Point mutational examination of eukaryotic initiation factor 4E (eIF4E), inclusion bodies analysis, and Barley Yellow Dwarf Virus (BYDV) exonuclease resistant RNA structure mapping and prediction
Eukaryotic initiation factor 4E (eIF4E) binds the 5’m7GTP cap structure of mRNA in order to facilitate effective translation. Recessive eIF4E alleles harboring naturally occurring point mutations have been associated with resistance to viral infection, particularly in members of the genus Potyvirus. Resistance is thought to be conferred by disrupting the binding of the 5’ viral-encoded protein (VPg), covalently attached to the 5’ end of the Potyvirus genomic RNA, to eIF4E. RNAs of members of the genus Panicovirus, some Carmoviruses and one Umbravirus however, bind eIF4E through a 3’ cap independent translation element (3’CITE) termed the PTE (Panicum mosaic virus-like translation enhancer). The PTE consists of a T shaped secondary structure with a C-rich region at the branch point between stem loops 1 and 2 and G-rich region in a bulge in the basal stem loop. The C-rich and G-rich regions form a pseudoknot where the G-rich region contains a flexible G that is thought to flip outward and potentially act as a 5’ m7GTP cap analog to bind eIF4E. Point mutations were introduced into wheat eIF4E in order to identify amino acids needed for PTE binding and were designed based on previous studies of recessive resistance-conferring eIF4E alleles, along with the crystal structure of wheat eIF4E bound to m7GDP. However, the chosen method of eIF4E purification involving a glutathione S-transferase (GST) tag repeatedly resulted GST-eIF4E inclusion bodies, which limited the ability to purify mutant eIF4E for binding studies. Therefore a large portion of this work is concerned with attempts to solubilize the GST-eIF4E fusion protein.
Additionally, the second portion of this work involves mapping and predicting the structure in Barley yellow dwarf virus RNA (BYDV) that blocks exonuclease Xrn1 to generate BYDV subgenomic RNA3. Deletion analysis places the Xrn1 resistant (xrRNA) structure within the first 5’ 67 nucleotides of BYDV sgRNA3. Dianthoviruses, related to luteoviruses, have recently been shown to contain an xrRNA structure of which a crystal structure has been obtained. Bioinformatics analysis using the programs INFERNAL and Dynalign suggest the BYDV sgRNA3 xrRNA structure may be different than that of dianthoviruses. A proposed secondary structure of BYDV sgRNA3 xrRNA was obtained by analysis with the programs DotKnot and RNAalifold.