ABSTRACT
Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.
Subject(s)
Antiviral Agents/pharmacology , Interferon Type I/pharmacology , Viruses/drug effects , Amino Acid Sequence , Animals , Chlorocebus aethiops , Humans , Interferon Type I/chemistry , Interferon Type I/genetics , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Sequence Alignment , Nicotiana/genetics , Vero CellsABSTRACT
The Red clover necrotic mosaic virus movement protein (MP) is essential for cell-to-cell movement. Eight previously characterized alanine-scanning mutants of the MP were fused to the green fluorescent protein (GFP) and expressed from viral infectious transcripts. Inoculated plants were assayed for movement and intracellular accumulation of MP by confocal laser-scanning microscopy. A strict correlation was observed between the targeting to the cell wall (presumably the plasmodesmata) and cell-to-cell movement. Complementation of dysfunctional MP mutants with either wild-type MP or other null mutants in some cases rescued intracellular targeting and movement. The data suggest the presence of distinct domains in the MP for virus movement (near residues 27-31), complementarity (near residues 122 and 128), and intracellular localization (near residue 161). These data support a model of MP interacting cooperatively with itself to bind viral RNA, localize to and modify plasmodesmata and effect virus movement.
