Spectroscopic Investigation of the Kinetic Mechanism Involved in the Association of Potyviral VPg with the Host Plant Translation Initiation Factor eIF4E.

The infectious cycle of potyviruses requires the formation of a complex between the viral genome-linked protein VPg and the host eukaryotic translation initiation factor 4E, eIF4E. Mutations associated with plant resistance to potyviruses were previously mapped at the eIF4E surface, while on the virus side, mutations leading to plant resistance breaking were identified within the VPg. In the present study, fluorescence spectroscopy was used to probe the contribution of the VPg intrinsically disordered region bearing amino acids determinant of the resistance breaking, to the VPg-eIF4E binding mechanism.

Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex.

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors.

Comparative analysis of mutational robustness of the intrinsically disordered viral protein VPg and of its interactor eIF4E.

Conformational intrinsic disorder is a feature present in many virus proteins. Intrinsically disordered regions (IDRs) have weaker structural requirement than ordered regions and mutations in IDRs could have a lower impact on the virus fitness. This could favor its exploration of adaptive solutions.

First Experimental Assessment of Protein Intrinsic Disorder Involvement in an RNA Virus Natural Adaptive Process.

Intrinsic disorder (ID) in proteins is defined as a lack of stable structure in physiological conditions. Intrinsically disordered regions (IDRs) are highly abundant in some RNA virus proteomes. Low topological constraints exerted on IDRs are expected to buffer the effect of numerous deleterious mutations and could be related to the remarkable adaptive potential of RNA viruses to overcome resistance of their host.

Toward the Reconstitution of a Two-Enzyme Cascade for Resveratrol Synthesis on Potyvirus Particles.

The highly ordered protein backbone of virus particles makes them attractive candidates for use as enzyme nano-carriers (ENCs). We have previously developed a non-covalent and versatile approach for adhesion of enzymes to virus particles. This approach makes use of z33, a peptide derived from the B-domain of Staphylococcus aureus protein A, which binds to the Fc domain of many immunoglobulins.

Electrochemical atomic force microscopy imaging of redox-immunomarked proteins on native potyviruses: from subparticle to single-protein resolution.

We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles.

General strategy for ordered noncovalent protein assembly on well-defined nanoscaffolds.

Here we develop a novel approach allowing the noncovalent assembly of proteins on well-defined nanoscaffolds such as virus particles. The antibody-binding peptide Z33 was genetically fused to the monomeric yellow fluorescent protein and 4-coumarate:CoA-ligase 2. This Z33 "tag" allowed their patterning on the surface of zucchini yellow mosaic virus by means of specific antibodies directed against the coat protein of the virus.

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein.

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro.

Intrinsic disorder in Viral Proteins Genome-Linked: experimental and predictive analyses.

Background: VPgs are viral proteins linked to the 5' end of some viral genomes. Interactions between several VPgs and eukaryotic translation initiation factors eIF4Es are critical for plant infection. However, VPgs are not restricted to phytoviruses, being also involved in genome replication and protein translation of several animal viruses.

Involvement of the cylindrical inclusion (CI) protein in the overcoming of an eIF4E-mediated resistance against Lettuce mosaic potyvirus.

The capacity of Lettuce mosaic virus to overcome the lettuce resistance conferred by the mo1(1) and mo1(2) alleles of the gene for eukaryotic translation initiation factor 4E (eIF4E) was analysed using reverse genetics. Mutations in the virus genome-linked protein (VPg) allowed mo1(1) only to be overcome, but mutations in the C-terminal portion of the cylindrical inclusion (CI) protein allowed both alleles to be overcome. Site-directed mutagenesis pinpointed a key role of the amino acid at position 621 in the virulence.

Lettuce mosaic virus: from pathogen diversity to host interactors.

Taxonomy: Lettuce mosaic virus (LMV) belongs to the genus Potyvirus (type species Potato virus Y) in the family Potyviridae.

Physical Properties: The virion is filamentous, flexuous with a length of 750 nm and a width of 15 nm. The particles are made of a genomic RNA of 10 080 nucleotides, covalently linked to a viral-encoded protein (the VPg) at the 5' end and with a 3' poly A tail, and encapsidated in a single type of capsid protein.

Mutational analysis of plant cap-binding protein eIF4E reveals key amino acids involved in biochemical functions and potyvirus infection.

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.

The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue.

The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV).