HR2 Peptide Research Articles

Design of recombinant protein-based SARS-CoV entry inhibitors targeting the heptad-repeat regions of the spike protein S2 domain

Entry of SARS-CoV into a target cell is initiated by binding of the S1 domain of spike protein to a receptor, followed by conformational changes of the spike protein S2 domain, resulting in the formation of a six-helix bundle by the heptad-repeat (HR1 and HR2) regions. Our previous studies have demonstrated that peptides derived from HR2 region could inhibit SARS-CoV entry. However, synthesis of these peptides is at high cost. In this study, we designed two recombinant proteins, one containing two HR1 and one HR2 peptides (denoted HR121), and the other consisting of two HR2 and one HR1 peptides (designated HR212). These two proteins could be easily purified with the low cost of production, exhibiting high stability and potent inhibitory activity on entry of the HIV/SARS pseudoviruses with IC 50 values of 4.13 and 0.95 μM, respectively. These features suggest that HR121 and HR212 can serve as potent inhibitors of SARS-CoV entry.

Characterization of BIV Env core: Implication for mechanism of BIV-mediated cell fusion

Entry of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), requires folding of two heptad repeat regions (HR1 and HR2) of gp41 into a trimer-of-hairpins, which subsequently brings virus and cell membrane into fusion. This motif is a generalized feature of viral fusion proteins and has been exploited in generating antiviral fusion agents. In the present paper, we report structural characters of Env protein from another lentivirus, bovine immunodeficiency virus (BIV), which contributes to a good animal model of HIV. BIV HR1 and HR2 regions are predicted by two different programs and expressed separately or conjointly in Escherichia coli. Biochemical and biophysical analyses show that the predicted HRs of BIV Env can form a stable trimer-of-hairpins or six-helix bundle just like that formed by feline immunodeficiency virus Env. Cell fusion assay demonstrates that the HR2 peptide of BIV can efficiently inhibit the virus-mediated cell fusion.

Structure and function study of paramyxovirus fusion protein heptad repeat peptides

Paramyxovirus might adopt a molecular mechanism of membrane fusion similar to that of other class I viruses in which the heptad repeat (HR) regions of fusion protein (F) HR1 and HR2 form a six-helix bundle structure inducing membrane fusion. In this study, we examined the structure and function of HR1 and HR2 from the avian paramyxovirus-2 (APMV-2) F protein. The study showed that APMV-2 HR1 and HR2 formed a stable six-helix bundle. Only a soluble APMV-2 HR2 peptide showed potent and specific virus-cell fusion inhibition activity. Cross-inhibiting activity with APMV-1 (Newcastle disease virus, NDV) was not found. A possible mechanism of membrane fusion inhibition by the paramyxovirus HR2 peptide is discussed.

Structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion protein.

A coronavirus (CoV) has recently been identified as the causative agent of the severe acute respiratory syndrome (SARS) in humans. CoVs enter target cells through fusion of viral and cellular membranes mediated by the viral envelope glycoprotein S. We have determined by x-ray crystallography the structure of a proteolytically stable core fragment from the heptad repeat (HR) regions HR1 and HR2 of the SARS-CoV S protein. We have also determined the structure of an HR1-HR2 S core fragment, containing a shorter HR1 peptide and a C-terminally longer HR2 peptide that extends up to the transmembrane region. In these structures, three HR1 helices form a parallel coiled-coil trimer, whereas three HR2 peptides pack in an oblique and antiparallel fashion into the coiled-coil hydrophobic grooves, adopting mixed extended and alpha-helical conformations as in postfusion paramyxoviruses F proteins structures. Our structure positions a previously proposed internal fusion peptide adjacent to the N-terminus of HR1. Peptides from the HR2 region of SARS-CoV S have been shown to inhibit viral entry and infection in vitro. The structures presented here can thus open the path to the design of small-molecule inhibitors of viral entry and candidate vaccine antigens against this virus.

Heptad Repeat 2-Based Peptides Inhibit Avian Sarcoma and Leukosis Virus Subgroup A Infection and Identify a Fusion Intermediate

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.

An inducible HIV type 1 gp41 HR-2 peptide-binding site on HIV type 1 envelope gp120.

