Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.flu vaccine ͉ glycan binding ͉ glycosylation T he highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1) viruses have caused global outbreaks and raised a great concern that further changes in the viruses may occur to bring about a deadly pandemic (1, 2). Important contributions to our understanding of influenza infections have come from the studies on hemagglutinin (HA), a viral coat glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract, allowing the virus to enter the cell (3-6). To cross the species barrier and infect the human population, avian HA must change its receptorbinding preference from a terminally sialylated glycan that contains ␣2,3 (avian)-linked to ␣2,6 (human)-linked sialic acid motifs (7), and this switch could occur through only two mutations, as in the 1918 pandemic (8). Understanding the factors that affect influenza binding to glycan receptors is thus critical for developing methods to control any future crossover influenza strains that have pandemic potential.HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region (3). Both regions carry N-linked oligosaccharides (9), which affect the functional properties of HA (10, 11). Among different subtypes of influenza A viruses, there is extensive variation in the glycosylation sites of the head region, whereas the stem oligosaccharides are more conserved and required for fusion activity (11). Glycans near antigenic peptide epitopes interfere with antibody recognition (12), and glycans near the proteolytic ...

Apicomplexa exhibit a unique form of substrate-dependent gliding motility central for host cell invasion and parasite dissemination. Gliding is powered by rearward translocation of apically secreted transmembrane adhesins via their interaction with the parasite actomyosin system. We report a conserved armadillo and pleckstrin homology (PH) domain-containing protein, termed glideosome-associated connector (GAC), that mediates apicomplexan gliding motility, invasion, and egress by connecting the micronemal adhesins with the actomyosin system. TgGAC binds to and stabilizes filamentous actin and specifically associates with the transmembrane adhesin TgMIC2. GAC localizes to the apical pole in invasive stages of Toxoplasma gondii and Plasmodium berghei, and apical positioning of TgGAC depends on an apical lysine methyltransferase, TgAKMT. GAC PH domain also binds to phosphatidic acid, a lipid mediator associated with microneme exocytosis. Collectively, these findings indicate a central role for GAC in spatially and temporally coordinating gliding motility and invasion.

e Dengue virus (DENV) causes dengue fever, a major health concern worldwide. We identified an amphipathic helix (AH) in the N-terminal region of the viral nonstructural protein 4A (NS4A). Disruption of its amphipathic nature using mutagenesis reduced homo-oligomerization and abolished viral replication. These data emphasize the significance of NS4A in the life cycle of the dengue virus and demarcate it as a target for the design of novel antiviral therapy. Dengue virus (DENV) infection is a growing public health threat, with more than one-third of the world population at risk (1). DENV is a positive single-strand RNA virus. Its genome is translated into a single polyprotein, which is cleaved to produce structural (components of the mature virus) and nonstructural (NS) proteins. In addition, the NS proteins generate the viral replication complexes (RC) (2). DENV replicates its RNA genome in association with modified intracellular membranes; the details of the assembly of these complexes are incompletely understood.NS4A, a transmembrane endoplasmic reticulum (ER) resident protein, is thought to induce the host membrane modifications that harbor the viral RC (3). A similar function for NS4A was reported in other flaviviruses (4, 5). To further understand the role of NS4A, we analyzed its cytosolic N-terminal region (amino acids 1 to 48) using sequence alignment of the four DENV serotypes. Within this sequence, amino acids that differed in their identity maintained their biochemical properties, suggesting the presence of a conserved structural motif with a potential functional significance (Fig. 1A). Secondary structure algorithms (6) indicated that this segment is predicted to fold into an ␣-helix (Fig. 1B). Helical wheel projections of amino acids 3 to 20 indicated a conserved polar-nonpolar asymmetry indicative of an amphipathic helix (AH) (Fig. 1C). To experimentally examine the conformation of the NS4A N terminus, a recombinant peptide comprising amino acids 1 to 48 was prepared. Codon-optimized DENV2 NS4A 1-48 was cloned into pGEV2 (7) with an N-terminal fusion to the immunoglobulin binding domain of streptococcal protein G (GB1). A tobacco etch virus (TEV) protease cleavage site (ENLYFQ) was introduced into the beginning of the NS4A coding sequence. Due to difficulties in separating NS4A from GB1 after TEV protease cleavage, an N-terminal glutathione S-transferase (GST) affinity tag was added to GB1-TEV-NS4A(1-48) by cloning it into pGEX4T-2. Protein expression and purification of the GST-GB1-NS4A(1-48) fusion were performed as described previously (8), except that an on-column cleavage was performed by adding TEV protease. The flowthrough was concentrated and subjected to size exclusion chromatography (HiLoad 16/60 Superdex 75) yielding pure NS4A(1-48). The resulting peptide was characterized using far-UV circular dichroism (CD) (Fig. 1D). In aqueous buffer, NS4A(1-48) showed limited solubility and a CD spectrum typical of a random coil conformation with an ␣-helix content below 10%. Addition of various memb...

Dengue virus (DENV) infection is a growing public health threat with more than one-third of the world's population at risk. Non-structural protein 4A (NS4A), one of the least characterized viral proteins, is a highly hydrophobic transmembrane protein thought to induce the membrane alterations that harbor the viral replication complex. The NS4A N-terminal (amino acids 1-48), has been proposed to contain an amphipathic α-helix (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization. Nuclear magnetic resonance (NMR) spectroscopy was used to characterize the N-terminal cytoplasmic region (amino acids 1-48) of both wild type and mutant NS4A in the presence of SDS micelles. Binding of the two N-terminal NS4A peptides to liposomes was studied as a function of membrane curvature and lipid composition. The NS4A N-terminal was found to contain two AHs separated by a non-helical linker. The above mentioned mutations did not significantly affect the helical secondary structure of this domain. However, they reduced the affinity of the N-terminal NS4A domain for lipid membranes. Binding of wild type NS4A(1-48) to liposomes is highly dependent on membrane curvature.