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. 2008 Oct;36(17):5451-61.
doi: 10.1093/nar/gkn519. Epub 2008 Aug 21.

Viral AlkB proteins repair RNA damage by oxidative demethylation

Erwin van den Born  1 , Marina V Omelchenko, Anders Bekkelund, Vibeke Leihne, Eugene V Koonin, Valerian V Dolja, Pål Ø Falnes
Affiliations

Viral AlkB proteins repair RNA damage by oxidative demethylation

Erwin van den Born et al. Nucleic Acids Res. 2008 Oct.

Abstract

Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

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Figures

Figure 1.
Figure 1.
Phylogenetic analysis of viral AlkB proteins. (A) The overall tree of the AlkB family. For clarity, individual viruses are grouped and major groups of bacteria and eukaryotes are collapsed, colour-coded and labelled with the name of the group. The complete tree is given in Figure S2 and the complete alignment used for tree construction is given in Figure S1. Numbers indicate bootstrap support for central tree nodes. The scale bar represents the number of substitutions per 100 residues. (B) Phylogenetic tree of selected viral AlkB proteins rooted by proteobacteria (Prote) outgroup (E. coli, Ralstonia metallidurans and Caulobacter vibrioides). Dots indicate tree nodes with bootstrap support ≥70%. Members of different viral families are colour-coded: blue, Flexiviridae (Flexi); orange, Closteroviridae (Clost); green, Potyviridae (Potyv); magenta, Sadwavirus (Picornavirales, Picor). Species studied in this work are underlined and inactivated AlkBs are indicated with an asterisk. Numbers represent Genbank Identifier (gi) numbers.
Figure 1.
Figure 1.
Phylogenetic analysis of viral AlkB proteins. (A) The overall tree of the AlkB family. For clarity, individual viruses are grouped and major groups of bacteria and eukaryotes are collapsed, colour-coded and labelled with the name of the group. The complete tree is given in Figure S2 and the complete alignment used for tree construction is given in Figure S1. Numbers indicate bootstrap support for central tree nodes. The scale bar represents the number of substitutions per 100 residues. (B) Phylogenetic tree of selected viral AlkB proteins rooted by proteobacteria (Prote) outgroup (E. coli, Ralstonia metallidurans and Caulobacter vibrioides). Dots indicate tree nodes with bootstrap support ≥70%. Members of different viral families are colour-coded: blue, Flexiviridae (Flexi); orange, Closteroviridae (Clost); green, Potyviridae (Potyv); magenta, Sadwavirus (Picornavirales, Picor). Species studied in this work are underlined and inactivated AlkBs are indicated with an asterisk. Numbers represent Genbank Identifier (gi) numbers.
Figure 2.
Figure 2.
Sequence alignment of a part of the nucleotide recognition lid region from various AlkB proteins. The region shown corresponds to aa 63–98 of EcAlkB (gi|113638). Filled arrows indicate residues Trp69 and Tyr76 in EcAlkB, which have been shown to be involved in coordinating 1-meA (30). Dotted arrows indicate residues that are conserved in viral, vertebrate and bacterial AlkB proteins belonging to group 1B, but not to group 1A (29). BlScV, Blueberry scorch virus; BVY, Blackberry virus Y; GVA, Grapevine virus A; LChV-2, Little cherry virus 2; CLBV, Citric leave blotch virus, CVB, Chrysanthemum virus B; LSV, Lily symptomless virus; ASPV, Apple stem pitting virus; GLV, garlic latent virus; ZVX, Zygocactus virus X; Eco, E. coli; Sme, Sinorhizobium meliloti; Rme, Ralstonia metallidurans; Ppu, Pseudomonas putida; Vvu, Vibrio vulnificus; Pae, Pseudomonas aeruginosa; Xca, Xanthomonas campestris; Son, Shewanella oneidensis; Hs, Homo sapiens; Gg, Gallus gallus; Dr, Danio rerio.
Figure 3.
Figure 3.
Viral AlkB proteins used in the present study. A sequence alignment was generated from 23 viral AlkB protein sequences, and the five sequences shown were extracted from this alignment. The shading pattern of this 23 sequence alignment has been maintained (explaining why some residues are shaded without being conserved among the five sequences shown). Arrows indicate the borders of the AlkB-coding regions included in the various expression constructs. The solid line indicates the ‘AlkB core’, i.e. the region which displays homology to other members of the AlkB family, and extensive sequence homology within the subfamily of viral AlkB proteins. The dotted line indicates a region where sequence conservation is observed within the group of 23 viral AlkB sequences (but not shared by other AlkB family members): (1) CXC in 14 sequences; (2) I/L/V/M-X-I/L/V in 15 sequences; 12 sequences adhered to the consensus CXCX3-I/L/V/M-X-I/L/V.
Figure 4.
Figure 4.
Reactivation of MMS-treated bacteriophages by expression of AlkB proteins in E. coli. (A) Reactivation of methylated ssRNA phage MS2 by AlkB proteins from GVA, BVY, BlScV, LChV-2 or CLBV. Reactivation of methylated MS2 by different variants of AlkB from (B) GVA, (C) BlScV and (D) BVY. (E) Reactivation of methylated ssDNA phage M13 by GVA-36, BVY-36 and BlScV-94. (F) Reactivation of methylated MS2 by BVY-94 and mutants BVY-94-H59A and BVY-94-D61A. Expression plasmid pJB658 without insert was used as control. Error bars in B–D represent the standard deviation of triplicate measurements.
Figure 5.
Figure 5.
AlkB-mediated decarboxylation of 2-oxoglutarate. (A) Purified recombinant His-tagged AlkB proteins used in this study. Proteins were visualized by coomassie staining of a 15% SDS–PAGE gel. (B) Reaction mixtures containing [5-14C]2-oxoglutarate, 100 pmol of EcAlkB and 500 pmol of GVA-36, BlScV-94, BVY-94, BVY-94-H59A or BVY-94-D61A were incubated for 30 min at 37°C, and the released [1-14C]succinate was measured by scintillation counting. Cofactor Fe2+ and substrate-mimic 1-methyladenosine (m1A) were present as indicated. Error bars represent the standard deviation of duplicate measurements.
Figure 6.
Figure 6.
Activity of AlkB proteins on various [3H]methylated RNA and DNA substrates. (A) [3H]methylated oligonucleotides were incubated with varying amounts of EcAlkB, GVA-36, BlScV-94 or BVY-94 and the ethanol soluble radioactivity released was measured by scintillation counting. (B) [3H]methylated poly(A) was incubated with 100 pmol of BVY94, BVY-94-H59A or BVY-94-D61A. Fe2+ and 2OG were present as indicated. Error bars represent the standard deviation of duplicate measurements.
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