Many plant virus genera encode a triple gene block (TGB), an especially evolutionarily conserved gene module involved in the cell-to-cell and long-distance movement of viruses. The TGB-based transport system exploits the coordinated action of three polypeptides to deliver viral genomes into plasmodesmata and to accomplish virus entry into neighboring cells [34, 35]. TGB-encoded proteins are referred to as TGBp1, TGBp2 and TGBp3, according to the positions of their cistrons . All three proteins are essential for virus movement. TGBp1 was widely studied and besides its role in movement, it has been shown that generally functions as an RNA silencing suppressor in members of the genus Potexvirus . At the same time, little is know about additional function of TGBp2 and TGBp3. In agreement with sequence analysis and in vitro studies predicting that TGBp2 and TGBp3 are integral membrane proteins  cell fractionation of plant tissues expressing these proteins demonstrates predominant association of both proteins with the P1 and P30 membranous fractions as well as with the cell wall [38, 39]. Understanding the molecular evolutionary biology of the various proteins expressed by viral genomes and their functions is a prerequisite for the control of virus propagation and the elaboration of efficient and durable antiviral strategies. In our previous study we have shown that TGBp3 is involved in host-pathogen interactions during PepMV infection. Experiments with PepMV TGBp3 mutants revealed that one single mutation K67E was required for converting a mild pathotype into a necrotic one . Mutant viruses of mild PepMV strain induced necrosis on Datura inoxia and Solanum lycopersicum. Symptoms of viral infection strongly depended on the inoculated host plant and result from species-specific host-pathogen interactions.
Secondary and tertiary TGBp3 structure predictions showed that the protein consists of three α-helices and two β-strands. The secondary structure prediction placed amino acid 67 in one of the α-helices. Our tridimensional structural predictions revealed that the region encompassing amino acid 67 in isolates P19, P22 and DB1 is located on the surface of the protein and thus mutations in this region, specially when the physical properties dramatically change as it is the case of mutation K67E, must have a strong impact on the ability of TGBp3 to interact with other protein. It is well known that in many cases amino acids can be replaced without impairing protein function, even if these are of quite different physico-chemical characteristics. However, the change of a positively charged K by a negatively charged E may change the local property of the protein surface, jeopardizing its ability to establish the correct interactions with other viral proteins or cell components. Supporting the existence of such functionality in this surface region, we have identified nucleotide sites 187-198, spanning amino acids 63-66 that were under the action of negative selection in seven branches of the phylogenetic tree describing the evolutionary history of PepMV. In particular, the PEVL motif is highly conserved in all isolates analyzed.
In general, functionally essential protein parts are negatively selected for (conserved), while other parts can be positively selected for. The ω mean value obtained for the TGBp3 cistron strongly suggests a predominant action of purifying selection. In good agreement with this average value, three different approaches detected strong signal of purifying selection for particular codons. Nevertheless, most codons are evolutionarily neutral. However, perhaps the most interesting result from the analyses of selective constraints if that amino acid K67 has been identified as under positive selection in the branch leading to necrotic isolates, suggesting that necrosis may be an adaptive trait. Since it is close to the region of amino acids 63-66, which is (i) under negative selection (and hence likely has an important function) and (ii) predicted to be in the surface of the folded protein, we can speculate that it may be involved in the formation of protein-protein complex that determine the development of symptoms. The results obtained with ConSurf indicated that amino acids 61-66 played functional and structural roles in the protein. A protein function is however, the results of the functional and structural communication between sites and, therefore, the ability of a given site to change depends on the interactions it must establish with other residues of the molecule. Mutations at either nearby sites (like K67E), or functionally related distant sites in the structure, will change the selective constraints . Functional sites, like binding domains, are less prone to amino acid changes than less important protein regions. Furthermore, some classes of proteins evolve faster than others .
The analysis of the TGB1 gene in PepMV populations clearly provides a mechanism for its rapid evolution and adaptation to the ever-changing environments . In the light of what is so far known about PepMV evolutionary dynamics  it seems that TGBp3 evolves mostly by the action of purifying selection operating over several sites, highlighting its functional role during PepMV infection. Gómez et al.  showed only one amino acid under purifying selection in the TGBp3 of CH2 genotype also using the methods implemented in the DATAMONKEY server. We have analyzed TGBp3 sequences representing different genotypes and we used more sophisticated methods to establish selection pressure acting on TGBp3. We were able to identify more codons under action of purifying selection, moreover amino acid 67 was predicted to be under positive selection. It seems that the particular pathotypes achieved an advantage over others especially in Europe. Recently, more aggressive pathotypes (causing necrosis or severe yellowing) of CH2 genotype have become dominant in Poland. This shift in the Polish PepMV population reveals a dynamic interplay between the different PepMV genotypes and their host. Necrotic isolates, however, were described in both the EU and CH2 genotypes, and it seems that the selective pressures act in the direction of increasing the virulence of isolates, less temperature dependent which cause significant losses in quality and quantity of yield. Preliminary data (Hasiów-Jaroszewska, unpublished results) have shown that necrotic isolates acquired the ability to infect a larger number of Solanum tuberosum varieties and of causing more severe symptoms in shorter time. Probably, the key feature of these isolates are faster replication or accumulation. It seems that molecular evolution is leading to higher variability among CH2 genotype in comparison to others genotypes. It has been suggested that the CH2 genotype has a biological advantage over the EU genotype, as it seems to spread more rapidly within a crop . Recent study on the evolutionary dynamics of the PepMV population in Spain using RT-quantitative PCR analyses in inoculated tomato plants showed that a CH2 isolate (PS5) accumulated more rapidly and to higher viral loads than an EU isolate (Sp13) . The TGBp3 is responsible for virus movement and further research will be performed to shed the light between particular mutation in TGBp3 and virus fitness.
The present in silico study opens new research avenues for researches interested in experimentally exploring the in vivo interactions between TGBp3 and host factors. Moreover, we report the first tridimensional structure of TGBp3, obtained with de novo folding methods followed by careful accuracy assessments. This model may serve as a platform for further sequence, structural and function analysis and will stimulate new experiment advances.