Caracterización enzimática de la RNA helicasa CI del virus de la sharka (PPV) y su función en el ciclo de infección viral

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 31-03-2004Potyviruses are positive-strand RNA viruses that encode nine or more mature proteins fmm a polyprotein expression strategy. One unique feature of the potyvims infection is the accumulation of characteristic pinwheel-shaped cytoplasmic inclusion bodies. The plum pox potyvirus (PPV) protein CI is an RNA helicase which is involved in replication and cell to cell spread of the virus. However, relationships among the enzymatic activities, assembly in inclusion bodies, and CI functions in the viral cycle, are not well established. The CI protein contains seven conserved sequence motifs Srpical of RNA helicases of the superfamily SF2. There is great controversy about the function of the VI motif in the enzymatic activity of the protein. We have introduced several individual point mutations into the sequence that coda for the motif VI of the PPV CI protein and we have expressed these mutant proteins in Escherichia coli as maltose binding protein fusion products. Our results seem to indicate that this motif is implicated in the direct interaction with the NTP instead that with the nucleic acid interaction. Moreover we also show that the protein exists in solution as a group of different aggregated forms and that the oligomerization of the protein is required for an efficient RNA helicase activity. Inhibition of the CI wild type RNA helicase activity by negative mutants of the protein, it gives a further evidence about the importance of oligomerization in the helicase of the protein. The systemic infection of the plant by viruses generally requires the virus to be able to move from cell to cell through plasmodesmata and to long distances through the phloem. Normally, the cell-to-cell movement requires specialized proteins w), which facilitate intra- and inter-cellular transport of the virus or nucleoprotein complexes. Several types of MP have been characterized: (i) Some viruses, such as tobacco mosaic virus and red clover necrotic mosaic virus, encode single dedicated MPs that modify plasmodesmata and facilitate transport of themselves and nucleic acids through the modified channel; (ii) Several families of viruses contain a set of movement genes called the triple gene block, which encodes three proteins that are proposed to function co-ordinately to transport genomes through plasmodesmata; (iii) Finally, some other groups of viruses encode MPs that form plasmodesmal associated tubules through which virus or transport complexes travel. In contrast to most other viruses, the potyviruses do not encode a dedicated MP. Rather, potyvinis movement involves vira1 proteins that perform additional roles in the viral infection cycle. It is known that cellto- cell transport requires the assembly of competent capsid protein (CP), suggesting that intercellular transport involves Wion formation. At early times post-infection, the CI protein is associated with cone-shaped structures anchored to the cell wall or with plasma membrane in close proximity to plasmodesmata. In addition to the CI protein, these plasmodesmal-associated structures contain CP and viral RNA. So these observations led to propose that the CI protein could facilitate the cell-to-cell movement of the virus through direct interactions or modifications of plasmodesmata. Fially genetic analysis data demonstrated the involvement of the CI protein of TEV in cell-tocell movement. Regardless of wether or not the helicase activity of the CI protein is required or not for the cell-to-cell movement, in this study, we show strong evidence about an structural implication of the CI protein on the vira1 cell-to-cell movement