Inhibition of human immunodeficiency virus type 1 infection of human CD4+ T cells by microbial HSP70 and the peptide epitope 407-426

Human immunodeficiency virus type 1 (HIV-1) virions contain heat shock proteins (HSP), but these proteins have received limited attention. The objectives of this study were to establish if the microbial 70-kDa HSP exerts an inhibitory effect on the HIV-1 infection of human CD4+ T cells, to identify an inhibitory peptide epitope within the sequence of HSP70, and to evaluate the kinetic features of any inhibitory activity. The results of these studies suggest that microbial HSP70 exerts dose-dependent inhibition on CCR5 (R5) strains of clades B, C, and D of HIV-1 infecting human CD4+ T cells. The site of the HIV-1-inhibitory function was identified within the C-terminal peptide binding domain of HSP70, and the function is expressed by the peptide epitope comprising amino acids 407 to 426. The mechanism of inhibition of HIV-1 infectivity by HSP70 is blocking of the CCR5 coreceptors directly and indirectly by inducing CC chemokines and APOBEC3G. The inhibitory effect of HSP70, its C-terminal fragment, or peptide 407-426 may make HSP70 useful as a microbicidal agent. A potentiating noncognate inhibition of HIV-1 infectivity by combined treatment with HSP70 and monoclonal or polyclonal antibody to CCR5 was demonstrated. This novel strategy may be utilized in therapeutic immunization against HIV-1 infection.     

From receptor recognition mechanisms to bioinspired mimetic antagonists in HIV-1/cell docking

Understanding the ways in which two or more proteins interact may give insight into underlying binding and activation mechanisms in biology, methods for protein separation and structure-based antagonism. This review describes ways in which protein recognition has been explored in our laboratory for the HIV-1/cell entry process. Initial contact between an HIV-1 virion particle and a human cell occurs between gp120 (an HIV-1 envelope protein) and CD4 (a human extracellular signaling protein). This interaction leads to a sequence of events which includes a conformational change in gp120, fusion of the HIV-1 and cellular membranes and eventual infection of the cell. Using an optical biosensor and a reporter antibody, we have been able to measure the conformational change in gp120 that occurs upon CD4 binding. We also have used this biosensor system to characterize CD4 mimetics, obtained by peptide synthesis in miniprotein scaffolds. Phage display techniques have been employed to identify novel miniprotein sequences. The combination of biosensor interaction kinetics analysis and phage display provides a useful approach for understanding the recognition mechanisms involved in the HIV/cell docking process. This approach may also be useful in investigating other protein complexes of importance in health and disease.     

Synthesis,Characterization and Conformational Analysis of gp120-derived Synthetic Peptides That Specifically Enhance HIV-1 Infectivity

A series of peptides patterned on the principal neutralizing domain of the HIV-1 envelope glycoprotein gp120 have been synthesized by solid-phase techniques. Interestingly, in vitro experiments have shown that some of these peptides specifically interact with CD4 and, in particular, that the peptide corresponding to the sequence 307–330 of the HIV-1 MN isolate was able to enhance infection in a dose- specific and not a strain-restricted way. To bypass problems observed in preliminary runs, several peptides were synthesized by both Fmoc and Boc chemistry. Comparison of the two strategies has allowed the set up of convenient protocols for the preparation of the target peptides in good yield, and with the high-purity grade needed for biological and physicochemical studies. Since the biological effects were present in the carboxyl-free C-terminal linear peptide but not in the amidated C-terminal analogue, preliminary conformational studies by circular dichroism and nuclear magnetic resonance techniques were also performed in an attempt to correlate these effects with possible contributions of structured conformations as predicted by theoretical calculations. The possibility of a β-turn structure for the crucial Gly-Pro-Gly-Arg sequence has been confirmed by 2D NMR experiments. Ongoing studies suggest the exploitation of the activating properties of the MN-derived peptides to design a more sensitive and innovative serological test based on the virus itself and not on anti-HIV antibodies, as is the case for the large majority of tests currently in use. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Broad and Cross-Clade CD4+ T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4⁺ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4⁺ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4⁺ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA^b and IA^d. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4⁺ and CD8⁺ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4⁺ and CD8⁺ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.     

Human immunodeficiency virus (HIV) type 1 proviral hypermutation correlates with CD4 count in HIV-infected women from Kenya

APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the vpu/env region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral vif. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.     

Effects of CD4 synthetic peptides on HIV type I envelope glycoprotein function.

Benzylated derivatives of a peptide (CD4(81-92)) representing the CDR3-like region of CD4 were previously found to inhibit gp120 binding, HIV-1 infectivity, and syncytium formation. These results have been interpreted to indicate a role for the corresponding CD4 region in these processes. The peptide (TbYICbEbVEDQKAcEE) is the prototype of a series of similar CD4(81-92) derivatives. We report that this peptide noncompetitively inhibits binding to CD4 of both gp120 and a mAb (MAX.16H5), both of which recognize the CDR2-like region of CD4. The binding of an antibody (Leu 3a) that is directed against a different area of the D1 domain of CD4 was also inhibited. The peptide derivative inhibited both HIV-1- and HTLV-1-mediated syncytium formation in the same concentration range. Nonbenzylated cyclic and linear peptides representing the CDR3-like region of CD4 (CD4(84-101)) had only minor effects on gp120 binding which were not sequence specific. The results of this study suggest that the effects of benzylated CD4(81-92) derivatives on HIV-1 binding or fusion should not be used to reach conclusions about the function of the corresponding CD4 region.     

A functional, discontinuous HIV-1 gp120 C3/C4 domain-derived, branched, synthetic peptide that binds to CD4 and inhibits MIP-1alpha chemokine binding.

This paper describes a branched synthetic peptide [3.7] that incorporates sequence discontinuous residues of HIV-1 gp120 constant regions. The approach was to bring together residues of gp120 known to interact with human cell membranes such that the peptide could fold to mimic the native molecule. The peptide incorporates elements of both the conserved CD4 and CCR5 binding sites. The 3.7 peptide, which cannot be produced by conventional genetic engineering methods, is recognized by antiserum raised to native gp120. The peptide also binds to CD4 and competitively inhibits binding of QS4120 an antibody directed against the CDR2 region of CD4. When preincubated with the CD4+ve MM6 macrophage cell line, which expresses mRNA for the CCR3 and CCR5 chemokine receptors, both 3.7 and gp120 inhibit binding of the chemokine MIP-1alpha. The peptide also inhibits infection of primary macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate peptide for a vaccine against HIV-1 and represents a novel approach to the rational design of peptides that can mimic complex sequence discontinuous ligand binding sites of clinically relevant proteins.     

Inhibition of HIV-1 infection by an intramolecular antisense peptide to T20 in gp160

Antisense amino acids are amino acids which can be translated from the corresponding anti-codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV-1 and has been demonstrated to be a potent inhibitor of HIV-1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA-1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA-1) to T20, was shown to inhibit HIV-1(IIIB) infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. The TA-1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV-1, especially at TA-1 and in the amino acids flanking the C terminus. Although the TA-1 sites of 18 out of 30 HIV-1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV-1(MN), were not. However, TA-1L inhibited infection by HIV-1(MN), which has no antisense peptide in T20 corresponding to TA-1, although the inhibitory effect was weaker. TA-1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA-1.     

Apelin, the natural ligand of the orphan seven-transmembrane receptor APJ, inhibits human immunodeficiency virus type 1 entry

In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolates from different clades into cells expressing CD4 and APJ. Analysis of apelin analogues demonstrated that potent and specific antiviral activity was retained by a 13-residue, arginine-rich peptide. Antiviral potency was influenced by the integrity of methionine 75, which contributes to APJ-binding affinity, and by the retention of apelin residues 63 to 65. These studies demonstrate the ability of a small peptide ligand to block the function of APJ as an HIV-1 coreceptor, identify apelin sequences important for the inhibition, and provide new reagents for the investigation of the significance of APJ to HIV-1 infection and pathogenesis.     

