Bioinformatic mapping of AlkB homology domains in viruses

Abstract

Background: AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily. In Escherichia coli the protein protects RNA and DNA against damage from methylating agents. 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form. Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins. Similar domains have also been observed in certain plant viruses. The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants. We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g. in Eukaryotes.

Results: Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family. Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations. This pattern seems to be more consistent with increased environmental pressure, e.g. from methylating pesticides, than with interaction with the PTGS system.

Conclusions: The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment.

Figure 1

PSI-Blast search results shown as a Venn diagram. Initial searches using methyltransferase, helicase and RdRp domains retrieved 163, 175 and 237 sequences, respectively. A total of 146 sequences contained all three domains, and 22 of these also contained an AlkB domain.

Figure 2

Unrooted phylogenetic tree for Flexiviridae 1 and 2, Tymoviridae and Closteroviridae. Sequences are labelled with genus and NCBI gi accession number. Bootstrap values ≥ 80 are shown. Sequences with AlkB domains are indicated with black dots.

Figure 3

Multiple alignment of sequence regions corresponding to the AlkB domains. The alignment was generated with ClustalX. The residues involved in coordination of the essential Fe²⁺ ion are completely conserved, except in one of the Vitivirus sequences. These residues are the HxD motif, a single H, and the first R in the RxxxxxR motif. The function of the remaining conserved residues is unclear, but at least some of them may be involved in coordination of the substrate [10].

Figure 4

Pairwise distances between sequence regions corresponding to methyltransferase (MT), RdRp and AlkB domains. Each data point corresponds to e.g. RP-RP and MT-MT distances for the same pair of sequences, and sequences showing similar evolutionary distance in these two regions will fall on the diagonal. The pairwise distances were estimated from multiple alignments using the Blosum50 score matrix [47]. Trend lines were estimated with Excel. The trend line for AlkB vs. RdRp is heavily influenced by the point at (675, 670). It represents two Foveavirus sequences (NCBI gi3702789 and gi9630738), they are 98% identical over the full polyprotein sequence.

Figure 5

Location of Pfam domains in the variable region of Flexiviridae 2 sequences. The regions have been extracted directly from Pfam output, and sequences and regions are drawn to scale. The black bar at each end of a motif indicates that a full-length motif has been found, for partial motifs the bar at the truncated end would be missing.

Figure 6

Dot plots for Potato virus M (NCBI gi9626090) and Aconitum latent virus (NCBI gi14251191). To the left the full sequences are shown, using the program default for similarity threshold, and to the right the region with AlkB, OTU and peptidase integration, using a slightly lower (more sensitive) threshold for sequence similarity. The Pfam regions corresponding to MT (magenta), AlkB (red), OTU (green), peptidase (blue), HEL (yellow) and RdRp (cyan) domains are indicated.