Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.

[Synthesis of lipid A analogs. Obtaining conjugates of 6-phosphate 2-desoxy-2-(3-hydroxymyristoyl)amino-D-glucose with polymer carriers and their immunological properties]. / Sintez analogov lipida A. Poluchenie kon'iugatov 6-fosfata 2-dezoksi-2-(3-oksimiristoil) amino-D-gliukozy s polimernymi nositeliami i ikh immunologicheskie svoistva.

The derivatives of 2-acylamino-6-O-(2-aminoethyl) phosphono-3-deoxy-D-glucose acylated with acetic or D,L-3-hydroxytetradecanoic acid were obtained, and their 31P-and 13C-NMR spectra investigated. These haptens were bound with a polysaccharide (Ficoll) or proteins (albumins, bovine gamma-globulin). The protein conjugates were immunogenic in rabbits, specific antibodies against the hapten being revealed by two immunochemical methods. As shown by the enzyme-linked immunoadsorbent assay, the specific rabbit antiserum reacted with lipid A from Yersinia pseudotuberculosis.

Studies on glycoproteins in the human malaria parasite Plasmodium falciparum. Identification of a myristilated 45kDa merozoite membrane glycoprotein.

A murine monoclonal antibody has been used to characterise a 45,000 Da antigen that is associated with the surface membrane of merozoites of the human malaria parasite Plasmodium falciparum. The antigen is a glycoprotein and incorporates myristic acid.

A novel monoclonal antibody to N-myristoyl glycine moiety found a new N-myristoylated HIV-1 p28gag protein in HIV-1-infected cells.

A novel monoclonal antibody was raised against a synthetic N-myristoyl glycine that is characteristic of all N-myristoylated proteins. The immunoreaction suppressed in the presence of hemocyanin as well as albumin conjugated with N-myristoyl glycine and other N-myristoyl glycyl peptides, while underivatized and myristoyl amino acid proteins or various fatty acids other myristic acid exerted no effect. The antibody specifically reacted with N-myristoylated pp60c-src in human colon adenocarcinoma cells, N-myristoylated pp60v-src in Rous sarcoma virus-infected cells, and N-myristoylated Gag precursor protein Pr55gag in HIV-1 producing cells. Furthermore, the antibody immunoreacted with a new N-myristoylated p28gag derived from HIV-1 gag protein. The antibody is shown to be a very useful tool for identification of N-myristoylated proteins.

Antibodies to an NH2-terminal myristoyl glycine moiety can detect NH2-terminal myristoylated proteins in the retrovirus-infected cells.

Novel antibodies were raised against a synthetic NH2-terminal myristoyl glycine moiety which is characteristic of N-myristoyl-proteins. Antisera raised against N-myristoyl-Gly-hemocyanin reacted with N-myristoyl-Gly-[125I]albumin. The immunoreaction was competed for by albumin conjugated with N-myristoyl-glycine, while underivatized albumin had no effect. Of the [3H]myristate-labeled proteins detected, pp60v-src, which is a transforming protein of Rous sarcoma virus, and p19gag and p17gag, which are core proteins in the human T-cell leukemia virus and the human immunodeficiency virus, were identified as N-myristoylated proteins by the radioimmunoprecipitation analyses with the antibody.