Summary
     
     Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus within the family Tombusviridae and, as the remaining members of the group, possesses a monopartite positive-sense single-stranded (ss) RNA genome of about 4 kb, which is encapsidated into isometric particles of 30 nm in diameter. Like other carmoviruses, the genomic RNA of PFBV harbours five open reading frames (ORF) flanked by non-coding regions of 32 and 236 nt at the 5' and 3', respectively. The 5'-proximal ORF 1 encodes a protein of 27 kDa (p27) and terminates with an amber codon, which may be read-through into an in-frame ORF2 to generate a protein of 86 kDa (p86). Both products (p27 and p86) exhibit high homology with proteins involved in replication in other carmoviruses. Protein p86 contains sequence motifs characteristic of RNA-dependent RNA polymerases while p27 does not present any motif to explain its presumed involvement in replication. Two internal ORF (ORF 3 and 4) encode small proteins of 7 and 12 kDa (p7 and p12), that probably mediate the viral intra- and intercellular movement, and ORF 5 encodes a polypeptide of 37 kDa (p37) that fits the structural pattern of coat proteins (CP) of the genus Carmovirus and, in general, of the family Tombusviridae.

     The work presented here has been aimed at determining the step of the infectious cycle in which the different proteins of PFBV are involved and at analyzing structure-function relationships in these products, putting the focus on certain atypical features and/or properties poorly characterized in related viruses.

     Mutation of ORF 1 and 2 has indicated that products p27 and p86 are essential for virus replication. The expression in plants and yeast of PFBV replicases tagged with fluorescent proteins and its subsequent observation under a confocal microscope, have revealed that both molecules are targeted independently to mitochondria. The morphology or distribution of these organelles is not affected by the presence of p27 and/or p86, which, moreover, show colocalization patterns. Analysis of the signals that guide the mitochondrial targeting of p27 has pointed to the involvement of two regions, one of 29 residues located toward the N-terminus of the protein and other of 27 residues, which match with its C-terminus. An in silico analysis has predicted the formation of amphipathic alpha-helical regions that could mediate the association of p27 with mitochondrial membranes. Such association seems to be independent of many of the cellular factors related with protein import to mitochondria, since their absence does not affect the subcellular localization of p27, at least in yeast.

     Additional studies with protein p27 have shown that it is also capable of binding RNA in vitro, with high affinity and with positive cooperativity. Competition assays have indicated that the protein binds preferentially to ssRNAs and, particularly, to those derived from the viral genome; nonetheless, it can also bind other types of nucleic acids (dsRNAs, ssDNAs, dsDNAs), albeit with lower efficiency. The fact that the complex p27:ssRNA is stable at high salt concentrations, suggests that interactions other than ionic ones might contribute to its formation / stability. Assessment of truncated versions of the protein has allowed identification of three regions of 40, 30 and 70 residues, respectively, that contribute differentially to nucleic acid binding and in additive manner. Both, the mitochondrial localization and RNA binding properties observed for the smaller replicase of PFBV, suggest that it plays an important role during the replication of the pathogen, most likely by recruiting the RNA template and by targeting the replicative complex to mitochondrial membranes, the site where the viral RNA synthesis takes place.

     On the other hand, it has been confirmed that the two small polypeptides encoded by ORF 3 (p7) and ORF 4 (p12) are required for viral movement. In order to obtain information about the mode-of-action of at least one of these viral products, the subcellular localization of protein p12 has been determined. In addition, the potential functional relevance of some atypical features, such as a putative leucine zipper motif (LZ) and an N-t extension rich in basic residues, has been evaluated. The results have indicated that p12 associates to subcellular membranes, primarily to endoplasmic reticulum, which is in accordance with its hydrophobic profile and with that observed for others movement proteins, including those of the genus Carmovirus. Furthermore, we have determined that the putative LZ is necessary for pathogen cell-to-cell transport and that the basic N-t extension of p12 confers RNA-binding properties to this protein, which are mediated by positively charged residues. These latter characteristics are not shared by homologous proteins, suggesting that the movement of PFBV may mechanistically differ from that of related viruses.

     Finally, the ability of all proteins encoded by the PFBV to inhibit RNA silencing has been tested using a transient expression assay in plants. Our results have shown that protein p37 is a strong suppressor of silencing, in line with that reported for the CP of at least two members of the genus Carmovirus. It has also been observed that this protein is able to bind small interfering RNAs (siRNAs) in vitro. This capability is correlated, in vivo, with both silencing suppressor activity and enhancement of viral pathogenicity. Taken together, these results suggest that protein p37 inhibits the RNA silencing by sequestering siRNAs thus preventing their incorporation into an RNA-induced silencing complex, a mechanism commonly used by non-related suppressors.