A Species-Specific Amino Acid Difference in the Macaque CD4 Receptor Restricts Replication by Global Circulating HIV-1 Variants Representing Viruses from Recent Infection

HIV-1 replicates poorly in macaque cells, and this had hindered the advancement of relevant nonhuman primate model systems for HIV-1 infection and pathogenesis. Several host restriction factors have been identified that contribute to this species-specific restriction to HIV-1 replication, but these do not fully explain the poor replication of most strains of HIV-1 in macaque cells. Only select HIV-1 envelope variants, typically those derived from viruses that have been adapted in cell culture, result in infectious chimeric SIVs encoding HIV-1 envelope (SHIVs). Here we demonstrate that most circulating HIV-1 variants obtained directly from infected individuals soon after virus acquisition do not efficiently mediate entry using the macaque CD4 receptor. The infectivity of these viruses is ca. 20- to 50-fold lower with the rhesus and pig-tailed macaque versus the human CD4 receptor. In contrast, culture-derived HIV-1 envelope variants that facilitate efficient replication in macaques showed similar infectivity with macaque and human CD4 receptors (within ∼2-fold). The ability of an envelope to mediate entry using macaque CD4 correlated with its ability to mediate entry of cells expressing low levels of the human CD4 receptor and with soluble CD4 sensitivity. Species-specific differences in the functional capacity of the CD4 receptor to mediate entry mapped to a single amino acid difference at position 39 that is under strong positive selection, suggesting that the evolution of CD4 may have been influenced by its function as a viral receptor. These results also suggest that N39 in human CD4 may be a critical residue for interaction of transmitted HIV-1 variants. These studies provide important insights into virus-host cell interactions that have hindered the development of relevant nonhuman primate models for HIV-1 infection and provide possible markers, such as sCD4 sensitivity, to identify potential HIV-1 variants that could be exploited for development of better SHIV/macaque model systems.     

Magnitude of functional CD8+ T-cell responses to the gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma

The importance of CD8+ T-cell responses in the control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated, yet few studies have been able to correlate these responses with markers of HIV-1 disease progression. This study measured cell-mediated immune responses using peripheral blood mononuclear cells (PBMC) obtained from 27 patients with chronic HIV-1 infection, the majority of whom were off antiretroviral therapy. The ELISPOT assay was used to detect gamma interferon-secreting PBMC after stimulation with overlapping HIV-1 peptides spanning the Gag, Pol, Env, and Nef proteins in addition to the baculovirus-derived p24 and gp160 proteins. All volunteers had responses to at least one HIV-1-specific peptide. All but one of the subjects (96%) responded to the Gag peptide pool, and 86% responded to the Pol and/or Nef peptide pools. The magnitude and the breadth of T-cell responses directed to either the Gag or p24 peptide pools correlated inversely with viral load in plasma (r = -0.60, P < 0.001 and r = -0.52, P < 0.005, respectively) and directly with absolute CD4+ T-cell counts (r = 0.54, P < 0.01 and r = 0.39, P < 0.05, respectively) using the Spearman rank correlation test. Responses to the Pol and integrase peptide pools also correlated with absolute CD4+ T-cell counts (r = 0.45, P < 0.05 and r = 0.49, P < 0.01, respectively). No correlation with markers of disease progression was seen with specific T-cell responses directed toward the Env or Nef peptides. These data serve as strong evidence that major histocompatibility complex class I presentation of Gag peptides is an essential feature for any HIV-1 vaccine designed to elicit optimal CD8+ T-cell responses.     

A Synthetic Peptide Fragment Derived from RANTES is a Potent Inhibitor of HIV-1 Infectivity Despite a Surprising Lack of CCR5 Receptor Affinity

Summary CCR5 (CC-chemokine receptor 5) is a key co-receptor, in concert with CD4, for infectivity of HIV-1 (human immunodeficiency virus type-1) into healthy human cells, and RANTES, an endogenous ligand for CCR5, is a potent inhibitor of HIV-1 infectivity. In this structure-activity relationship (SAR) study, peptide fragments derived from RANTES were designed, synthesized and evaluated for their ability to inhibit HIV-1 infectivity. The goal was to determine the effect of peptide length on anti-HIV activity and to obtain an optimally sized RANTES peptide probe for further SAR studies. The analogue Ac[Ala^10,11]RANTES-(1–14)NH₂, AA14, was identified as an effective inhibitor of HIV-1 infectivity at 10 nM but despite the functional activity, surprisingly it did not exhibit any notable affinity for the CCR5 chemokine receptor. Further, increasing peptide size enhanced neither the inhibition of HIV-1 infectivity nor CCR5 receptor affinity. As a potent inhibitor of HIV-1 infectivity, the lead analogue most likely utilizes a different (and currently unknown) mechanism than interaction with CCR5 for anti-HIV activity.     

A Synthetic Peptide Fragment Derived from RANTES is a Potent Inhibitor of HIV-1 Infectivity Despite a Surprising Lack of CCR5 Receptor Affinity

CCR5 (CC-chemokine receptor 5) is a key co-receptor, in concert with CD4, for infectivity of HIV-1 (human immunodeficiency virus type-1) into healthy human cells, and RANTES, an endogenous ligandfor CCR5, is a potent inhibitor of HIV-1 infectivity. In this structure-activity relationship (SAR) study, peptide fragments derived from RANTES were designed, synthesized and evaluated fortheir ability to inhibit HIV-1 infectivity. The goal was to determine the effect of peptide length on anti-HIV activity and to obtain an optimally sized RANTES peptide probe for further SAR studies. The analogue Ac[Ala^10,11]RANTES-(1–14)NH₂, AA14, was identified as an effective inhibitor of HIV-1 infectivity at 10 nM but despite the functional activity, surprisingly it did not exhibit any notable affinity for the CCR5 chemokine receptor. Further, increasing peptide size enhanced neither the inhibition of HIV-1 infectivity nor CCR5 receptor affinity. As a potent inhibitor of HIV-1 infectivity,the lead analogue most likely utilizes a different (and currentlyunknown) mechanism than interaction with CCR5 for anti-HIV activity.     

Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.     

Evidence by peptide mapping that the region CD4(81-92) is involved in gp120/CD4 interaction leading to HIV infection and HIV-induced syncytium formation.

Peptide fragments of the CD4 molecule were compared in their ability to 1) inhibit CD4-dependent HIV-induced cell fusion; 2) inhibit CD4-dependent HIV infection in vitro; and 3) block gp120 envelope glycoprotein binding to CD4. Peptides from the region CD4(81-92), although inactive when underivatized, were equipotent inhibitors of CD4-dependent virus infection, cell fusion, and CD4/gp120 binding when derivatized via benzylation and acetylation. Peptides of identical chemical composition, but altered sequence and derivatization pattern that blocked gp120 binding to either CD4-positive cells or solubilized CD4, also blocked infection and fusion with similar potencies. Those that did not block gp120/CD4 interaction were also inactive in HIV-1 infection and cell fusion assays. No other peptide fragments of the CD4 molecule inhibited fusion, infection, or CD4/gp120 interaction. The peptide CD4(23-56), derived from a region of CD4 implicated in binding of CD4 antibodies that neutralize HIV infection and cell fusion, had no effect on CD4-dependent cell fusion, HIV-1 infection, or CD4/gp120 binding, but did reverse OKT4A and anti-Leu 3a blockade of gp120 binding to CD4. These data provide evidence that the 81-92 region of CD4 is directly involved in gp120 binding leading to CD4-dependent HIV infection and syncytium formation. Previous observations with structural mutants of CD4 suggest that the CDR2-homologous region of CD4 is also involved, either directly or indirectly, in binding of gp120 to CD4. The CDR2- and CDR3-like domains of CD4 may both contribute to the binding of the HIV envelope necessary for HIV-1 infection and HIV-1-induced cell fusion.     

Envelope glycoproteins of human immunodeficiency virus type 1: profound influences on immune functions.

Infection by human immunodeficiency virus type 1 (HIV-1) leads to progressive destruction of the CD4+ T-cell subset, resulting in immune deficiency and AIDS. The specific binding of the viral external envelope glycoprotein of HIV-1, gp120, to the CD4 molecules initiates viral entry. In the past few years, several studies have indicated that the interaction of HIV-1 envelope glycoprotein with cells and molecules of the immune system leads to pleiotropic biological effects on immune functions, which include effects on differentiation of CD34+ lymphoid progenitor cells and thymocytes, aberrant activation and cytokine secretion patterns of mature T cells, induction of apoptosis, B-cell hyperactivity, inhibition of T-cell dependent B-cell differentiation, modulation of macrophage functions, interactions with components of complement, and effects on neuronal cells. The amino acid sequence homologies of the envelope glycoproteins with several cellular proteins have suggested that molecular mimicry may play a role in the pathogenesis of the disease. This review summarizes work done by several investigators demonstrating the profound biological effects of envelope glycoproteins of HIV-1 on immune system cells. Extensive studies have also been done on interactions of the viral envelope proteins with components of the immune system which may be important for eliciting a "protective immune response." Understanding the influences of HIV-1 envelope glycoproteins on the immune system may provide valuable insights into HIV-1 disease pathogenesis and carries implications for the trials of HIV-1 envelope protein vaccines and immunotherapeutics.     

A synthetic peptide with sequence identity to the transmembrane protein GP41 of HIV-1 inhibits distinct lymphocyte activation pathways dependent on protein kinase C and intracellular calcium influx.

A synthetic peptide containing env amino acid (aa) sequence 581 to 597 of the transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) was tested for its effect on protein kinase C (PKC) and cytoplasmic free Ca2+ [( Ca2+]i) influx-dependent immune functions. We have previously shown that this peptide inhibits PKC-mediated phosphorylation and T-cell receptor-mediated [Ca2+]i influx as well as lymphoproliferation. In this study we demonstrate that the HIV-1 gp41 peptide aa581-597 inhibits lymphoproliferation stimulated via the distinct T-cell-activation molecules CD3, CD2, and CD28, as well as direct stimulation mediated by phorbol ester combined with ionomycin. Further, aa581-597 inhibits both PKC-dependent interleukin 2 (IL 2) production and the [Ca2+]i influx-dependent but PKC-independent induction of IL 2 receptor expression. The HIV-1 gp41 peptide also induces dramatic morphologic changes in lymphocytes, characterized by cytoplasmic ballooning and the acquisition of adherence to plastic, and these changes are dependent on both the length and the temperature of exposure. The results of this study suggest that the HIV-1 gp41 sequence aa581-597 acts at multiple sites to inhibit both PKC activity and [Ca2+]i influx, resulting in the abrogation of several distinct immune functions that are critical for an intact immune response and are defective in HIV-1-infected individuals.     

Comparison of the solution conformations of a cell-adhesive peptide LBE and its reverse sequence EBL

T-cell adhesion is mediated by an ICAM-1/LFA-1 interaction; this interaction plays a crucial role in T-cell activation during immune response. LBE peptide, which is derived from the beta-subunit of LFA-1, has been shown to inhibit ICAM-1/LFA-1-mediated T-cell adhesion. In this work, we studied the solution conformations of LBE peptide and its reverse sequence (EBL) by NMR, CD and molecular dynamics simulations. Reverse peptides have been used as controls in biological studies. The effect of reversing the sequence of LBE to EBL peptides on their respective conformations is important in understanding their biological properties in vitro or in vivo. The NMR studies for these peptides were carried out in water and in TFE/water solvent systems. In 40% TFE/water, both peptides exhibited helical conformation. CD studies suggested that the LBE exhibits 30% helical conformation, while the EBL exhibits 20% helical conformation. From the NMR and MD simulation studies, it was evident that the peptides exhibited a stable helical conformation; a stable helical structure was found at Leu6 to Leu15 for LBE and at Gly9 to Leu17 for EBL. The helical conformations of LBE and EBL may be in equilibrium with other possible conformers; the other conformers contain loop and turn structures. Both peptides bind to divalent cations because the LBE is derived from the cation-binding region of the LFA-1. This study shows that reversing the peptide sequence did not alter the secondary structure of the corresponding sequence. Hence, caution must be exercised when using reverse peptides as controls in biological studies. This report will improve our ability to design a better inhibitor of ICAM-1/LFA-1 interaction.