This report describes the first functional characterization of AlkB proteins from plant viruses. A sequence comparison with other AlkB proteins suggested that the viral AlkB proteins are involved in repairing methylation damage, and this indeed turned out to be the case. The three proteins studied in detail represent two diverse viral superfamilies, Alphavirus- and Picornavirus-like (34
), and they had very similar properties, indicating that they perform the same biological function in planta
. They all displayed a repair activity similar to EcAlkB, but preferred RNA over DNA, and are the first AlkB proteins with such substrate specificity. Moreover, the viral AlkB proteins displayed robust demethylation activity both on ssRNA and dsRNA, presumably enabling them to repair alkylation damage both in the ssRNA viral genome and in the dsRNA replication intermediates generated in the host cell during infection. Thus, viral AlkB represents the first example of virally encoded alkylation repair proteins.
It has been demonstrated that AlkB-mediated repair can functionally reactivate methylated tRNA and mRNA, indicating that RNA repair may be biologically relevant (17
). This notion was further supported by the observation that while mice lacking mABH2 displayed defective repair of 1-meA lesions in DNA, no such defect was observed in mice lacking the putative RNA repair enzyme mABH3 (35
). However, since the AlkB proteins reported to repair RNA are equally (ABH3) or more (EcAlkB) active on DNA, it has been difficult to rule out the possibility that AlkB-mediated RNA demethylation is an irrelevant side activity. On the other hand, since the AlkB-containing viruses have a positive-strand RNA genome, replicate in cytosolic membrane-bound compartments (36
), and the AlkB domain in many cases remains part of an unprocessed (cytosolic) replicase polyprotein, it is very difficult to imagine a DNA repair function for viral AlkB. Thus, the current finding that viral AlkB proteins are able to efficiently repair methyl lesions in RNA, and even prefer RNA over DNA, provides a very strong argument for the biological significance of AlkB-mediated RNA repair.
Only a few examples of viral RNA repair have so far been demonstrated. The 3'-end of viral RNAs is vulnerable to degradation by host RNases, and several mechanisms can mend the resulting truncated ends (37
). Also, when bacteria are infected with bacteriophage T4, a host response is mounted involving the cleavage of the Lys-specific tRNA in the anticodon loop, thereby preventing the synthesis of proteins required for the phage infection to proceed. The T4 RNA ligase counteracts this response by repairing the cleaved tRNA (39
). Furthermore, mechanisms for removing aberrant bases from viral DNA genomes have been reported. Some DNA viruses encode photolyases or glycosylases to remove UV-induced pyrimidine dimers from the genome (41
), whereas others carry uracil-DNA glycosylases to remove uracils resulting from cytosine deamination or uracil misincorporation (43
). Several of these DNA repair mechanisms depend on an undamaged template strand (present in dsDNA). The AlkB-mechanism, on the other hand, is template-independent, making it well suited for RNA repair.
In plants, small interfering RNAs (siRNAs) are mediators of a host RNA interference (RNAi) response causing degradation of viral RNAs. It was originally suggested that the role of viral AlkB may be to counteract a host response involving RNA methylation (6
). Interestingly, it was subsequently shown that siRNAs are methylated at the 2′-OH of the 3′-terminal nucleotide by the methylase HEN1 (44
). Several virus-encoded suppressors of silencing have been shown to interfere with HEN1-mediated methylation (45
). Since the viral AlkBs displayed robust activity on 1-meA and contain key conserved residues involved in the coordination of this substrate, we find it less likely that the 2′-O
-methylated ribose of siRNAs is a substrate for viral AlkB. In agreement with this, we were unable to detect RNAi suppression activity of viral AlkBs using conventional transient in planta
assays or stable transformation of Arabidopsis thaliana
by viral AlkBs (Peremyslov,V.V. and Dolja,V.V., unpublished data). However, although our presented results clearly indicate a role for viral AlkB in RNA repair, one cannot completely rule out the possibility that they are involved in counteracting a yet undiscovered host antiviral defence system, involving the introduction of AlkB substrates into host or viral RNA.
Alkylating agents are found both in the environment and intracellularly, and it is believed that both these sources can contribute significantly to the cellular load of alkylation damage. It has been suggested that plant viruses may have acquired AlkB recently in response to the use of methylating agents, e.g. methylbromide, as pesticides (46
). We consider this unlikely, because many AlkB-containing viruses infect plant species not treated with such agents, and because the time elapsed since the relatively recent introduction of such pesticides is probably too short for the observed spread and phylogenetic divergence of viral AlkBs from their cellular ancestors. Viruses that have AlkB often infect woody or perennial plants, where they may survive for years in a hostile environment within the phloem sieve elements to ensure their eventual transmission to new plants. We favour the hypothesis that the viral genome may occasionally be subjected to considerable methylation damage in this environment, and that viral AlkBs substantially enhance virus propagation by repairing such damage upon virus entry into the host cell.
Viral AlkBs represent a remarkable example of viral proteins with readily identifiable homologues in cellular genomes. Based on the observation that viral AlkB proteins are more similar to AlkB homologues from bacteria than from plants, it appears most likely that the first viral AlkB was derived via horizontal gene transfer from a bacterial source, possibly, from a bacterial pathogen or symbiont of plants. The lack of clustering between viral AlkBs and any specific bacterial branch (A) could be explained by the well-known fast evolution of viral RNA sequences and/or by the absence of close relatives of the actual source bacterium in the current databases. With the exception of Flexiviridae, AlkB-containing viruses represent a minor proportion of viruses within their corresponding taxa, with BVY being the only known AlkB-containing member of the vast family Potyviridae. This distribution pattern suggests that AlkB was acquired relatively recently, likely by a virus within the family Flexiviridae where AlkB-containing viruses are more common than in other viral families, and then disseminated horizontally, via recombination between viruses including very distantly related ones such as BRNV. The spread of AlkB proteins to new viruses might have occurred through gene transfer between flexiviruses and viruses from other plant RNA virus families, which frequently occurs in mixed infections of the same, often woody or perennial host. Also, given the rapid evolution of RNA viruses, the very existence of a compact viral clade in the AlkB family tree (A) is best compatible with a horizontal mode of their spread among viruses.
It is an intriguing question why only a subset of the plant viruses within a given family possesses an AlkB homologue. Possibly, having an AlkB protein gives the virus a selective advantage under conditions of high methylation load, and viruses that do not experience such conditions may have disposed of, or never acquired AlkBs. The notion that viral AlkB proteins may be disposed of when no longer needed, e.g. when a virus evolves to infect a different host, is supported by the observation that a handful of plant viruses have AlkB domains where several of the residues thought to be important for enzymatic activity are mutated. The scattered presence of inactivated AlkBs among active ones in the phylogenetic tree is best compatible with multiple, independent inactivations of AlkB that potentially might have been linked to a switch to environments with lower alkylation pressure. Interestingly, three of the viruses that apparently have inactivated AlkBs, infect potato; a non-woody and non-perennial plant, where the evolutionary pressure for maintaining the AlkB function might be absent (B and S3
To further understand the role of AlkB in viral infection, it will be of interest to study the infectivity in planta of viruses in which the AlkB region has been inactivated by mutation, as well as to study the possible accumulation of methylation damage in wild-type and AlkB-mutated viral genomes during infection.