Synthetic peptides of sequences within the HIV-1 gp41 heptad repeat-regions (HR-1 and HR-2) can effectively inhibit cell fusion and viral entry. DP178 (T-20), an HR-2 peptide, acts by inhibiting the association between HR-1 and HR-2, thereby interfering with HIV-1 fusion and viral entry. HR-2 peptide binding is predicted to be an important indicator of the presence of Env gp41 fusion intermediate conformation. A stabilized HR-2/Env conjugate might be an HIV-1 vaccine candidate and have the potential for inducing antibodies against transiently exposed epitopes on HIV-1 Env. To explore the possibility of design of HR-2 stabilized-HIV-1 immunogens, we studied the ability of HIV-1 Env to bind to HR-2 peptides. Using surface plasmon resonance (SPR)-binding assays and precipitation of soluble Env gp120 proteins with HR-2 peptide DP178, we have found that there is an HR-2 peptide-binding site on soluble HIV-1 recombinant gp120. Binding of DP178 was induced by sCD4 and by the anti-gp120 human mAb A32. The induction of DP178 binding was inhibited > 80% by the HIV-1 coreceptor-binding site mAb 17b. Binding of DP178 to gp120 was also inhibited by gp120 C4 peptides with sequences that are centrally located within the HIV-1 coreceptor-binding site. Thus, in addition to interactions with the gp41 HR-1 region, the fusion inhibitor peptide DP178 binds to triggered soluble HIV-1 recombinant gp120 following its interaction with sCD4 or CD4 mimic mAb A32. This may prove to be an important consideration when designing an HIV vaccine that utilizes constrained HIV Env proteins.

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides.

The coronavirus SARS-CoV is the primary cause of the life-threatening severe acute respiratory syndrome (SARS). With the aim of developing therapeutic agents, we have tested peptides derived from the membrane-proximal (HR2) and membrane-distal (HR1) heptad repeat region of the spike protein as inhibitors of SARS-CoV infection of Vero cells. It appeared that HR2 peptides, but not HR1 peptides, were inhibitory. Their efficacy was, however, significantly lower than that of corresponding HR2 peptides of the murine coronavirus mouse hepatitis virus (MHV) in inhibiting MHV infection. Biochemical and electron microscopical analyses showed that, when mixed, SARS-CoV HR1 and HR2 peptides assemble into a six-helix bundle consisting of HR1 as a central triple-stranded coiled coil in association with three HR2 alpha-helices oriented in an antiparallel manner. The stability of this complex, as measured by its resistance to heat dissociation, appeared to be much lower than that of the corresponding MHV complex, which may explain the different inhibitory potencies of the HR2 peptides. Analogous to other class I viral fusion proteins, the six-helix complex supposedly represents a postfusion conformation that is formed after insertion of the fusion peptide, proposed here for coronaviruses to be located immediately upstream of HR1, into the target membrane. The resulting close apposition of fusion peptide and spike transmembrane domain facilitates membrane fusion. The inhibitory potency of the SARS-CoV HR2-peptides provides an attractive basis for the development of a therapeutic drug for SARS.

Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors

SummaryBackgroundStudies on the fusion-inhibitory peptides derived from the heptad repeat 1 and 2 (HR1 and HR2) regions of the HIV-1 envelope glycoprotein gp41 provided crucial information on the viral fusogenic mechanism. We used a similar approach to study the fusogenic mechanism of severe-acute-respiratory-syndrome-associated coronavirus (SARS-CoV).MethodsWe tested the inhibitory activity against infection of two sets of peptides corresponding to sequences of SARS-CoV spike protein HR1 and HR2 regions and investigated the interactions between the HR1 and HR2 peptides by surface plasmon resonance, sedimentation equilibration analysis, circular dichroism, native polyacrylamide-gel electrophoresis, size exclusion high-performance liquid chromatography, and computer-aided homology modelling and molecule docking analysis.FindingsOne peptide, CP-1, derived from the HR2 region, inhibited SARS-CoV infection in the micromolar range. CP-1 bound with high affinity to a peptide from the HR1 region, NP-1. CP-1 alone had low -helicity and self-associated to form a trimer in phosphate buffer (pH 7·2). CP-1 and NP-1 mixed in equimolar concentrations formed a six-helix bundle, similar to the fusogenic core structure of HIV-1 gp41.InterpretationAfter binding to the target cell, the transmembrane spike protein might change conformation by association between the HR1 and HR2 regions to form an oligomeric structure, leading to fusion between the viral and target-cell membranes. At the prefusion intermediate state, CP-1 could bind to the HR1 region and interfere with the conformational changes, resulting in inhibition of SARS-CoV fusion with the target cells. CP-1 might be modifiable to increase its anti-SARS-CoV activity and could be further developed as an antiviral agent for treatment or prophylaxis of SARS-CoV infection.

