- Email: [email protected]
SIV to Peptidic Fusion Inhibitors
doi:10.1016/j.jmb.2004.04.027
J. Mol. Biol. (2004) 340, 9–14
COMMUNICATION
The Stability of the Intact Envelope Glycoproteins is a Major Determinant of Sensitivity of HIV/SIV to Peptidic Fusion Inhibitors Stephen A. Gallo1†, Kelly Sackett2†, Satinder S. Rawat1, Yechiel Shai2 and Robert Blumenthal1* 1
Membrane Structure and Function Section, LECB CCR, National Cancer Institute-Frederick, NIH Frederick, MD 21702, USA 2
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100 Israel
C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC50 , 10 nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC50 . 100 nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: HIV; SIV; membrane fusion; entry inhibitors; gp41
Membrane fusion between human/simian immunodeficiency virus (HIV/SIV) and target cells is mediated by the trimeric envelope protein hetero-complex of gp120 and gp41.1 Upon binding of this complex with cellular CD4 receptor2,3 and chemokine co-receptor,4 conformational changes † S.A.G. & K.S. contributed equally to this work. Abbreviations used: HIV-1, human immunodeficiency virus type 1; HIV-2, human immunodeficiency virus type 2; SIV, simian immunodeficiency virus; N-HR, N-terminal heptad repeat; C-HR, C-terminal heptad repeat; CD, circular dichroism; PBS, phosphate-buffered saline; Env, envelope glycoprotein. E-mail address of the corresponding author: [email protected]
are induced in both gp120 and gp41 which allow for their partial dissociation from each other and the insertion of the fusion peptide into the target or viral membrane.1,5 The partial dissociation of HIV-1 gp120 from gp41 leads to the exposure of the N-terminal heptad repeat (N-HR) and C-terminal heptad repeat (C-HR) regions of gp41 prior to their refolding into a six-helix bundle6 – 8 and membrane fusion.9 Several peptides that mimic the sequence of the N-HR and C-HR regions have been found to inhibit fusion by blocking of the interaction between the N-HR and C-HR regions and, thus, preventing the formation of the six-helix bundle fusogenic state of gp41.10 – 14 C-HR-derived peptides (C-peptides) inhibit HIV-1 Env-mediated cell fusion and are
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
10
HIV/SIV Sensitivity to Entry Inhibitors
Table 1. N36/C34 core formation and inhibition of HIV/SIV Env-mediated fusion by C34 HIV-1IIIBEnva
HIV-1IIIBC34a HIV-2SBLC34b SIV Mac251C34c
HIV-2SBLEnvb
SIV Mac251Envc
IC50 (nM)d
Tm (8C)e
IC50 (nM)d
Tm (8C)e
IC50 (nM)d
Tm (8C)e
5^4 9^3 9^1
61 ^ 1 52 ^ 1 57 ^ 1
3334 ^ 1392 164 ^ 74 543 ^ 266
Nof Nof 42 ^ 1
.10,000 .10,000 2100 ^ 1200
Nof Nof 36 ^ 1
a Sequences of HIV-1IIIB Env-derived peptides: N36, SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL; C34, WMEWDREINN YTSLIHSLIEESQNQQEKNEQELL. b Sequences of HIV-2SBL Env-derived peptides: N36, RGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVT; C34, WQEWEHKIR FLEANISESLEQAQIQQEKNMYELQ. c Sequences of SIVMac251 Env-derived peptides: N36, AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVT; C34, WQEWERKVD FLEENITALLEEAQIQQEKNMYELQ. d HIV/SIV Env-mediated fusion was measured using the dye transfer assay described in the text. SupT1 cells were used as targets for the X4-utilizing HIV-1 and HIV-2 strains, and TZM-bl cells were used as targets for the R5-utilizing SIV strain. Inhibitory concentration 50% (IC50), the concentration of C34, which reduced the fusion levels by 50% in comparison with untreated control, was determined by fitting the dose–response curves to a simple hyperbolic function using Sigmaplot. e Based on temperature denaturation of core complexes as indicated by CD spectra (Figure 2) of N36–C34 peptide mixtures. N and C peptide pairs that formed cores in PBS at 10 mM were melted over a range of 15 –95 8C in a capped quartz cell. Ellipticity at 222 nm were recorded every 2 deg. C, with ten seconds averaging time, following two minutes equilibrium. The cell was warmed at a rate of 2 deg. C/minute. Tm was derived from CD melting curves. f The mixtures of N and C peptides that failed to form cores showed a gradual linear thermal denaturation profile from 0 8C to 60 8C with no Tm , indicating no structure inducing interaction between the peptides.
