Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection

Abstract

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1 1 and mo1 2 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1 1 or mo1 2 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

“…These changes appear to have altered the ability of eIF4E1b/eIF4E1c to interact with eIF4G compared with eIF4E (see below). While mutations in eIF4E that confer viral resistance are naturally occurring (Robaglia and Caranta, 2006) and directed mutagenesis has further identified residues conferring virus resistance (German-Retana et al, 2008;Ashby et al, 2011), the 15 conserved flowering plant eIF4E residues differing in eIF4E1b-type proteins do not overlap with these residues, with the exception of a K78 mutation, which confers virus resistance in pea (Ashby et al, 2011). Interestingly, transfer DNA (T-DNA) insertion mutants for EIF4E1B or EIF4E1C do not have any effect on turnip mosaic virus infection in Arabidopsis (Gallois et al, 2010).…”

“…Residues marked # are conserved residues involved in interaction with eIF4G identified in yeast (Gross et al, 2003). Residues marked + are experimentally determined to be essential for cap binding (Yeam et al, 2007;German-Retana et al, 2008). Residues marked * are predicted to be involved in cap binding in plant eIF4E crystal structures (Monzingo et al, 2007;Ashby et al, 2011), and the residue marked^is predicted from the wheat eIF4E structure to form a salt bridge with the negatively charged phosphate backbone of the cap.…”

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

“…This Saccharomyces cerevisiae strain lacks its endogenous eIF4E gene and requires complementation with an external eIF4E to survive, for example the human eIF4E expressed from the pGAL-eIF4E-URA3 plasmid in a Gal-dependent manner (Altmann et al, 1989). This assay is a good indicator of all the eIF4E functions that affect yeast growth, including mRNA translation (Altmann et al, 1989;Charron et al, 2008;German-Retana et al, 2008;Ashby et al, 2011), and we have used it previously to test the effect of single Cm eIF4E mutations (Miras et al, 2017b). Almost all Cm Figure 6.…”

“…Plant virus resistance mutations modeled on the melon eIF4E-eIF4G 1003-1092 complex. A, The residues implicated in plant potyvirus and bymovirus resistance are shaded in green and magenta, respectively (Gao et al, 2004;Kang et al, 2005;Kanyuka et al, 2005;Ruffel et al, 2005;Stein et al, 2005;Nicaise et al, 2007;German-Retana et al, 2008;Ashby et al, 2011). The Cm eIF4E molecule is shown as a surface rendering and Cm eIF4G 1003-1092 is shown as a cartoon representation, both colored in gray and orange, respectively.…”

“…At least one amino acid substitution in the lateral surface of tomato eIF4E, G107R, disrupted in planta its interaction with VPg from Tobacco etch virus (Yeam et al, 2007). Moreover, analysis of mutations in the lateral surface of pea (Pisum sativum) and lettuce (Lactuca sativa) eIF4Es revealed that the mutant proteins could not complement viral infection of two potyviruses, Pea seed-borne mosaic virus and Lettuce mosaic virus (German-Retana et al, 2008;Ashby et al, 2011). Interestingly, several studies have suggested that potyviral VPg is able to inhibit cap-dependent translation (Khan et al, 2008;Miyoshi et al, 2008;Eskelin et al, 2011).…”