Previous comparative analysis of the BYV and CTV genomes revealed that proteins encoded by ORFs 1a and 1b possess conserved domains characteristic of the replicases of Sindbis virus-like viruses (4
). In contrast to the single-component genomes of BYV and CTV, the genome of lettuce infectious yellows closterovirus is divided among two RNAs (33
). RNA 1 harbors ORFs 1a and 1b and an additional ORF that is unique to lettuce infectious yellows closterovirus. RNA 2 codes for homologs of BYV p6, HSP70h, p64, CP, CPm, and a protein with marginal similarity to p21 (33
). It was demonstrated recently that RNA 1 can replicate in the absence of RNA 2 but not vice versa (34
). This result further supported the involvement of the products of ORFs 1a and 1b in RNA replication, while the possible roles of several additional proteins encoded in one- or two-component closteroviruses remained elusive.
Generation of a full-length BYV clone permitted mapping of the genes involved in the replication and transcription of the 15.5-kb viral RNA. As expected, expression of intact ORFs 1a and 1b, encoding putative methyltransferase, RNA helicase, and RNA polymerase, was found to be essential for RNA replication. Deletion of the cleavage site located between L-Pro and the rest of the ORF 1a product abolished RNA replication, suggesting that autocatalytic release of L-Pro or maintenance of a proper protein structure around the cleavage site is critical for virus viability. In addition to the conserved papain-like proteinase domain, closterovirus L-Pro possesses a variable N-terminal domain (4
). A function of this domain was probed by using a series of deletion mutants. Analysis of the mutant phenotypes revealed that the N-terminal domain is required for efficient amplification of the BYV genome. This function was separable from proteinase activity, since some mutants that exhibited no detectable processing defects accumulated reduced levels of RNA. The ability of an L-Pro mutant lacking most of the N-terminal domain to replicate, albeit inefficiently, suggested that the function of this domain in genome amplification is accessory rather than essential.
The possibility cannot be excluded that at least some of the deletions in the L-Pro-encoding region disturbed the RNA elements (e.g., RNA polymerase recognition signals) that are directly involved in the RNA accumulation process. These elements, however, are normally distinct for the plus and minus RNA strands. Since analyzed L-Pro mutations affected the accumulation of both strands to similar extents, it seems more likely that the observed effects were mediated by encoded protein products rather than by the RNA itself.
Interestingly, mutational analysis of the helper component proteinase (HC-Pro) encoded in plant potyviruses revealed a similar functional profile. Processing activity of the C-terminal papain-like proteinase domain of HC-Pro (8
) was found to be indispensable for RNA replication (30
), whereas the central domain functioned as a replicational enhancer (6
). Recent experiments suggested that HC-Pro activates RNA synthesis indirectly, possibly disarming the host defense system that limits the accumulation of viral RNA (31
Both potyvirus HC-Pro and closterovirus L-Pro belong to a large class of papain-like leader proteinases that provide various accessory functions in viral reproduction (19
). Additional examples include the aphthovirus L-proteinase that is involved in the shutoff of host protein synthesis and in virus pathogenicity (43
) and p29, a symptom determinant of the hypovirulence-associated virus of chestnut blight fungus (10
). It appears that diverse plant, animal, and fungal viruses recruit papain-like proteinases to manipulate host functions in the course of an infection.
Six genes located in the 3′-terminal region of the BYV genome were found to be dispensable for virus RNA accumulation at the single-cell level. It seems likely that corresponding products p6, HSP70h, p64, CPm, CP, and p20 contribute to the processes of virion assembly, cell-to-cell and long-distance transport, and aphid transmission of BYV. On the other hand, p21, encoded by the 3′-most BYV gene, was identified as an enhancer of RNA amplification. The mutant lacking the p21 start codon exhibited a fivefold reduction in the accumulation of both the plus and minus strands of genomic RNA. The proteins related to p21 are conserved in other one-component closteroviruses, such as CTV and beet yellow stunt virus (17
, and data not shown). Although a database search did not reveal any non-closterovirus homologs of p21, the γb protein encoded in barley stripe mosaic hordeivirus seems to perform an analogous accessory function in RNA replication (44
). It should be emphasized that none of the mutations in p21, in L-Pro, or in products of ORFs 2 through 7 specifically affected the synthesis of sgRNAs. These data suggest that the only closterovirus proteins required for genome transcription are the products of ORFs 1a and 1b.
Interesting analogies were observed in the organization and expression of closterovirus and coronavirus genomes (17
). These analogies included unusually large replicases encoded in ORFs 1a and 1b, with ORF 1b expressed via translational frameshift; the presence of papain-like proteinases in the 5′-proximal part of ORF 1a; and the formation of multiple sgRNAs required for expression of non-replicase genes (11
). The functional analysis presented here revealed that closteroviruses, similar to coronavirus-like viruses (32
), in addition to replicase, do require a product of the 3′-most ORF for efficient RNA amplification. Further mechanistic studies employing full-length cDNA clones of coronavirus-like viruses (49
) and closteroviruses should explain why these two phylogenetically and biologically diverse virus groups convergently adopted this particular combination of genome replication and expression strategies in the course of evolution towards the largest known “RNA chromosomes.”