thought to act through binding to the hydrophobic grooves that line the internal N-terminal trimeric coiled-coil core of the gp41 ectodomain.15 – 18 C34 comprises residues that interact specifically with the deep hydrophobic pocket on the surface of the N-terminal internal trimeric coiled coil of gp41.14 T20 (also referred to as DP178, enfuvirtide and fuzeon), which has extensive overlap with C34, has been found to be effective in vivo and has been recently granted FDA approval as an anti-HIV therapeutic†. C-peptides derived from HIV-1 are poor inhibitors of HIV-2 entry.16,19 By contrast, C34 derived from either SIV or HIV-1 has been found to inhibit HIV-1 envelope glycoprotein (Env)-mediated fusion equally well.20 To understand the basis for the sensitivity/resistance of HIV/SIV to these entry inhibitors, we have conducted peptide inhibition studies on HIV-1, HIV-2 and SIV Envmediated fusion using C34 derived from HIV-1, HIV-2 or SIV as well as biophysical studies on possible complex formation between these peptides and their N36 counterparts. Fusion was assayed using a dye transfer system as described.21 In brief, CV-1 cells (ATCC, Rockville, MD) were plated on 96-well plates (Costar, Cambridge, MA) and infected with vaccinia recombinants (kindly provided by Drs Pat Earl and Bernard Moss) VPE16, for IIIB strain of HIV-1,22 VSC50 for the SBL/ISY strain of HIV-223 and WR194 for SIVMAC24 overnight at multiplicity of infection of 10. The HeLa-derived cell line, TZM-bl,25 which expresses high levels of CD4 and CCR5 along with endogenously expressed CXCR4, was obtained through the AIDS Research and Reference Reagent Program, Division † http://www.fuzeon.com/
of AIDS, NIAID, NIH from Dr John C. Kappes, Dr Xiaoyun Wu and Tranzyme Inc. SupT1 cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr James Hoxie. Vaccinia-infected CV-1 cells labeled with 5-(and-6) (((4chloromethyl)benzoyl)amino)tetramethylrhodamine (494/517, red) were co-cultured at 37 8C with SupT1 cells or TZM-bl cells labeled with Calcein (541/565, green). The fluorescent dyes were obtained from Molecular Probes (Eugene, OR). The fusion inhibitors (see Table 1), obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, were dissolved in 10% NH4OH in 200 mM stock solutions and added to the cells at the beginning of the co-culture. To examine the susceptibility of HIV/SIV Envmediated fusion to inhibition by peptides, dose response curves were generated from the cell – cell fusion assay described above. Table 1 shows that HIV-1, HIV-2 and SIV C34 inhibited HIV-1 Envmediated cell – cell fusion with approximately equal efficiency with IC50 , 10 nM. All of these peptides inhibit HIV-2 Env-mediated fusion with far less efficacy (IC50 . 100 nM), and only SIV C34 inhibited SIV Env-mediated fusion. In addition, HIV-1 C34 never fully inhibited HIV-2SBL Envmediated fusion, only reaching 50% inhibition at very high concentrations. To examine the ability of the C34 peptides to form stable six-helix bundles with their N36 counterparts, we performed circular dichroism (CD) experiments using an Aviv 202 CD spectrometer. Peptides were dissolved in PBS (pH 7.4) to a final concentration of 10 mM. Spectral contribution from the PBS solvent was subtracted from that of peptide(s) dissolved in solvent. The wavelength dependence of ellipticity was monitored at 25 8C in 1 nm increments using ten seconds
Figure 1. Circular dichroism studies of complex formation between N36 and C34 pairs. HIV-2SBL, HIV-1IIIB or SIVMAC N36 peptide (10 mM) was incubated with either HIV-2SBL, HIV-1IIIB or SIVMAC C34 peptide (10 mM) and the spectra were recorded. Shown is the actual reading of the peptide pairs versus the theoretical addition of the individual peptide spectra. The unit of CD on the Y-axis is elipticity (mdeg.) and the unit of wavelength on the X-axis is nm. Since all the pairs have comparable molecular masses, the values on the Y-axis reflect the relative elipticities of all of them. Although HIV-1 N36 forms complexes with all three C34 peptides, both HIV-2 and SIV N36 only form complexes with SIV C34.
