear, particularly in dose response studies where some survivors were often present and is notable in Figure 3A, where survivors from treatment with the Nef2-22 are seen to be mainly small white colonies. The peptide treatment does not induce the formation of petites (like some drug treatments): instead we see the petites as being the last to die. The slight advantage that these respiratory deficient mutants have over the wild-type could be due to many factors, such as their respiration state, their smaller size, or an altered state of some of their membranes. Cell permeabilization by Nef peptides. The loss of colony formation was investigated to see whether permeabilization was a likely cause of cell death: yeast provides an advantage for this kind of examination because the cell wall remains intact even when the mem-
Nef peptides kill E. coli cells. Nef peptides were added to a suspension of E. coli cells in water to determine whether they would be capable of killing prokaryotic microbial cells. The Nef peptides killed E. coli cells in a manner comparable to yeast (Fig. 5). Peptide cytotoxicity could be delayed by suspending the cells in buffer (50 mM HEPES/2% glucose) so that complete killing was not observed until 20 h. incubation at 307C. Taken together with the yeast data, these results suggest that extracellular Nef-induced toxicity for eukaryotic and prokaryotic cells is exerted through a common effect on cell membranes. DISCUSSION This study shows that the peptides comprising the N-terminal portion of Nef kills yeast cells through permeabilization of the cell membrane. The Nef peptides caused changes that allowed rapid uptake of PI and increased side scatter by flow cytometry, phenomena usually correlated with cell membrane damage or
FIG. 4. Cellular propidium iodide (PI) uptake analysed by flow cytometric analysis. Cells of S. cerevisiae were incubated with peptides essentially as described in the legend to Fig. 1, except that the cell concentration was increased to Ç107 cells/ml and the peptide concentration raised to 33 mM. The peptides were myrNef31-50 (myrControl), myrNef2-22 (myrNef), and Nef2-22 (Nef). An arbitrary gate was set such that 5% of untreated cells were classified as stained.
709
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells by contact. In this regard it is of interest that when the C-terminal portion of Nef is exposed on the surface of insect cells killing of bystander lymphocytes can be observed (17). Alternatively, these studies may have relevance to the passage of Nef through the cell membrane or to the killing of cells producing high Nef levels. Whatever the role of the cytotoxicity of the Nterminus of Nef, it is an advantage to reproduce the effect in a microbial system. Apart from allowing the mechanism of cytotoxicity to be investigated, it should enable methods for the convenient, rapid screening of inhibitors of such an activity to be developed. ACKNOWLEDGMENTS FIG. 5. Effect of peptides on E. coli colony formation. Peptides were added to E. coli cells suspended in water. After 30 min cells were spread onto solidified 21 YT and plates were examined after overnight incubation at 377C.
death (21). Peptides of three other HIV-1 proteins, Vpr, Vpu and gp41, also affect membrane permeability, a property that is regarded as an important mechanism for replication in many animal viruses (reviewed in 22). Our work suggests that yeast may be convenient for assaying effects of other membrane perturbing proteins and peptides. In fact a study on HIV-1 Vpr using a yeast model has been highly informative in analysing Vpr cytotoxicity (23). Cell killing was also observed for bacterial cells, consistent with the study on artificial membranes (18), suggesting that the N-terminus of Nef can have a general interaction with cell membranes. The degree of cell killing was greatly increased by myristylation of Nef, although myristylation of an externally added control peptide did not of itself cause toxicity. The cell killing appeared to be effective at submicromolar levels, a property common to other toxic peptides which cause membrane permeabilization (reviewed in 24). As well being myristylation dependent, the cell killing was Nef-peptide dose-dependent and dependent on the cell concentration. If the ratio of cells to Nef peptide greatly exceeds the ratios employed in this study there may be no toxicity. The biological relevance of the current findings to HIV-1 pathogenesis is still unclear. Cell death is a feature noted in HIV-1 infection (25-28) and such death could be mediated by a transmitted factor, or a factor on the cell surface. The possibility that such a factor could be Nef makes this study all the more relevant. The appropriate presentation of Nef in vivo still needs to discovered. For example, certain breakdown products of Nef would be expected to have the cell killing activity described here. Several other peptides, including myrNef2-20 and myrNef2-26 produce effects similar to myrNef2-22 in yeast (data not shown) and human cells (20). In addition, presentation of the N-terminus of Nef on a cell surface could be mechanism of killing
We gratefully acknowledge the gifts of E. coli and yeast strains from Drs. Malcolm Casadaban, Hiroshi Fukuhara, Paul Nurse, David Stillman, John Warmington, and Dennis Winge. We thank Alan Kirkpatrick and Phil Strike for the provision of peptides and Drs. Morry Frenkel and Don Rivett for critical reading of the manuscript.
REFERENCES 1. Kirchoff, F., Greenough, T. C., Brettler, D. B., Sullivan, J. L., and Desrosiers, R. C. (1995) N. Engl. J. Med. 332, 228–232. 2. Deacon, N. J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D. J., McPhee, A., Greenway, A. L., Ellett, A., Chatfield, C., Lawson, V. A., Crowe, S., Maerz, A., Sonza, S., Learmont, J., Sullivan, J. S., Cunningham, A., Dwyer, D., Dowton, D., and Mills, J. (1995) Science 270, 988–991. 3. Kestler, H. W., III., Ringler, D. J., Mori, K., Panicali, D. L., Sehgal, P. K., Daniel, M. D., and Desrosiers R. C. (1991) Cell 65, 651–662. 4. Culmann, B., Gomard, E., Kieny, M.-P., Guy, B., Dreyfus, F., Saimot, A. G., Sereni, D., and Levy, J. P. (1989) Eur. J. Immunol. 19, 2383–2386. 5. Culmann, B., Gomard, E., Kieny, M.-P., Guy, B., Dreyfus, F., Saimot, A. G., Sereni, D., Sicard, D., and Levy, J. P. (1991) J. Immunol. 146, 1560–1565. 6. Reiss, P., de Ronde, A., Lange, J. M. A., de Wolfe, F., Dekker, J., Debouck, C., and Goudsmit, J. (1989) AIDS 3, 227–233. 7. Reiss, P., de Wolf, F., Kuiken, C. L., de Ronde, A., Dekker, J., Boucher, C. A., Debouck, C., Lange, J. M., and Goudsmit, J. (1991) J. AIDS 4, 165–172. 8. Bahraoui, E., Benjouad, A., Sabatier, J. M., Allain, J. P., Laurian, Y., Montagnier, L., and Bluckman, J. C. (1990) Blood 76, 257–264. 9. Koenig, S., Fuerst, T. R., Wood, L. V., Woods, R. M., Suzich, J. A., Jones, G. M., de la Cruz, V. F., Davey, R. T., Jr., Venkatesan, S., Moss, B., Biddison, W. E., and Fauci, A. S. (1990) J. Immunol. 145, 127–135. 10. Gilmour, J. E., Read, S. J., Eglin, R., Ryan, C., Burns, N. R., Graff, N., Stenner, N., Kingsman, S. M., Kingsman, A. J., and Adams, S. E. (1990) AIDS 4, 967–973. 11. Wieland, U., Kuhn, J. E., Jassoy, C., Rubsamen-Waigmann, H., Wolber, V., and Braun, R. W. (1990) Med. Micro. Immun. 179, 1–11. 12. Kienzle, N., Broker, M., Harthus, H. P., Enders, M., Erfle, V., Buck, M., and Muller-Lantzsch, N. (1991) Arch. Virol. 118, 29– 41. 13. Hadida, F., Parrot, A., Kieny, M.-P., Sadat-Sowti, B., Mayaud, C., Debre, P., and Autran, B. (1992) J. Clin. Invest. 89, 53–60.
710
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
14. Fujii, Y., Nishino, Y., Nakaya, T., Tokunaga, T., and Ikuta, K. (1993) Vaccine 11, 1240–1246. 15. Guy, B., Riviere, Y., Dott, K., Regnault, A., and Kieny, M. P. (1990) Virology 176, 413–425. 16. Macreadie, I. G., Castelli, L. A., Lucantoni, A., and Azad, A. A. (1995) Gene 162, 239–243. 17. Fujii, Y., Otake, K., Tashiro, M., and Adachi, A. (1996) J. Gen. Virol. 77, 2943–2951. 18. Curtain, C. C., Separovic, F., Rivett, D., Kirkpatrick, A., Waring, A. J., Gordon, L. M., and Azad, A. A. (1994) AIDS Res. Human Retroviruses 10, 1231–1240. 19. Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284–291. 20. Curtain, C. C., Lowe, M. G., Arunagiri, C. K., Mobley, P. W., Macreadie I. G., and Azad, A. A. (1997) AIDS Res. Human Retroviruses, in press.
21. Shapiro, H. (1994) Practical Flow Cytometry, 3rd ed., Wiley– Liss, New York. 22. Carrasco, L. (1995) Adv. Virus Res. 45, 61–112. 23. Macreadie, I. G., Arunagiri, C. K., Hewish, D. R., White, J. F., and Azad, A. A. (1996) Mol. Micro. 19, 1185–1192. 24. Nicolas, P., and Mor, A. (1995) Annu. Rev. Microbiol. 49, 277– 304. 25. Levy, J. A. (1993) Microbiol. Rev. 57, 183–289. 26. Wei, X., Ghosh, S. K., Taylor, M. E., Johnson, V. A., Emini, E. A., Deutsch, P., Lifson, J. D., Bonhoeffer, S., Nowak, M. A., Hahn, B. H., Saag, M. S., and Shaw, G. M. (1995) Nature 373, 117– 122. 27. Ho, D. D., Neumann, A. U., Perelson, A. S., Chen, W., Leonard, J. M., and Markowitz, M. (1995) Nature 373, 123–126. 28. Finkel, T. H., Tudor-Williams, G., Bandra, N. K., Cotton, M. F., Curiel, T., Monks, C., Baba, T. W., Ruprecht, R. M., and Kupfer, A. (1995) Nature Med. 1, 129–134.
711
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
232, 707–711 (1997)
RC976349
Cytotoxicity Resulting from Addition of HIV-1 Nef N-Terminal Peptides to Yeast and Bacterial Cells Ian G. Macreadie,*,1 Melinda G. Lowe,* Cyril C. Curtain,* Dean Hewish,† and A. A. Azad* *Biomolecular Research Institute, Parkville, Victoria; and †Division of Biomolecular Engineering, Commonwealth Scientific and Industrial Research Organisation, Parkville, Victoria, Australia
Received February 7, 1997
The Nef protein of human immunodeficiency type 1 (HIV-1) has been implicated in diverse intracellular functions; however, extracellular functions have been less studied. Nef and the N-terminus of Nef possess membrane-perturbing and fusogenic activities in artificial membranes that also cause cytotoxicity to human cells, including lymphocytes. The present study investigates the toxicity of HIV-1 Nef peptides employing yeast and bacterial cells. The N-terminal portion of Nef was found to cause cell killing in Escherichia coli and in a variety of yeast cells. This activity was enhanced by myristylation of the Nef N-terminus, a modification that did not lead to toxicity in a control peptide. Cell death in yeast was due to permeabilization of the cell membrane as determined by the propidium iodide uptake of peptide-treated cells. Extracellular Nef, or its breakdown products, may have effects similar to the Nef peptides described here and could be responsible, at least in part, for the death of cells in lymphoid tissues during AIDS. Assays using yeast or bacteria are convenient, inexpensive, and robust and should be useful in further analysis and screening of inhibitors of this activity associated with HIV-1 Nef. q 1997 Academic Press
The genomes of all primate lentiviruses contain nef, a highly conserved gene that overlaps the 3* LTR. Recent evidence suggests that the nef gene product, a myristylated protein of 205 aa (Fig. 1A), is involved in AIDS pathogenesis since mutations in HIV-1 nef leading to the loss of Nef correlate with no progression to patho1 To whom correspondence should be addressed at Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia. Fax: (61-3) 9342 4301. E-mail: [email protected]. Abbreviations: aa, amino acids; AIDS, aquired immunodeficiency syndrome; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; LTR, long terminal repeat; myr, myristylated; PBS, phosphate buffered saline; PI, propidium iodide; SIV, simian immunodeficiency virus.
