RESUMO
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. Synthetic peptides encompassing the VPg88-120 central region were found to tightly bind to eIF4E. Fluorescence energy transfer experiments show that, upon binding to eIF4E, the N and C termini of the VPg88-111 fragment move closer to one another, at a distance compatible with a α-helix folding. When the VPg112-120 region, which contains amino acids associated with resistance breakdown, is appended to VPg88-111, the complex formation with eIF4E switches from a single-step to a two-step kinetic model. This study revisits a recent investigation of the VPg-eIF4E complex by specifying the contribution of the VPg central helix and its appended disordered region to VPg association with eIF4E.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Doenças das Plantas/genética , Proteínas de Plantas/química , Potyvirus/genética , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Fator de Iniciação 4E em Eucariotos/genética , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , Cinética , Defesa das Plantas contra Herbivoria/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Potyvirus/química , Potyvirus/patogenicidade , Ligação Proteica/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The viral protein genome-linked (VPg) of potyviruses is a protein covalently linked to the 5' end of viral RNA. It interacts with eIF4E, a component of the cellular translation initiation complex. It has been suggested that the 5' RNA-linked VPg could mimic the cellular mRNA cap, promoting synthesis of viral proteins. Here, we report evidence for recruitment of the plant eIF4E by Lettuce mosaic virus (LMV, potyvirus) particles via the 5' RNA-linked VPg. Analysis of the viral population was performed by enzyme-linked immunosorbent assay-based tests, either with crude extracts of LMV-infected tissues or purified viral particles. In both cases, LMV-VPg and LMV-eIF4E subpopulations could be detected. After reaching a maximum within the first 2 weeks postinoculation, these populations decreased and very few labeled particles were found later than 3 weeks postinoculation. The central domain of VPg (CD-VPg) was found to be exposed at the surface of the particles. Using a purified recombinant lettuce eIF4E and CD-VPg-specific antibodies, we demonstrate that the plant factor binds to the VPg via its central domain. Moreover, the plant eIF4E factor could be imaged at one end of the particles purified from LMV plant extracts, by immunoredox atomic force microscopy coupled to scanning electrochemical microscopy. We discuss the biological significance of these results.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Genoma Viral , Lactuca/virologia , Potyvirus/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Anticorpos , Proteínas do Capsídeo/metabolismo , Microscopia de Força Atômica , Oxirredução , Doenças das Plantas/virologia , Ligação Proteica , Recombinação Genética/genéticaRESUMO
Lettuce mosaic virus (LMV) isolates LMV-E and LMV-0 differ in their virulence on lettuce varieties carrying the mo1(2) resistance gene, which reduces viral accumulation and blocks the expression of symptoms after infection with avirulent isolates such as LMV-0. Previous work had indicated that reporter genes such as GUS or GFP affect the biological properties of recombinant LMV isolates in both susceptible and resistant lettuce varieties when fused to the N-terminus of the viral protein HC-Pro. The impact of the addition of a cleavage site for the NIa proteinase between the reporter gene and HC-Pro was evaluated, in an effort to recover the full spectrum of the biological properties of parental isolates. Symptoms, accumulation, cell-to-cell and long distance movement of the recombinant viruses containing the NIa cleavage site were studied in susceptible and mo1(2) lettuce varieties. Both LMV-0 and LMV-E recombinant viruses recovered the behaviour of their wild-type parent in susceptible plants upon addition of the NIa cleavage site. While the recombinant LMV-E modified in this way recovered the breaking properties of its wild-type counterpart in mo1(2) plants, similar modification of the LMV-0 derived recombinants failed to rescue a severe inhibition in systemic accumulation in mo1(2) plants, despite the fact that neither cell-to-cell movement nor phloem loading or unloading seemed to be severely affected.