12
averaging time. Each peptide was measured alone and then N36/C34 pairs were measured. Triplicate recordings were conducted for each peptide and peptide pair, with superimposing spectra. The N36/C34 pairs included homogenous pairs and heterogenous pairs (e.g. HIV-2 N36 and HIV-1 C34). The sum of the individual N-HR and C-HR peptide spectra were added together and presented graphically along with the spectra of the co-incubated peptide pairs, for comparison. Figure 1 shows that helical structure is induced upon co-incubation of HIV-1 N36 with C34 peptides derived from all three species, indicating core formation in each case. By contrast N36 peptides derived from HIV-2 and SIV did not induce a-helical structure when incubated with C34 peptides derived from HIV-1 or HIV-2. However, SIV C34 formed helical cores when incubated with N36 derived from all three species. In order to determine the comparative stability of the various C and N-peptide mixes with C34 inhibitory efficacy we determined the melting temperature ðTm Þ of the complexes. Table 1 shows the Tm values for all the C and N-peptide mixes that formed complexes. Figure 2 shows a semi-logarithmic plot of IC50 values against the Tm of the corresponding N36/C34 complexes. In contrast to data on inhibition of HIV-1 Env-mediated fusion by C34 variants with modified cavity-binding residues,14 there appears to be a lack of correlation between stability of N36/C34 HIV-1/HIV-2/SIV complexes and C34 inhibitory efficacy. In Figure 2 the data are clustered in three groups. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34 ðTm . 50 8CÞ, which all inhibited HIV-1 Envmediated fusion at IC50 , 10 nM. The second group yielded moderate Tm values (, 40 8C) and higher IC50 values (. 100 nM). The third group exhibited no complex-forming propensity ðTm , 0 8CÞ. HIV-2 N36/C34 belongs to this
HIV/SIV Sensitivity to Entry Inhibitors
group; yet HIV-2 C34 inhibits HIV-2 ENV-mediated fusion with greater efficacy than SIV C34, which makes a relatively stable complex with HIV-2 N36. Based on these data it is hard to explain the differences in inhibitory efficacy of the various C34-derived peptides for HIV-1, HIV-2 and SIV Env-mediated fusion. To examine whether the lower efficacy of HIV-2/ SIV Env-mediated fusion inhibition by C-peptides was due to kinetics,26 we reduced these fusion rates by lowering the incubation temperature. At 31 8C the time (t1/2) to reach half-maximal HIV-2 Env-mediated fusion was reduced from 15 minutes (at 37 8C) to 30 minutes. The t1/2 for HIV-1 Envmediated fusion at 37 8C was about the same as that for HIV-2 Env-mediated fusion at 31 8C. Yet the IC50 for inhibition by SIV C34 for HIV-2 Envmediated fusion at 31 8C was still an order of magnitude higher than that for inhibition of HIV-1 Env-mediated fusion at 37 8C. Since in this case kinetics does not appear to be the only determinant of entry inhibitor efficacy, we considered the issue of stability of the intact envelope glycoproteins. Although the sequence homology between HIV-1 and SIV in the gp41 ectodomain is relatively low, around 56% amino acid identity24, there is great similarity between the structures of the SIV (which has high sequence homology with HIV-2) and the HIV-1 gp41 ectodomain core.8,20 However, differences have been found in their thermodynamic properties as indicated by an , 30 deg. C lower melting temperature20,27,28 (see also Table 1) and a lower concentration of urea required for unfolding for SIV gp41.29 Moreover, in vitro studies indicate that folding into a six-helix bundle is much faster for HIV-1 than for SIV.29 Our results show that the propensity of C-terminal peptides to form stable bundles with N36 counterparts may not be predictive of inhibitory potency. However, it has been shown that destabilizing the gp41 core
Figure 2. Lack of correlation of C34 inhibitory potency with N36/ C34 stability. The C34 peptides listed in Table 1 were tested for inhibition of HIV/SIV Envmediated cell fusion. IC50 values (Table 1) for inhibition by HIV-1 C34 (circles), HIV-2 C34 (squares) and SIV C34 (triangles) are plotted on a logarithmic scale against the Tm of the corresponding N36/C34 complex. The Tm values for the mixtures of N and C-peptides that failed to form cores were set at 0 8C. The linear regression (line) yields an r2 value of 0.65, indicating a lack of correlation between C34 inhibitory potency with N36/C34 stability.
13
HIV/SIV Sensitivity to Entry Inhibitors
bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.
Acknowledgements
Figure 3. Inhibition by SIV C34 following priming with sCD4 and washing. Effector cells (E) expressing HIV-1IIIB SIVMAC or HIV-2SBL envelope were pre-incubated for one hour at 37 8C with 10 mg/ml of sCD4, with or without 30 mM of SIV C34. Effector cells were then washed and co-cultured with target cells (T) and fusion was assayed in two hours. In contrast to HIV-2/ SIV, HIV-1 Env-mediated fusion was inhibited in the prime-wash experiment.
results in stabilizing the gp120-gp41 envelope glycoprotein.30,31 We therefore surmize that it is the stability of the intact Env that renders it more resistant to C-terminal entry inhibitors. It has been shown by co-immunoprecipation16 and primewash9,21 experiments that sCD4 is sufficient to trigger conformational changes in HIV-1IIIB Env, which allow the exposure of N-HR bundles to C-terminal peptides. However, this may not be the case for HIV-2 or SIV Env. We therefore tested the stability of the three Envs by applying primewash experiments with sCD4 and SIV C34. Cells expressing HIV-1IIIB, SIVMAC or HIV-2SBL envelope were pre-incubated for one hour at 37 8C with 10 mg/ml of soluble CD4 (Immunodiagnostics, Inc., Woburn, MA), with or without 30 mM SIV C34, and then washed and co-cultured with target cells. Figure 3 shows that HIV-1 Env-mediated fusion is highly susceptible to inhibition by preincubation with CD4 and SIV C34, whereas no inhibition of HIV-2 or SIV Env-mediated fusion occurs under the same conditions. The CD4 and co-receptor-induced triggering events leading to HIV/SIV Env-mediated fusion are stochastic, leading to relatively slow fusion kinetics.5 The lack of synchrony in the activation of HIV/SIV Envs therefore provides an opportunity for the C-peptide inhibitors to bind to the pre-hairpin grooves, which in the case of HIV-1 Env become transiently exposed following CD4induced triggering. In the case of HIV-2/SIV Env these regions are not exposed following the interactions with sCD4 and therefore the C-terminal peptides have a much smaller window of opportunity to target that site. Our results therefore suggest that several factors, including six-helix
We are grateful to Dr Opendra Sharma for his help in procuring the N-HR and C-HR peptides through the NIH AIDS Research and Reference Reagent Program, which also supplied the TZM-bl and Sup-T1 cells. We thank Drs Pat Earl and Bernard Moss for their gift of vaccinia recombinants. We also thank the members of the Blumenthal laboratory for their helpful suggestions. This study was supported, in part, by the Minerva Foundation (to Y.S.).
References 1. Chan, D. C. & Kim, P. S. (1998). HIV entry and its inhibition. Cell, 93, 681– 684. 2. Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D. H., Greaves, M. F. & Weiss, R. A. (1984). The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature, 312, 763– 767. 3. Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., Hercend, T. et al. (1984). T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature, 312, 767– 768. 4. Berger, E. A. (1997). HIV entry and tropism: the chemokine receptor connection. AIDS, 11, S3– S16. 5. Gallo, S. A., Finnegan, C. M., Viard, M., Raviv, Y., Dimitrov, A., Rawat, S. S. et al. (2003). Env-mediated fusion reaction. Biochim. Biophys. Acta, 1614, 36 – 50. 6. Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J. & Wiley, D. C. (1997). Atomic structure of the ectodomain from HIV-1 gp41. Nature, 387, 426– 428. 7. Chan, D. C., Fass, D., Berger, J. M. & Kim, P. S. (1997). Core structure of gp41 from the HIV envelope glycoprotein. Cell, 89, 263– 273. 8. Caffrey, M., Cai, M., Kaufman, J., Stahl, S. J., Wingfield, P. T., Covell, D. G. et al. (1998). Threedimensional solution structure of the 44 kDa ectodomain of SIV gp41. EMBO J. 17, 4572– 4584. 9. Melikyan, G. B., Markosyan, R. M., Hemmati, H., Delmedico, M. K., Lambert, D. M. & Cohen, F. S. (2000). Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J. Cell Biol. 151, 413– 423. 10. Wild, C., Oas, T., McDanal, C., Bolognesi, D. & Matthews, T. (1992). A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc. Natl Acad. Sci. USA, 89, 10537– 10541. 11. Jiang, S., Lin, K., Strick, N. & Neurath, A. R. (1993). HIV-1 inhibition by a peptide. Nature, 365, 113. 12. Wild, C., Greenwell, T. & Matthews, T. (1993). A synthetic peptide from HIV-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion. AIDS Res. Hum. Retroviruses, 9, 1051– 1053. 13. Lu, M. & Kim, P. S. (1997). A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein. J. Biomol. Struct. Dynam. 15, 465– 471.