genesis, even though HIV-1 infection persists (1,2). Genetically engineered point and deletion mutations in molecularly cloned SIVmacnef also implicate Nef in AIDS pathogenesis in rhesus monkeys (3). Numerous studies have shown that Nef is implicated in a diverse range of activities within the cell. However, there has been little consideration of an extracellular mode of action, even though there is some evidence that Nef exists in an extracellular form. Antibodies to Nef appear early after HIV-1 infection (4-13), and the C-terminus of Nef has been observed on the surface of HIV-1 infected cells (14). Further, in a vaccinia expression system Nef was reported to be ‘secreted’ from cells into the medium (15), in a yeast expression system Nef was released from the cells under conditions of stress (16), and in a baculovirus expression system it was found to be present on the cell surface (17). In this study the properties of Nef in its extracellular form have been considered by employing peptides that correspond to native Nef N-terminus. Previously, it was noted that the N-terminus of Nef bore a similarity with bee venom melittin (see Fig. 1B) and that Nef, particularly Nef N-terminal peptides, could cause perturbation and fusion of artificial membranes (18). Subsequently these N-terminal peptides were shown to exhibit in vitro killing of human lymphocytes and red blood cells (20). In this study we determined that Nef N-terminal peptides could also kill yeast and bacterial cells, in a manner that is consistent with the killing observed in human cells. The microbial cell assays provide an additional means for the analysis of Nef function and for the convenient screening of inhibitors of such an activity. MATERIALS AND METHODS Strains and Growth. The yeast strains reported in this study were Saccharomyces cerevisiae stain DY150 (MATa ura3-52 leu23,112 trp1-1 ade2-1 his3-11 can1-100), Candida glabrata strain L5 (leu), Candida albicans clinical isolate JRW#5, Kluyveromyces lactis stain MW98-8c (MATa uraA arg lys) and Schizosaccharomyces
707
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pombe (h0ade ura leu1-32). Strains were grown in YEPD (1% yeast extract, 2% peptone, 2% glucose). Escherichia coli strain MC1061 (F0araD139 D(ara-leu)7696 / galE15 galK16 D(lac)X74 rpsL (Strr) hsdR2 (r0 KmK) mcrA mcrB1) was also employed for toxicity studies and plated onto 2 x YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl). Peptide synthesis. Peptides were synthesized on an Applied Biosystems 430A Peptide Synthesiser as previously described (18). Peptides were analysed for purity by aa composition, HPLC, mass spectrometry and/or sequence analysis. Peptides are myrNef2-22 myr, GGKWSKSSVIGWPAVRERMRR; Nef2-22 , GGKWSKSSVIGWPAVRERMRR; and myrNef31-50 , myrGAVSRDLEKHGAITSSNTAA. Peptide treatments. Peptides were purified to homogeneity by HPLC and then dissolved in water. Aliquots of peptide solutions were added to cells suspended in a final volume of 100 ml water, and after 1 h. or other specified time the mixture was plated onto the appropriate solidified medium. Plates were incubated for one to three days, depending on the strain, and the numbers of viable colonies were determined. Microscopy of yeast. PI uptake of treated cells was examined by microscopy immediately after the addition of PI to 1 mg/ml to cells in suspension. Fluorescence microscopy utilised an Olympus BH2RFCA microscope equipped with an excitation cube/filter B for PI fluorescence. Flow cytometry analysis. Cells were analysed by forward angle and 907 light scatter as well as PI fluorescence using a Coulter EPICS Elite flow cytometer. PI was added to 25 mg/ml and dye penetration was measured by the presence of fluorescence emission at 520 nm. Gating was adjusted such that the population defined as ‘‘PI stained’’ comprised 5% of the control cell population.
RESULTS The N-terminal portion of Nef kills yeast cells. Because of the convenience of a microbial model system, we investigated a peptide, myrNef2-22 , corresponding to the first 21 aa of the native Nef protein, for its effects on yeast cells. The myrNef2-22 peptide was added to cells suspended in water, held for 60 min. and then plated onto rich growth media (YEPD) to determine
FIG. 1. Nef and its comparison to mellittin. (A) Structure of the 206 aa Nef primary translation product from pNL4-3 (19) that is subsequently processed to remove the N-terminal methionine and myristylate the new N-terminus. The sequences underlined correspond to the chemically-synthesized parts of Nef that have been used in this study. (B) Comparison (modified from 18) of melittin (24 of the 26 residues) and the myrNef2-22 used in this study. Identical residues are boxed.
FIG. 2. Effect of myrNef2-22 on yeast colony formation. Yeast cells were suspended to a density of Ç104 cells/ml in water and myrNef2-22 was added to a final concentration of 10 mM. After 60 min, equal aliquots of untreated and treated cells were spread onto solidified YEPD and plates were examined after incubation at 287C. The photograph shows the colonies formed after 1 day for C. albicans and C. glabrata, 2 days for K. lactis and S. cerevisiae, and 3 days for Sz. pombe without peptide treatment. With peptide treatment all plates were identical—no colonies formed.
