“Functional analysis and subcellular localization of the proteins involve in the intra and intercellular movement of the Melon necrotic spot virus (MNSV)”

   Plant viruses constitute a serious threat for numerous crops. In spite of the numerous researches carried out, it exist still a great ignorance of the routes and the mechanisms that operate in the viral invasion of the corresponding hosts. It is important to emphasize that the blockade in the viral movement constitutes one of the more frequent mechanisms of natural resistance to virus. So, the progress in the knowledge of these stages of the viral cycle can lead to antiviral strategies. In the present work, it have been characterized the structure and the function of the 5 proteins codified by the MNSV genome, doing special support in characterizing the factors, both viral and cellular, that intervene in the transport intra and intercellular of the virus.
   As previous and necessary step, in the Chapter 1 of the present Thesis, an infectious clone of the MNSV has been obtained, pMNSV, from which can be generated in vitro transcripts capable of reproducing the same symptomatology that the virus after its mechanical inoculation on the natural host (melon). This infectious clone constitutes an indispensable molecular tool that has allowed to carrying a functional analysis of the viral genes. This way, by pMNSV clone oligonucleotide-directed mutagenesis, it has been obtained mutants that prevent the expression of each one of the virus genome ORFs. In addition, the same modifications have been effected on the recombinant clone, pMNSV-?cp-GFP, where the p42 ORF from the full-length clone has been replaced by the green fluorescence protein (GFP) reporter gene. The results revealed that the proteins 29 and 89 would be involved in the genome replication. On the other hand, the p7A and the p7B would take part in the virus cell to cell movement. Finally, the p42 ó CP, besides its structural role, is necessary for the virus systemic invasion, being capable of accentuating the symptomatology. Likewise, the proteins 7B and CP, among all the MNSV encoded proteins, were able to delay the RNA silencing in transient expression assays. Finally, the p42 favoured the virus local movement. 
   Once determined the functions of each one of the proteins codified by the MNSV, this work was focus on elucidate the movement strategy followed by the virus. In the Chapter 2, it was carried out a biochemical and structural study of the MNSV movement proteins. Carmovirus movement proteins present common characteristics in their secondary structure that might be the reason of its functional similarity. The 7A is hydrophilic protein that contains numerous basic residues and that binds preferably simple chain RNA in a cooperative form and without apparent sequence specificity. Nevertheless, unlike the p7 of the Carnation mottle virus that has the functional motif constricted in the central??-helix, there has been demonstrated that the whole sequence of p7A is involved in the RNA binding, with a major participation of the central region in the process. In this way, a synthetic peptide from the p7A ?-helix central region bound RNA with a dissociation constant (Kd) significantly higher than the entire protein (9mM vs 25,7 ?M). On the other hand, in vitro the p7B behaved as an integral membrane protein when it was expressed in transcription/translation experiments in presence of ER-derived microsomal membranes. Later, it was assayed a collection of p7A and p7B mutants concerning different structural properties of this MNSV movement proteins. In case of the p7A, this study revealed that the basic residues, specially those placed in the central region of the protein, are directly involved in the local movement of the virus, existing a direct relationship between the presence or not of the above mentioned residues and the RNA binding ability. On the other hand, the decrease in the p7B hydrophobic profile, the unfolding of its transmembrane ?-helix, as well as changes on both the aromatic amino acids and the charge balance on both sides of its hydrophobic domain affect negatively in the MNSV cell to cell movement.
   Finally, with the purpose of obtaining information about the intracelular route followed by the pathogen, there has been studied the subcellular location of the MNSV MPs. To approach these studies, there have been obtained recombinant constructs of both p7A and p7B with the fluorescent proteins GFP or RFP. These constructs have been transiently expressed in N. benthamiana plants in presence of cellular organelle markers. The subcellular distribution pattern of p7B shows dynamism in the time and in the space that, given its hydrophobic properties, coincides always with membranous compartments of the cell. Initially, p7B is located in the cortical network of the endoplasmic reticulum (ER) and the external membrane of the nucleus. Later, the protein forms part of mobile particles that have been identified like dictiosomes of the Golgi apparatus. These particles move within the cell cytoplasm by the actin/miosin microfilaments. Finally, the p7B is located in the cell plasmodesmata (PD). On the other hand, although the p7A presents characteristics of soluble protein, it forms attaches in the cell cytoplasm, likewise, it is capable of joining to the dictiosomes of the Golgi apparatus and to the PD. Moreover, the absence of other viral factors does not modify the subcellular distribution pattern of the p7A, by what, given the properties of this protein, some cellular factor must be involved in the same one. Concerning the p7B, the treatment with Brefeldin A, an inhibitor of the secretory route, prevents the exit of this protein to Golgi apparatus, remaining retained in the RE and not coming to the PD. This result shows that this one might be the route used by the same one to reach the periphery of the cell during an infection. Likewise, the subcellular location of different p7B mutants with the topology, the hydrophobity and the membrane insertion of the same one modified, show a clear correlation between the MNSV cell to cell movement of and the subcellular distribution of this constructs. Thus, it has been found that when p7B is not located in the dictiosomes of Golgi apparatus the virus is not capable of moving from cell to cell. Finally, the advance of the viral infection slows down in presence of BrA, suggesting the implication of the secretory route in the intra and intercellular transport of the virus. The information obtained in the present Thesis has allowed the formulation of a model for the intracelular movement of the Carmovirus, in which the secretory route across Golgi apparatus is directly involved, being the first time that this mechanism is described for the intracellular movement of a plant virus.
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