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14. Chan, D. C., Chutkowski, C. T. & Kim, P. S. (1998). Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target. Proc. Natl Acad. Sci. USA, 95, 15613– 15617. 15. Chen, C. H., Matthews, T. J., McDanal, C. B., Bolognesi, D. P. & Greenberg, M. L. (1995). A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion. J. Virol. 69, 3771–3777. 16. Furuta, R. A., Wild, C. T., Weng, Y. & Weiss, C. D. (1998). Capture of an early fusion-active conformation of HIV-1 gp41. Nature Struct. Biol. 5, 276– 279. 17. Rimsky, L. T., Shugars, D. C. & Matthews, T. J. (1998). Determinants of human immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides. J. Virol. 72, 986– 993. 18. Kilgore, N. R., Salzwedel, K., Reddick, M., Allaway, G. P. & Wild, C. T. (2003). Direct evidence that C-peptide inhibitors of human immunodeficiency virus type 1 entry bind to the gp41 N-helical domain in receptor-activated viral envelope. J. Virol. 77, 7669– 7672. 19. Wild, C. T., Shugars, D. C., Greenwell, T. K., McDanal, C. B. & Matthews, T. J. (1994). Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc. Natl Acad. Sci. USA, 91, 9770– 9774. 20. Malashkevich, V. N., Chan, D. C., Chutkowski, C. T. & Kim, P. S. (1998). Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides. Proc. Natl Acad. Sci. USA, 95, 9134– 9139. 21. Gallo, S. A., Puri, A. & Blumenthal, R. (2001). HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process. Biochemistry, 40, 12231– 12236. 22. Earl, P. L., Koenig, S. & Moss, B. (1991). Biological and immunological properties of human immunodeficiency virus type 1 envelope glycoprotein: analysis of proteins with truncations and deletions
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Edited by I. Wilson (Received 5 February 2004; received in revised form 7 April 2004; accepted 7 April 2004)
J. Mol. Biol. (2004) 340, 9–14
COMMUNICATION
The Stability of the Intact Envelope Glycoproteins is a Major Determinant of Sensitivity of HIV/SIV to Peptidic Fusion Inhibitors Stephen A. Gallo1†, Kelly Sackett2†, Satinder S. Rawat1, Yechiel Shai2 and Robert Blumenthal1* 1
Membrane Structure and Function Section, LECB CCR, National Cancer Institute-Frederick, NIH Frederick, MD 21702, USA 2
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100 Israel
C-peptides derived from the HIV envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (C-HR) region are potent HIV fusion inhibitors. These peptides interact with the gp41 N-terminal heptad repeat (N-HR) region and block the gp41 six-helix bundle formation that is required for fusion. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by comparing the ability of C34, derived from HIV-1, HIV-2 and SIV gp41, to inhibit HIV-1, HIV-2 and SIV envelope-mediated fusion and the ability of these peptides to form stable six-helix bundles with N36 peptides derived from gp41 of these three viruses. The ability to form six-helix bundles was examined by circular dichroism spectroscopy, and HIV/SIV Env-mediated membrane fusion was monitored by a dye transfer assay. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34, which all inhibited HIV-1 Env-mediated fusion at IC50 , 10 nM. The three C34 peptides were poor inhibitors of HIV-2 and SIV fusion (IC50 . 100 nM), although HIV-2 and SIV N36 formed stable helix bundles with SIV C34. Priming experiments with sCD4 indicate that, in contrast to HIV-1, HIV-2 and SIV Env do not expose their N-HR region to SIV C34 following CD4 binding, but rapidly proceed to co-receptor engagement and six-helix bundle formation resulting in fusion. Our results suggest that several factors, including six-helix bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: HIV; SIV; membrane fusion; entry inhibitors; gp41
Membrane fusion between human/simian immunodeficiency virus (HIV/SIV) and target cells is mediated by the trimeric envelope protein hetero-complex of gp120 and gp41.1 Upon binding of this complex with cellular CD4 receptor2,3 and chemokine co-receptor,4 conformational changes † S.A.G. & K.S. contributed equally to this work. Abbreviations used: HIV-1, human immunodeficiency virus type 1; HIV-2, human immunodeficiency virus type 2; SIV, simian immunodeficiency virus; N-HR, N-terminal heptad repeat; C-HR, C-terminal heptad repeat; CD, circular dichroism; PBS, phosphate-buffered saline; Env, envelope glycoprotein. E-mail address of the corresponding author: [email protected]
are induced in both gp120 and gp41 which allow for their partial dissociation from each other and the insertion of the fusion peptide into the target or viral membrane.1,5 The partial dissociation of HIV-1 gp120 from gp41 leads to the exposure of the N-terminal heptad repeat (N-HR) and C-terminal heptad repeat (C-HR) regions of gp41 prior to their refolding into a six-helix bundle6 – 8 and membrane fusion.9 Several peptides that mimic the sequence of the N-HR and C-HR regions have been found to inhibit fusion by blocking of the interaction between the N-HR and C-HR regions and, thus, preventing the formation of the six-helix bundle fusogenic state of gp41.10 – 14 C-HR-derived peptides (C-peptides) inhibit HIV-1 Env-mediated cell fusion and are
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
10
HIV/SIV Sensitivity to Entry Inhibitors
Table 1. N36/C34 core formation and inhibition of HIV/SIV Env-mediated fusion by C34 HIV-1IIIBEnva
HIV-1IIIBC34a HIV-2SBLC34b SIV Mac251C34c
HIV-2SBLEnvb
SIV Mac251Envc
IC50 (nM)d
Tm (8C)e
IC50 (nM)d
Tm (8C)e
IC50 (nM)d
Tm (8C)e
5^4 9^3 9^1
61 ^ 1 52 ^ 1 57 ^ 1
3334 ^ 1392 164 ^ 74 543 ^ 266
Nof Nof 42 ^ 1
.10,000 .10,000 2100 ^ 1200
Nof Nof 36 ^ 1
a Sequences of HIV-1IIIB Env-derived peptides: N36, SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL; C34, WMEWDREINN YTSLIHSLIEESQNQQEKNEQELL. b Sequences of HIV-2SBL Env-derived peptides: N36, RGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVT; C34, WQEWEHKIR FLEANISESLEQAQIQQEKNMYELQ. c Sequences of SIVMac251 Env-derived peptides: N36, AGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQTRVT; C34, WQEWERKVD FLEENITALLEEAQIQQEKNMYELQ. d HIV/SIV Env-mediated fusion was measured using the dye transfer assay described in the text. SupT1 cells were used as targets for the X4-utilizing HIV-1 and HIV-2 strains, and TZM-bl cells were used as targets for the R5-utilizing SIV strain. Inhibitory concentration 50% (IC50), the concentration of C34, which reduced the fusion levels by 50% in comparison with untreated control, was determined by fitting the dose–response curves to a simple hyperbolic function using Sigmaplot. e Based on temperature denaturation of core complexes as indicated by CD spectra (Figure 2) of N36–C34 peptide mixtures. N and C peptide pairs that formed cores in PBS at 10 mM were melted over a range of 15 –95 8C in a capped quartz cell. Ellipticity at 222 nm were recorded every 2 deg. C, with ten seconds averaging time, following two minutes equilibrium. The cell was warmed at a rate of 2 deg. C/minute. Tm was derived from CD melting curves. f The mixtures of N and C peptides that failed to form cores showed a gradual linear thermal denaturation profile from 0 8C to 60 8C with no Tm , indicating no structure inducing interaction between the peptides.
thought to act through binding to the hydrophobic grooves that line the internal N-terminal trimeric coiled-coil core of the gp41 ectodomain.15 – 18 C34 comprises residues that interact specifically with the deep hydrophobic pocket on the surface of the N-terminal internal trimeric coiled coil of gp41.14 T20 (also referred to as DP178, enfuvirtide and fuzeon), which has extensive overlap with C34, has been found to be effective in vivo and has been recently granted FDA approval as an anti-HIV therapeutic†. C-peptides derived from HIV-1 are poor inhibitors of HIV-2 entry.16,19 By contrast, C34 derived from either SIV or HIV-1 has been found to inhibit HIV-1 envelope glycoprotein (Env)-mediated fusion equally well.20 To understand the basis for the sensitivity/resistance of HIV/SIV to these entry inhibitors, we have conducted peptide inhibition studies on HIV-1, HIV-2 and SIV Envmediated fusion using C34 derived from HIV-1, HIV-2 or SIV as well as biophysical studies on possible complex formation between these peptides and their N36 counterparts. Fusion was assayed using a dye transfer system as described.21 In brief, CV-1 cells (ATCC, Rockville, MD) were plated on 96-well plates (Costar, Cambridge, MA) and infected with vaccinia recombinants (kindly provided by Drs Pat Earl and Bernard Moss) VPE16, for IIIB strain of HIV-1,22 VSC50 for the SBL/ISY strain of HIV-223 and WR194 for SIVMAC24 overnight at multiplicity of infection of 10. The HeLa-derived cell line, TZM-bl,25 which expresses high levels of CD4 and CCR5 along with endogenously expressed CXCR4, was obtained through the AIDS Research and Reference Reagent Program, Division † http://www.fuzeon.com/
