ABSTRACT
Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.
Subject(s)
Cysteine/metabolism , Glycoproteins/metabolism , Sindbis Virus/physiology , Viral Envelope Proteins/metabolism , Virus Assembly , Amino Acid Sequence , Amino Acid Substitution/genetics , Cysteine/genetics , Glycoproteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Alignment , Sindbis Virus/genetics , Viral Envelope Proteins/geneticsABSTRACT
Anacardic acid (2-hydroxy-6-alkylbenzoic acid) produced and secreted from glandular trichomes of zonal geranium (Pelargonium x hortorum; Geraniaceae family) provides resistance to small pests (aphids and spider mites). To assess the potential bioactivity of anacardic acid against larger insect pests and to determine if an alternate mode of application (ingestion rather than the topical application) could impart resistance to pests, the effects of anacardic acid consumption on the development of Colorado potato beetle larvae were tested. Analysis of dose-response curves indicated a significant effect on weight gain and mortality. Assessment of food preference (treated versus untreated) indicated larvae avoid food containing anacardic acid and have a lower feeding rate on food containing anacardic acid. On the basis of these results, it is suggested that anacardic acid, applied as a chemical spray or through bioengineering production in crop plants, may provide a new tool in the arsenal to minimize damage to plants caused by pests.
