The major objective of this study was to define the complete functional profile of the BYV L-Pro. Early work revealed that L-Pro possesses two principal domains, a nonconserved N-terminal domain and a conserved C-terminal domain that is related to papain-like proteinases (1
). To assess the functional significance of these putative domains, we conducted an extensive genetic study using alanine-scanning mutagenesis and gene swapping. This study revealed that L-Pro provides an illuminating example of a multifunctional viral protein that enables progression of virus infection through its major phases.
Initial analyses showed that the C-terminal domain is both essential and sufficient for autocatalytic release of L-Pro from the viral polyprotein (23
). The subsequent gene-swapping experiments suggested that in addition to proteolysis per se, this domain is required for efficient RNA amplification (24
). The extensive mutational analysis presented here supported this suggestion and indicated that the roles played by C-terminal domain in proteolysis and genome amplification are genetically separable. A previous study of the N-terminal domain revealed that although it is not essential for basal-level replication, its deletion results in a dramatic reduction in RNA accumulation (23
). It is yet to be determined if the functions of the two principal L-Pro domains in genome amplification are independent or interdependent. The dramatic phenotype of mutant A13, which is competent in proteolysis but deficient in RNA amplification, seems to support the latter possibility. Since this mutation is located near the junction of L-Pro domains, it could abolish L-Pro function by affecting the relative orientation of two domains.
Our finding of a novel function of L-Pro in BYV long-distance transport provides an example of activity that requires an entire L-Pro molecule. Indeed, the mutations that affect virus transport were found in each of the L-Pro domains. L-Pro, however, is not the only BYV protein that plays the role of an LTF. Very recently we demonstrated that p20 is also required for systemic spread of BYV (29
). Although the defects in virus spread due to mutations in L-Pro and p20 are phenotypically similar, the underlying mechanisms could be different for the following reasons. First, p20 is tightly associated with the BYV virions (29
), whereas L-Pro is not. Even though we cannot exclude transient or weak binding of L-Pro to virions, the nature of this interaction is certainly different from that of p20. Second, the regulation and functional profiles of the two BYV LTFs are very distinct. L-Pro is expressed via translation of the genomic RNA from the very onset of infection, whereas p20 is translated from a subgenomic mRNA only late in infection (14
). Furthermore, p20 is not involved in the amplification of the BYV RNA (26
), whereas L-Pro acts as a strong enhancer of this process (23
Is it possible to reconcile the major roles played by L-Pro in BYV replication and transport? Since long-distance transport involves virus passage through and replication within the phloem (21
), it seems plausible that L-Pro provides a specific activity required for efficient replication of BYV in this plant tissue. The ability of some mutants (e.g., A2 and A15 [Table ]) to establish a productive infection in only a fraction of the inoculated plants suggests interference of L-Pro with the host defense response, the extent of which may vary between the individual plants. Because these same mutants did not exhibit defects in their ability to invade the leaf mesophyll tissue and to move from cell to cell, this putative defense response could be specific to phloem tissue.
Genetic requirements for long-distance transport vary between plant viruses. Some viruses, exemplified by Tobacco mosaic virus
, require only a cell-to-cell movement protein and a capsid protein to spread systemically (9
). Other plant viruses, such as potyviruses, code for an LTF, a protein with a specialized function in long-distance transport (7
). BYV is unusual in that its long-distance transport requires the action of at least two LTFs, p20 and L-Pro. While the functional profile of L-Pro is unique, it partially overlaps that of the potyviral LTF HC-Pro. Both L-Pro and HC-Pro are leader proteinases that are required for efficient genome amplification. However, the amplification and long-distance transport activities of HC-Pro correlate with each other and are empowered by its ability to suppress RNA silencing (18
). In contrast, we show here that the L-Pro functions in genome amplification and virus transport are genetically separable. These differences in the functional profiles point to mechanistic distinctions between HC-Pro and L-Pro that are underscored by the inability of HC-Pro to rescue L-Pro function (24
). Furthermore, HC-Pro, but not L-Pro, is capable of suppressing RNA silencing that is induced by transient expression of the double-stranded RNA (30
In conclusion, we demonstrated that the BYV L-Pro functions include polyprotein processing, genome amplification, virus invasion into plant tissues, and virus long-distance transport via the phloem. Thus, L-Pro provides activities that are essential for integration of the distinct phases of the viral life cycle from genome expression and amplification to virus dissemination through the infected host.