No abstract

The membrane destabilizing and fusogenic properties of the synthetic peptide , corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

Wheat blast disease, caused by Magnaporthe oryzae (anamorph Pyricularia oryzae), produces severe damage to wheat production in South America. It was observed that many resistant cultivars contain the 2NS/2AS translocation from Triticum ventricosum. In this study, we evaluate the presence of the 2NS/2AS translocation in 57 advanced breeding lines and one variety ‘Caninde 1’ from Paraguayan wheat germplasm, using VENTRIUP‐LN2 primers. The germplasm ‘Caninde 1 and 22’ of the breeding lines, found positive for the presence of 2NS/2AS translocation, were inoculated with a single aggressive Magnaporthe pathotype P14‐039, to assess their response to wheat blast infection under controlled conditions. Based on the disease infection score, ten of the breeding lines, ‘Caninde 1’ and ‘Milan’ (positive control), were classified as resistant. Three of the remaining breeding lines were classified as moderately resistant, five as moderately susceptible and other four as susceptible. Our results show that the expression of 2NS/2AS‐based blast resistance is more dependent on genetic background of the inserted germplasm than previously envisioned.

VP3(110-121), a peptide from the exposed VP3 capsid protein of hepatitis A virus, has been shown earlier to bind at pH 7.4 to bilayered vesicles containing the major phospholipids found in the membrane of hepatocytes and to cause aggregation and fusion of these vesicles by subsequent acidification. To deepen the understanding of the interaction of VP3(110-121) with the lipid membrane, in this paper we report a detailed study of the miscibility properties and behavior of mixed monomolecular films of these major lipid components at the air/water interface. The miscibility of these components in mixed monolayers was determined with the Langmuir film technique and the excess free energy of mixing (∆GM ex ) and interaction energies were calculated. Results show that all the components are miscible in all ratios and indicate the presence of interactions of low energy between them at certain mole fractions. Next, we have measured the insertion of VP3(110-121) into spread monolayers of these components at mole ratios that closely resemble the hepatocyte membrane. Insertion was measured as a function of the lateral packing of the monolayer. Three compositions with different net charge were selected: 1-palmitoyl-2-oleoylphosphatidylcholine:cholesterol:sphingomyelin:1,2-dipalmitoylphosphatidylethanolamine (POPC/CH/SM/DPPE) (35: 24:31:10), a mole ratio similar to the hepatocyte membrane, and its mixtures with a cationic lipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) POPC/CH/SM/DPPE/DOTAP (32:21:28:9:10) and with an anionic lipid (L-R-phosphatidyl-L-serine from bovine brain, PS) POPC/CH/SM/DPPE/PS (32:21:28:9: 10). Results indicate that the interaction of VP3(110-121) has both hydrophobic and electrostatic components, with insertion in all three monolayers but with a stronger interaction with the cationic interface, with higher increases in surface pressure and faster insertion rates. The lowest affinity of binding corresponds to anionic PS-containing films, whereas interaction with zwitterionic POPC/CH/ SM/DPPE films is intermediate. This is expected due to the anionic net charge of this amphiphilic peptide at the pH of the experiments.