colony-forming ability. Four species of budding yeast, Candida glabrata, Candida albicans, Kluyveromyces lactis, and Saccharomyces cerevisiae, as well as the fission yeast, Schizosaccharomyces pombe, were tested. All yeast species responded similarly to the peptide treatments. Figure 2 shows that treatment with a 10 mM concentration of myrNef2-22 caused the loss of colony formation of the entire cell population. Although further experiments were performed on the above strains and numerous other strains, results below are restricted to those involving the S. cerevisiae strain, DY150, since cell killing was observed as a general phenomenon in all yeast strains tested. Effects of Nef myristylation on activity. The importance of N-terminal myristylation of the Nef peptide was examined using two additional peptides. myrNef31-50 is a peptide that contains an internal portion of Nef that was unnaturally myristylated at its N-terminus. Addition of 1 mM myrNef31-50 peptide to cells caused no loss of colony formation (Fig. 3A), showing that myristylation per se is not a cause of peptide toxicity. The second peptide, Nef2-22 , is the non-myristylated version of myrNef2-22 . As shown in Figure 3A, treatment with a 1 mM amount of Nef2-22 resulted in 80% loss of colony formation, while the same concentration of myrNef2-22 caused total loss of colony formation. To compare the myrNef2-22 and Nef2-22 in more quantitative terms, dose response studies were performed. In a 30 min. interval the lowest myrNef2-22 concentration for complete cell killing was about 1 mM (Fig. 3B) while the Nef2-22 peptide required about three times that level to cause a similar effect. Concentrations of myrNef2-22
708
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
branes are permeabilized. Following peptide treatment cells were examined for staining with PI. By fluorescence microscopy, cells treated with myrNef2-22 were stained with PI within 30 min. of the peptide addition while untreated cells did not stain (data not shown). PI staining is useful to distinguish unstained viable cells or cells with an intact membrane from those that are dead or have a compromised membrane (21). Quantitation of the peptide-induced permeabilization was investigated by flow cytometry (Fig. 4). Essentially all of the cells treated with myrNef2-22 were stained with PI after a 30 min. treatment. In contrast Nef2-22 caused about a third of cells to be permeabilized, while the treatment with the control peptide, myrNef31-50 , produced about 5% PI-staining, similar to the untreated cells. These data indicate that the Nef N-terminal peptides disrupt the plasma membrane and other cell membranes leading to cell death.
FIG. 3. Effect of myristylation of Nef peptides on yeast cell killing. (A) S. cerevisiae cells were treated with a 1 mM concentration of the Nef peptides indicated on the figure, according to the procedures described in the legend to Fig. 2. (B) Dose responses for treatments of 30 min with myrNef2-22 and Nef2-22 .
down to 50 nM were partially effective but below this concentration there was no effect. Thus the myristylated end group either adds stability or enhances the activity of the Nef2-22 peptide. It is worthy of comment that in these studies petite mutants (naturally arising mutants of S. cerevisae that delete parts of their mitochondrial genome to become respiratory deficient) had a subtle preferential survival over respiration competent cells. This observation was clear, particularly in dose response studies where some survivors were often present and is notable in Figure 3A, where survivors from treatment with the Nef2-22 are seen to be mainly small white colonies. The peptide treatment does not induce the formation of petites (like some drug treatments): instead we see the petites as being the last to die. The slight advantage that these respiratory deficient mutants have over the wild-type could be due to many factors, such as their respiration state, their smaller size, or an altered state of some of their membranes. Cell permeabilization by Nef peptides. The loss of colony formation was investigated to see whether permeabilization was a likely cause of cell death: yeast provides an advantage for this kind of examination because the cell wall remains intact even when the mem-
Nef peptides kill E. coli cells. Nef peptides were added to a suspension of E. coli cells in water to determine whether they would be capable of killing prokaryotic microbial cells. The Nef peptides killed E. coli cells in a manner comparable to yeast (Fig. 5). Peptide cytotoxicity could be delayed by suspending the cells in buffer (50 mM HEPES/2% glucose) so that complete killing was not observed until 20 h. incubation at 307C. Taken together with the yeast data, these results suggest that extracellular Nef-induced toxicity for eukaryotic and prokaryotic cells is exerted through a common effect on cell membranes. DISCUSSION This study shows that the peptides comprising the N-terminal portion of Nef kills yeast cells through permeabilization of the cell membrane. The Nef peptides caused changes that allowed rapid uptake of PI and increased side scatter by flow cytometry, phenomena usually correlated with cell membrane damage or
FIG. 4. Cellular propidium iodide (PI) uptake analysed by flow cytometric analysis. Cells of S. cerevisiae were incubated with peptides essentially as described in the legend to Fig. 1, except that the cell concentration was increased to Ç107 cells/ml and the peptide concentration raised to 33 mM. The peptides were myrNef31-50 (myrControl), myrNef2-22 (myrNef), and Nef2-22 (Nef). An arbitrary gate was set such that 5% of untreated cells were classified as stained.
