Structural and Mechanistic Evidence for Calcium Interacting Sites in the HIV Transmembrane Protein gp41 Involved in Membrane Fusion.

The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR-NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.

PTX80, a novel compound targeting the autophagy receptor p62/SQSTM1 for treatment of cancer.

Cancer cells are dependent on protein quality-control mechanisms, including protein chaperones, the ubiquitin-proteasome system, and autophagy. The p62 receptor is a classical, ubiquitously expressed receptor, involved in many signal transduction pathways. Upregulation and/or reduced degradation of p62 have been implicated in tumor formation and resistance to therapy. PTX80 is a first-in-class novel inhibitor of protein degradation, developed by Pi Therapeutics for the treatment of cancer. PTX80 binds to p62, inducing a decrease in soluble p62 and formation of insoluble p62 aggregates, and failure of polyubiquitinated proteins to colocalize with p62. PTX80 induces proteotoxic stress and activation of unfolded protein response, which, in turn, leads to apoptosis. Targeting p62, which is a major protein degradation hub, may serve as a novel and beneficial strategy for the treatment of cancer.

Switching Bond: Generation of New Antimicrobial Peptides via the Incorporation of an Intramolecular Isopeptide Bond.

Antimicrobial peptides (AMPs), which can be modified to kill a broad spectrum of microoganisms or a specific microorganism, are considered as promising alternatives to combat the rapidly widespread, resistant bacterial infections. However, there are still several obstacles to overcome. These include toxicity, stability, and the ability to interfere with the immune response and bacterial resistance. To overcome these challenges, we herein replaced the regular peptide bonds with isopeptide bonds to produce new AMPs based on the well-known synthetic peptides Amp1L and MSI-78 (pexiganan). Two new peptides Amp1EP and MSIEP were generated while retaining properties such as size, sequence, charge, and molecular weight. These new peptides have reduced toxicity toward murine macrophage (RAW 264.7) cells, human monocytic (THP-1) cells, and human red blood cells (hRBCs) and enhanced the stability toward proteolytic degradation. Importantly, the new peptides do not repress the pro-inflammatory cytokine and hence should not modulate the immune response. Structurally, the new peptides, Amp1EP and MSIEP, have a structure of random coils in contrast to the helical structures of the parental peptides as revealed by circular dichroism (CD) analysis. Their mode of action, assessed by flow cytometry, includes permeabilization of the bacterial membrane. Overall, we present here a new approach to modulate AMPs to develop antimicrobial peptides for future therapeutic purposes.

The HIV gp41 Fusion Protein Inhibits T-Cell Activation through the Lentiviral Lytic Peptide 2 Motif.

The human immunodeficiency virus enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41-derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat, and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion, and cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT-based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism that can take place post-membrane fusion.

The HTLV-1 gp21 fusion peptide inhibits antigen specific T-cell activation in-vitro and in mice.

The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait.

Mapping out the intricate relationship of the HIV envelope protein and the membrane environment.

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

The Transmembrane Domain of HIV-1 gp41 Inhibits T-Cell Activation by Targeting Multiple T-Cell Receptor Complex Components through Its GxxxG Motif.

To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg(696)) residue and a GxxxG motif, making them ideal candidates for mediators of TMD-TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg(696) does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41's TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.

The HIV gp41 pocket binding domain enables C-terminal heptad repeat transition from mediating membrane fusion to immune modulation.

For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.