Structure and Immunogenicity of a Peptide Vaccine,Including the Complete HIV-1 gp41 2F5 Epitope: IMPLICATIONS FOR ANTIBODY RECOGNITION MECHANISM AND IMMUNOGEN DESIGN*

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund''s adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.     

Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity

Cellulose biosynthesis is a vital but yet poorly understood biochemical process in Oomycetes. Here, we report the identification and characterization of the cellulose synthase genes (CesA) from Saprolegnia monoica. Southern blot experiments revealed the occurrence of three CesA homologues in this species and phylogenetic analyses confirmed that Oomycete CesAs form a clade of their own. All gene products contained the D,D,D,QXXRW signature of most processive glycosyltransferases, including cellulose synthases. However, their N-terminal ends exhibited Oomycete-specific domains, i.e. Pleckstrin Homology domains, or conserved domains of an unknown function together with additional putative transmembrane domains. Mycelial growth was inhibited in the presence of the cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile or Congo Red. This inhibition was accompanied by a higher expression of all CesA genes in the mycelium and increased in vitro glucan synthase activities. Altogether, our data strongly suggest a direct involvement of the identified CesA genes in cellulose biosynthesis.     

Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5

The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization.     

Phagocytosis of apoptotic and necrotic thymocytes is inhibited by PAF-receptor antagonists and affects LPS-induced COX-2 expression in murine macrophages

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.     

Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism

Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.Respiratory virus infections cause considerable morbidity and mortality worldwide; it was recently reported that hospitalizations due to respiratory syncytial virus (RSV) exceed 2 million per year in the Unites States alone (16). An H1N1 swine influenza pandemic took place during the 2009-2010 winter season (14), and there is the lingering threat of an H5N1 avian influenza pandemic, with mortality due to direct bird-to-human H5N1 infection in hospitalized patients between 30 and 100% (3). The severe acute respiratory syndrome (SARS)-associated coronavirus, isolated in 2003, resulted in devastating respiratory tract infections with few treatment options (40). For most common respiratory viruses, treatment is symptomatic, and for pathogens such as influenza viruses for which specific treatments are available, oseltamivir (Tamiflu)- and amantidine-resistant strains are emerging and being transmitted globally (33).All functions within a cell are triggered and regulated by cell signaling cues. Since viruses are obligate intracellular parasites, they rely upon cell signaling to regulate all processes within the cell that drive virus replication. In this study we investigated the effects of kinase inhibitors as a therapeutic strategy and to investigate the roles played by some kinases during virus replication. The extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs) have been shown by us and others to play important roles during virus replication in vitro (19, 20, 26, 30, 42), and we have recently reported that inhibition of p38 MAPK activation is an effective and novel antiviral strategy in vivo (29). The significance of p38 MAPK activity in vivo is such that inadvertent and coincident activation of this kinase by some pharmaceutical agents enhances virus replication (29). Antiviral strategies may exist whereby inhibition of host cell kinases may stem the spread and replication of numerous different viral species. Such broad antiviral strategies would permit administration of kinase inhibitors to patients suspected of having respiratory viral infection, and to health care workers or inhabitants within the locale of a viral outbreak, prior to the availability of results from laboratory diagnostic testing.The activation of p38 MAPK by pattern recognition receptors (PRRs) has been studied in the context of the antiviral immune response (reviewed in reference 22). We report here that viruses usurp these responses for the benefit of virus replication through activation of p38 MAPK, mediated by a PRR (Toll-like receptor 4 [TLR4]) and MyD88, providing the basis for a broad-spectrum antiviral.     

1H, 13C and 15N resonance assignments of the Onconase FL-G zymogen

Onconase^® FL-G zymogen is a 120 residue protein produced by circular permutation of the native Onconase^® sequence. In this construction, the wild type N- and C-termini are linked by a 16 residue segment and new N- and C-termini are generated at wild type positions R73 and S72. This novel segment linking the native N- and C-termini is designed to obstruct Onconase’s^® active site and encloses a cleavage site for the HIV-1 protease. As a first step towards the resolution of its 3D structure and the study of its structure–function relationships, we report here the nearly complete NMR ¹H, ¹³C and ¹⁵N resonance chemical shift assignments at pH 5.2 and 35°C (BMRB deposit no 17973). The results presented here clearly show that the structure of the wild type Onconase^® is conserved in the FL-G zymogen.