709
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells by contact. In this regard it is of interest that when the C-terminal portion of Nef is exposed on the surface of insect cells killing of bystander lymphocytes can be observed (17). Alternatively, these studies may have relevance to the passage of Nef through the cell membrane or to the killing of cells producing high Nef levels. Whatever the role of the cytotoxicity of the Nterminus of Nef, it is an advantage to reproduce the effect in a microbial system. Apart from allowing the mechanism of cytotoxicity to be investigated, it should enable methods for the convenient, rapid screening of inhibitors of such an activity to be developed. ACKNOWLEDGMENTS FIG. 5. Effect of peptides on E. coli colony formation. Peptides were added to E. coli cells suspended in water. After 30 min cells were spread onto solidified 21 YT and plates were examined after overnight incubation at 377C.
death (21). Peptides of three other HIV-1 proteins, Vpr, Vpu and gp41, also affect membrane permeability, a property that is regarded as an important mechanism for replication in many animal viruses (reviewed in 22). Our work suggests that yeast may be convenient for assaying effects of other membrane perturbing proteins and peptides. In fact a study on HIV-1 Vpr using a yeast model has been highly informative in analysing Vpr cytotoxicity (23). Cell killing was also observed for bacterial cells, consistent with the study on artificial membranes (18), suggesting that the N-terminus of Nef can have a general interaction with cell membranes. The degree of cell killing was greatly increased by myristylation of Nef, although myristylation of an externally added control peptide did not of itself cause toxicity. The cell killing appeared to be effective at submicromolar levels, a property common to other toxic peptides which cause membrane permeabilization (reviewed in 24). As well being myristylation dependent, the cell killing was Nef-peptide dose-dependent and dependent on the cell concentration. If the ratio of cells to Nef peptide greatly exceeds the ratios employed in this study there may be no toxicity. The biological relevance of the current findings to HIV-1 pathogenesis is still unclear. Cell death is a feature noted in HIV-1 infection (25-28) and such death could be mediated by a transmitted factor, or a factor on the cell surface. The possibility that such a factor could be Nef makes this study all the more relevant. The appropriate presentation of Nef in vivo still needs to discovered. For example, certain breakdown products of Nef would be expected to have the cell killing activity described here. Several other peptides, including myrNef2-20 and myrNef2-26 produce effects similar to myrNef2-22 in yeast (data not shown) and human cells (20). In addition, presentation of the N-terminus of Nef on a cell surface could be mechanism of killing
We gratefully acknowledge the gifts of E. coli and yeast strains from Drs. Malcolm Casadaban, Hiroshi Fukuhara, Paul Nurse, David Stillman, John Warmington, and Dennis Winge. We thank Alan Kirkpatrick and Phil Strike for the provision of peptides and Drs. Morry Frenkel and Don Rivett for critical reading of the manuscript.
REFERENCES 1. Kirchoff, F., Greenough, T. C., Brettler, D. B., Sullivan, J. L., and Desrosiers, R. C. (1995) N. Engl. J. Med. 332, 228–232. 2. Deacon, N. J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D. J., McPhee, A., Greenway, A. L., Ellett, A., Chatfield, C., Lawson, V. A., Crowe, S., Maerz, A., Sonza, S., Learmont, J., Sullivan, J. S., Cunningham, A., Dwyer, D., Dowton, D., and Mills, J. (1995) Science 270, 988–991. 3. Kestler, H. W., III., Ringler, D. J., Mori, K., Panicali, D. L., Sehgal, P. K., Daniel, M. D., and Desrosiers R. C. (1991) Cell 65, 651–662. 4. Culmann, B., Gomard, E., Kieny, M.-P., Guy, B., Dreyfus, F., Saimot, A. G., Sereni, D., and Levy, J. P. (1989) Eur. J. Immunol. 19, 2383–2386. 5. Culmann, B., Gomard, E., Kieny, M.-P., Guy, B., Dreyfus, F., Saimot, A. G., Sereni, D., Sicard, D., and Levy, J. P. (1991) J. Immunol. 146, 1560–1565. 6. Reiss, P., de Ronde, A., Lange, J. M. A., de Wolfe, F., Dekker, J., Debouck, C., and Goudsmit, J. (1989) AIDS 3, 227–233. 7. Reiss, P., de Wolf, F., Kuiken, C. L., de Ronde, A., Dekker, J., Boucher, C. A., Debouck, C., Lange, J. M., and Goudsmit, J. (1991) J. AIDS 4, 165–172. 8. Bahraoui, E., Benjouad, A., Sabatier, J. M., Allain, J. P., Laurian, Y., Montagnier, L., and Bluckman, J. C. (1990) Blood 76, 257–264. 9. Koenig, S., Fuerst, T. R., Wood, L. V., Woods, R. M., Suzich, J. A., Jones, G. M., de la Cruz, V. F., Davey, R. T., Jr., Venkatesan, S., Moss, B., Biddison, W. E., and Fauci, A. S. (1990) J. Immunol. 145, 127–135. 10. Gilmour, J. E., Read, S. J., Eglin, R., Ryan, C., Burns, N. R., Graff, N., Stenner, N., Kingsman, S. M., Kingsman, A. J., and Adams, S. E. (1990) AIDS 4, 967–973. 11. Wieland, U., Kuhn, J. E., Jassoy, C., Rubsamen-Waigmann, H., Wolber, V., and Braun, R. W. (1990) Med. Micro. Immun. 179, 1–11. 12. Kienzle, N., Broker, M., Harthus, H. P., Enders, M., Erfle, V., Buck, M., and Muller-Lantzsch, N. (1991) Arch. Virol. 118, 29– 41. 13. Hadida, F., Parrot, A., Kieny, M.-P., Sadat-Sowti, B., Mayaud, C., Debre, P., and Autran, B. (1992) J. Clin. Invest. 89, 53–60.
710
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC
Vol. 232, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
14. Fujii, Y., Nishino, Y., Nakaya, T., Tokunaga, T., and Ikuta, K. (1993) Vaccine 11, 1240–1246. 15. Guy, B., Riviere, Y., Dott, K., Regnault, A., and Kieny, M. P. (1990) Virology 176, 413–425. 16. Macreadie, I. G., Castelli, L. A., Lucantoni, A., and Azad, A. A. (1995) Gene 162, 239–243. 17. Fujii, Y., Otake, K., Tashiro, M., and Adachi, A. (1996) J. Gen. Virol. 77, 2943–2951. 18. Curtain, C. C., Separovic, F., Rivett, D., Kirkpatrick, A., Waring, A. J., Gordon, L. M., and Azad, A. A. (1994) AIDS Res. Human Retroviruses 10, 1231–1240. 19. Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284–291. 20. Curtain, C. C., Lowe, M. G., Arunagiri, C. K., Mobley, P. W., Macreadie I. G., and Azad, A. A. (1997) AIDS Res. Human Retroviruses, in press.
21. Shapiro, H. (1994) Practical Flow Cytometry, 3rd ed., Wiley– Liss, New York. 22. Carrasco, L. (1995) Adv. Virus Res. 45, 61–112. 23. Macreadie, I. G., Arunagiri, C. K., Hewish, D. R., White, J. F., and Azad, A. A. (1996) Mol. Micro. 19, 1185–1192. 24. Nicolas, P., and Mor, A. (1995) Annu. Rev. Microbiol. 49, 277– 304. 25. Levy, J. A. (1993) Microbiol. Rev. 57, 183–289. 26. Wei, X., Ghosh, S. K., Taylor, M. E., Johnson, V. A., Emini, E. A., Deutsch, P., Lifson, J. D., Bonhoeffer, S., Nowak, M. A., Hahn, B. H., Saag, M. S., and Shaw, G. M. (1995) Nature 373, 117– 122. 27. Ho, D. D., Neumann, A. U., Perelson, A. S., Chen, W., Leonard, J. M., and Markowitz, M. (1995) Nature 373, 123–126. 28. Finkel, T. H., Tudor-Williams, G., Bandra, N. K., Cotton, M. F., Curiel, T., Monks, C., Baba, T. W., Ruprecht, R. M., and Kupfer, A. (1995) Nature Med. 1, 129–134.
711
AID
BBRC 6349
/
6924$$$321
03-10-97 10:15:37
bbrcg
AP: BBRC