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex.

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus.

Menangle virus is a novel paramyxovirus isolated in Australia in 1997, but its classification position has not yet been finally settled. Here by using a computational program, LearnCoil-VMF, we determined the heptad repeat (HR) regions (HR1 and HR2) of Menangle virus F protein. Subsequently the HR1 and HR2 peptides were expressed as a single chain (named 2-Helix) connected by a six amino-acid linker as a GST fusion protein with an E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of alpha-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials.

The hydrophobic pocket contributes to the structural stability of the N-terminal coiled coil of HIV gp41 but is not required for six-helix bundle formation.

In models of HIV fusion, the glycoprotein gp41 is thought to form a six-helix bundle during viral fusion with the target cell. This bundle is comprised of three helical regions (from the heptad repeat 2, or HR2, region of gp41) bound to an inner, trimeric, coiled-coil core (from the HR1 region). Although much has been learned about the structure and thermodynamics of this complex, the energetics of the isolated HR1 self-associated oligomer remain largely unknown. By systematically studying self-association through a series of truncations based on a 51-mer HR1 peptide (T865), we have identified amino acid segments which contribute significantly to the stability of the oligomeric HR1 complex. Biophysical characterization of C-terminal truncations of T865 identifies a 10-15-amino acid region that is essential for HR1 oligomerization. This region coincides with a hydrophobic pocket that provides important contacts for the interaction of HR2 helices. Complete removal of this pocket abolishes HR1 oligomerization. Despite the dramatic reduction in stability, the monomeric HR1 peptides are still able to form stable six-helix bundles in the presence of HR2 peptides. Truncations on the N-terminal side of T865 have little effect on oligomerization but significantly reduce the stability of the HR1-HR2 six-helix bundle. Unlike the HR2 binding site, which extends along a hydrophobic groove on the HR1 oligomer, the residues that are critical for HR1 oligomerization are concentrated in a 10-15-amino acid region. These results demonstrate that there are localizations of binding energy, or "hot spots", in the self-association of peptides derived from the HR1 region of gp41.

The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide, T20.

A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. One of them, T20 (aa 638-673), was found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (HR1) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from HR1, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action consistent with a previous fluorescence study. It was also found that peptides capable of solubilizing T20 dispersion have a high content of helix, as well as beta-strand, conformation in aqueous solution. Specificity of T20/HR1-derived peptide binding was ascertained by using a scrambled sequence of a T20-active peptide and a plateau in scattering reduction of T20 suspension with variation in the concentration of a T20-active HR1 peptide. Implications on the mechanism of T20 inhibition and the sequence of folding of the gp41 core structure are discussed.

Peptides derived from the heptad repeat region near the C-terminal of Sendai virus F protein bind the hemagglutinin-neuraminidase ectodomain

Previously, we showed that Sendai virus fusion protein (F) acts as an inhibitor of neuraminidase activity of hemagglutinin-neuraminidase (HN) protein. Here we report that synthetic peptides derived from the heptad repeat region proximal to the transmembrane domain (HR2) of Sendai virus F inhibit fusion and enhance the enzymatic activity of the HN. This occurs on the virus-bound HN and on its soluble globular head. The enhancing effect on virus-bound HN is reversible and depends on the presence of F. The data indicate that, by binding to the HN ectodomain, the HR2 peptides abolish the F inhibition of HN and disrupt the communication between the F and HN essential to promote virus–cell fusion.

Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex.

The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques. Two heptad-repeat (HR) regions within F(1) were shown to interact. Proteinase-K digestion experiments highlight the HR1 region (located proximal to the fusion peptide sequence) of the F(1) protein to which an HR2-derived (located proximal to the membrane-spanning domain) peptide binds, thus protecting both the protein and peptide from digestion. Solution-phase analysis of HR1-derived peptides shows that these peptides adopt helical secondary structure as measured by circular dichroism. Sedimentation equilibrium studies indicate that these HR1 peptides self-associate in a monomer/trimer equilibrium with an association constant of 5.2 x 10(8) M(-2). In contrast, HR2-derived peptides form random monomers in solution. CD analysis of mixtures containing peptides from the two regions demonstrate their propensity to interact and form a very stable (T(m) = 87 degrees C), helical (86% helicity) complex comprised of three HR1 and three HR2 members.

Interaction of peptides with sequences from the Newcastle disease virus fusion protein heptad repeat regions.

Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix.