The leader proteinases of the positive-strand RNA viruses from the families Potyviridae
have similar plans of organization: they possess a variable N-terminal domain and a conserved C-terminal papain-like domain. In addition to being involved in polyprotein processing, these proteinases were implicated in efficient genome amplification (21
), and it has been suggested that they have similar functional profiles (12
). To test this possibility, we replaced the L-Pro of BYV, a closterovirus, with HC-Pro of TEV, a potyvirus. Although the chimeric polyprotein was efficiently processed, the hybrid BYV was nonviable, suggesting that the functions of L-Pro and HC-Pro in genome amplification are mechanistically different. It was proposed recently that the HC-Pro role in genome amplification is mediated by the suppression of RNA silencing (6
). The BYV L-Pro, however, lacks detectable silencing suppression activity (J. Reed, K. D. Kasschau, J. C. Carrington, and V. V. Dolja, unpublished data).
We further asked whether the functional specialization of L-Pro and HC-Pro is provided solely by their unrelated N-terminal domains. A chimeric protein in which the N-terminal domain of the BYV L-Pro was fused to the papain-like domain of the TEV HC-Pro failed to support genome amplification of BYV, suggesting that, despite their homology, the papain-like domains of the closteroviruses and potyviruses are functionally distinct. It should be noted that functional differences between L-Pro and HC-Pro cannot be attributed to differences in the host ranges of BYV and TEV since these viruses readily infect N. benthamiana
and have several other common hosts. We can also exclude the possibility that the expression of the TEV proteinase domain or insertion of the corresponding RNA exerted an inhibitory effect on BYV amplification. Indeed, this domain was expressed from several locations within the BYV genome without affecting the viability of the resulting hybrid variants (16
To examine functional specialization of the closterovirus leader proteinases, we replaced BYV genes with the corresponding genes of CTV and LIYV, which belong to two distinct evolutionary lineages within the family Closteroviridae. Among these viruses, BYV and LIYV possess only one leader proteinase, whereas CTV possesses two, L1 and L2. Phylogenetic analysis of the proteinase domains (Fig. ) suggested that the corresponding gene tandem in CTV evolved via a duplication event. It was not, however, known if L1 and L2 are functionally distinct and, if so, which of them is more similar to the leader proteinases of BYV and LIYV. The ability of the CTV L1 and LIYV P-Pro to substitute for the BYV L-Pro in genome amplification indicated that these three leader proteinases belong to the same functional class. In contrast, failure of the CTV L2 to support amplification of the chimeric genome suggested that L2 function had diverged from that of L1, L-Pro, and P-Pro. This assumption was further confirmed by a phenotype of the BYV chimera that expressed both L1 and L2. This chimera amplified almost twice as efficiently as the original BYV, suggesting the synergistic mode of action for L1 and L2, and providing a remarkable example of a hybrid virus that outperformed its parent.
The transient-expression experiments revealed distinct patterns of subcellular localization of the leader proteinases fused with the GFP reporter. Most of the CTV L1 and L2 proteinases were uniformly distributed in the cytoplasm and nucleus, whereas the LIYV P-Pro almost exclusively localized to nuclei. In contrast, the BYV L-Pro was observed predominantly in cytoplasmic inclusion bodies. The possibility that the localization of the leader proteinases in the context of the virus-infected cell might be different from that observed in the transient-expression experiments cannot be excluded. Nevertheless, our results suggest that the leader proteinases of CTV, BYV, and LIYV possess distinct intrinsic signals for interaction with the cell environment and may function in different compartments of the infected cell.
Duplication and functional divergence of the leader proteinase genes are not unique to closteroviruses. A tandem arrangement of the leader proteinases is found among several animal viruses from the order Nidovirales
). Although Closteroviridae
are phylogenetically dissimilar, they are the most complex positive-strand RNA viruses of plants and animals, respectively (26
). Apparently independent duplication of the leader proteinases in these viruses may be interpreted as one of the means to facilitate evolution of the larger and more-complex genomes. In accord with this speculation, acquisition of the second leader proteinase gene in the ~20-kb CTV genome is accompanied by three additional genes that have no homologs in the otherwise closely related ~15-kb BYV genome.
The gene swapping experiments revealed an unexpected degree of functional specialization of the papain-like domains of the closterovirus proteinases. Each of these domains efficiently processed the chimeric polyprotein. However, the papain-like domains of the CTV L1 and L2 supported relatively low levels of BYV genome amplification, whereas the corresponding domain of the LIYV P-Pro was completely nonfunctional (Fig. and Table ). Although the mechanistic basis for this specialization is unknown, it seems possible that the proper function of the leader proteinases requires structural compatibility between the N-terminal and C-terminal domains.
Perhaps the most important outcome of this work is a better understanding of the multifunctional nature of the closterovirus proteinases. In addition to the primary role in the autocatalytic processing, each of the studied four proteinases functions in activation of genome amplification. Because the L1, L1-L2, and P-Pro chimeras amplified to severalfold-higher levels in the N. benthamiana protoplasts than in N. tabacum protoplasts (Table ), this activation appears to work in a host-specific manner. Furthermore, at least L-Pro is critical for the ability of BYV to establish infection in the initially inoculated cells (virus invasiveness) and to translocate from cell to cell (Table ).
The cell-to-cell movement of plant viruses proceeds through plasmodesmata and is activated by the movement proteins (27
). In BYV, as many as five proteins that are encoded by a conserved gene block were implicated in virus movement. These proteins include three dedicated movement proteins (p6, HSP70 h, and p64) and two capsid proteins (2
). Since virion assembly is a prerequisite for BYV cell-to-cell movement (2
), it was possible that the debilitated movement of the chimeric BYV-GFP variants was due to defective assembly. However, analysis of the chimeric virus progeny from the transfected protoplasts revealed normal virion assembly (data not shown). It should be noted that L-Pro by no means could be considered the movement protein since its primary functions are in proteolysis and virus genome amplification. Nevertheless, the fact that variant CTV-L1-L2 accumulates in N. benthamiana
protoplasts almost twice as much RNA as the wild type (Table ) yet does not move from cell to cell even in the leaves of N. benthamiana
(Table ) clearly indicates that L-Pro plays an essential role in cell-to-cell movement. This role, albeit indirect, suggests the need for coordination between the processes of genome amplification and virus translocation. Intriguingly, the leader proteinase of Foot-and-mouth disease virus
, an aphtovirus, was recently implicated in virus spread within infected animals (7
). Although the mechanisms of virus transport in plants and animals are different, this functional parallelism highlights the evolutionary plasticity of the viral papain-like proteinases that provide a structural platform for a variety of functions.
The mechanistic basis of the multifunctionality and specialization of the closterovirus leader proteinases is yet to be determined. These proteinases may act via cleavage of or via interaction with the particular viral or host target proteins. The host-dependent mode of activation of genome amplification and its role in virus invasiveness suggest that the intracellular targets of the closterovirus leader proteinases may include host factors. In conclusion, the gene swapping approach allowed us to reveal novel functions of the leader proteinases encoded in diverse representatives of the family Closteroviridae in genome amplification and virus invasiveness and spread. We also characterized the autonomous subcellular distribution and evolutionary relations of these leader proteinases and generated capable interviral hybrids. Further study of these hybrids will provide an insight into molecular mechanisms underlying multiple activities of leader proteinases and help to design more-efficient viral gene expression vectors.