of AIDS, NIAID, NIH from Dr John C. Kappes, Dr Xiaoyun Wu and Tranzyme Inc. SupT1 cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr James Hoxie. Vaccinia-infected CV-1 cells labeled with 5-(and-6) (((4chloromethyl)benzoyl)amino)tetramethylrhodamine (494/517, red) were co-cultured at 37 8C with SupT1 cells or TZM-bl cells labeled with Calcein (541/565, green). The fluorescent dyes were obtained from Molecular Probes (Eugene, OR). The fusion inhibitors (see Table 1), obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, were dissolved in 10% NH4OH in 200 mM stock solutions and added to the cells at the beginning of the co-culture. To examine the susceptibility of HIV/SIV Envmediated fusion to inhibition by peptides, dose response curves were generated from the cell – cell fusion assay described above. Table 1 shows that HIV-1, HIV-2 and SIV C34 inhibited HIV-1 Envmediated cell – cell fusion with approximately equal efficiency with IC50 , 10 nM. All of these peptides inhibit HIV-2 Env-mediated fusion with far less efficacy (IC50 . 100 nM), and only SIV C34 inhibited SIV Env-mediated fusion. In addition, HIV-1 C34 never fully inhibited HIV-2SBL Envmediated fusion, only reaching 50% inhibition at very high concentrations. To examine the ability of the C34 peptides to form stable six-helix bundles with their N36 counterparts, we performed circular dichroism (CD) experiments using an Aviv 202 CD spectrometer. Peptides were dissolved in PBS (pH 7.4) to a final concentration of 10 mM. Spectral contribution from the PBS solvent was subtracted from that of peptide(s) dissolved in solvent. The wavelength dependence of ellipticity was monitored at 25 8C in 1 nm increments using ten seconds
Figure 1. Circular dichroism studies of complex formation between N36 and C34 pairs. HIV-2SBL, HIV-1IIIB or SIVMAC N36 peptide (10 mM) was incubated with either HIV-2SBL, HIV-1IIIB or SIVMAC C34 peptide (10 mM) and the spectra were recorded. Shown is the actual reading of the peptide pairs versus the theoretical addition of the individual peptide spectra. The unit of CD on the Y-axis is elipticity (mdeg.) and the unit of wavelength on the X-axis is nm. Since all the pairs have comparable molecular masses, the values on the Y-axis reflect the relative elipticities of all of them. Although HIV-1 N36 forms complexes with all three C34 peptides, both HIV-2 and SIV N36 only form complexes with SIV C34.
12
averaging time. Each peptide was measured alone and then N36/C34 pairs were measured. Triplicate recordings were conducted for each peptide and peptide pair, with superimposing spectra. The N36/C34 pairs included homogenous pairs and heterogenous pairs (e.g. HIV-2 N36 and HIV-1 C34). The sum of the individual N-HR and C-HR peptide spectra were added together and presented graphically along with the spectra of the co-incubated peptide pairs, for comparison. Figure 1 shows that helical structure is induced upon co-incubation of HIV-1 N36 with C34 peptides derived from all three species, indicating core formation in each case. By contrast N36 peptides derived from HIV-2 and SIV did not induce a-helical structure when incubated with C34 peptides derived from HIV-1 or HIV-2. However, SIV C34 formed helical cores when incubated with N36 derived from all three species. In order to determine the comparative stability of the various C and N-peptide mixes with C34 inhibitory efficacy we determined the melting temperature ðTm Þ of the complexes. Table 1 shows the Tm values for all the C and N-peptide mixes that formed complexes. Figure 2 shows a semi-logarithmic plot of IC50 values against the Tm of the corresponding N36/C34 complexes. In contrast to data on inhibition of HIV-1 Env-mediated fusion by C34 variants with modified cavity-binding residues,14 there appears to be a lack of correlation between stability of N36/C34 HIV-1/HIV-2/SIV complexes and C34 inhibitory efficacy. In Figure 2 the data are clustered in three groups. HIV-1 N36 formed stable helix bundles with HIV-1, HIV-2 and SIV C34 ðTm . 50 8CÞ, which all inhibited HIV-1 Envmediated fusion at IC50 , 10 nM. The second group yielded moderate Tm values (, 40 8C) and higher IC50 values (. 100 nM). The third group exhibited no complex-forming propensity ðTm , 0 8CÞ. HIV-2 N36/C34 belongs to this
HIV/SIV Sensitivity to Entry Inhibitors
group; yet HIV-2 C34 inhibits HIV-2 ENV-mediated fusion with greater efficacy than SIV C34, which makes a relatively stable complex with HIV-2 N36. Based on these data it is hard to explain the differences in inhibitory efficacy of the various C34-derived peptides for HIV-1, HIV-2 and SIV Env-mediated fusion. To examine whether the lower efficacy of HIV-2/ SIV Env-mediated fusion inhibition by C-peptides was due to kinetics,26 we reduced these fusion rates by lowering the incubation temperature. At 31 8C the time (t1/2) to reach half-maximal HIV-2 Env-mediated fusion was reduced from 15 minutes (at 37 8C) to 30 minutes. The t1/2 for HIV-1 Envmediated fusion at 37 8C was about the same as that for HIV-2 Env-mediated fusion at 31 8C. Yet the IC50 for inhibition by SIV C34 for HIV-2 Envmediated fusion at 31 8C was still an order of magnitude higher than that for inhibition of HIV-1 Env-mediated fusion at 37 8C. Since in this case kinetics does not appear to be the only determinant of entry inhibitor efficacy, we considered the issue of stability of the intact envelope glycoproteins. Although the sequence homology between HIV-1 and SIV in the gp41 ectodomain is relatively low, around 56% amino acid identity24, there is great similarity between the structures of the SIV (which has high sequence homology with HIV-2) and the HIV-1 gp41 ectodomain core.8,20 However, differences have been found in their thermodynamic properties as indicated by an , 30 deg. C lower melting temperature20,27,28 (see also Table 1) and a lower concentration of urea required for unfolding for SIV gp41.29 Moreover, in vitro studies indicate that folding into a six-helix bundle is much faster for HIV-1 than for SIV.29 Our results show that the propensity of C-terminal peptides to form stable bundles with N36 counterparts may not be predictive of inhibitory potency. However, it has been shown that destabilizing the gp41 core
Figure 2. Lack of correlation of C34 inhibitory potency with N36/ C34 stability. The C34 peptides listed in Table 1 were tested for inhibition of HIV/SIV Envmediated cell fusion. IC50 values (Table 1) for inhibition by HIV-1 C34 (circles), HIV-2 C34 (squares) and SIV C34 (triangles) are plotted on a logarithmic scale against the Tm of the corresponding N36/C34 complex. The Tm values for the mixtures of N and C-peptides that failed to form cores were set at 0 8C. The linear regression (line) yields an r2 value of 0.65, indicating a lack of correlation between C34 inhibitory potency with N36/C34 stability.
13
HIV/SIV Sensitivity to Entry Inhibitors
bundle stability and the ability of CD4 to destabilize the envelope glycoprotein, serve as determinants of sensitivity to entry inhibitors.
Acknowledgements
Figure 3. Inhibition by SIV C34 following priming with sCD4 and washing. Effector cells (E) expressing HIV-1IIIB SIVMAC or HIV-2SBL envelope were pre-incubated for one hour at 37 8C with 10 mg/ml of sCD4, with or without 30 mM of SIV C34. Effector cells were then washed and co-cultured with target cells (T) and fusion was assayed in two hours. In contrast to HIV-2/ SIV, HIV-1 Env-mediated fusion was inhibited in the prime-wash experiment.
results in stabilizing the gp120-gp41 envelope glycoprotein.30,31 We therefore surmize that it is the stability of the intact Env that renders it more resistant to C-terminal entry inhibitors. It has been shown by co-immunoprecipation16 and primewash9,21 experiments that sCD4 is sufficient to trigger conformational changes in HIV-1IIIB Env, which allow the exposure of N-HR bundles to C-terminal peptides. However, this may not be the case for HIV-2 or SIV Env. We therefore tested the stability of the three Envs by applying primewash experiments with sCD4 and SIV C34. Cells expressing HIV-1IIIB, SIVMAC or HIV-2SBL envelope were pre-incubated for one hour at 37 8C with 10 mg/ml of soluble CD4 (Immunodiagnostics, Inc., Woburn, MA), with or without 30 mM SIV C34, and then washed and co-cultured with target cells. Figure 3 shows that HIV-1 Env-mediated fusion is highly susceptible to inhibition by preincubation with CD4 and SIV C34, whereas no inhibition of HIV-2 or SIV Env-mediated fusion occurs under the same conditions. The CD4 and co-receptor-induced triggering events leading to HIV/SIV Env-mediated fusion are stochastic, leading to relatively slow fusion kinetics.5 The lack of synchrony in the activation of HIV/SIV Envs therefore provides an opportunity for the C-peptide inhibitors to bind to the pre-hairpin grooves, which in the case of HIV-1 Env become transiently exposed following CD4induced triggering. In the case of HIV-2/SIV Env these regions are not exposed following the interactions with sCD4 and therefore the C-terminal peptides have a much smaller window of opportunity to target that site. Our results therefore suggest that several factors, including six-helix
We are grateful to Dr Opendra Sharma for his help in procuring the N-HR and C-HR peptides through the NIH AIDS Research and Reference Reagent Program, which also supplied the TZM-bl and Sup-T1 cells. We thank Drs Pat Earl and Bernard Moss for their gift of vaccinia recombinants. We also thank the members of the Blumenthal laboratory for their helpful suggestions. This study was supported, in part, by the Minerva Foundation (to Y.S.).
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Edited by I. Wilson (Received 5 February 2004; received in revised form 7 April 2004; accepted 7 April 2004)