Viral AlkB proteins repair RNA damage by oxidative demethylation

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.

Cassava Brown Streak Virus ( Potyviridae ) Encodes a Putative Maf/HAM1 Pyrophosphatase Implicated in Reduction of Mutations and a P1 Proteinase That Suppresses RNA Silencing but Contains No HC-Pro

“…This virus is the only Potyviridae member besides CBSV and EuRSV that is known to carry an insertion of cellular origin. AlkB domains in the aforementioned viral proteins are functional in repairing methylation damage of nucleic acids by oxidative demethylation, as also shown for the homologous proteins in cellular organisms (42). The AlkB proteins of viruses have not been found to suppress RNA silencing (42), which was also the case with CBSV HAMh1 in this study.…”

“…AlkB domains in the aforementioned viral proteins are functional in repairing methylation damage of nucleic acids by oxidative demethylation, as also shown for the homologous proteins in cellular organisms (42). The AlkB proteins of viruses have not been found to suppress RNA silencing (42), which was also the case with CBSV HAMh1 in this study. Since a large proportion of the AlkBcontaining viruses infect perennial, often woody, host plants, it is anticipated that RNA repair by AlkB proteins may be advantageous for the stability of viruses in hosts infected for long periods of time under various environmental conditions (24).…”

Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

“…Our recent work already revealed novel roles for proteases that affect GLRaV-2 invasiveness in grapevine (33) and suggested such roles for the AlkB RNA repair enzyme encoded by many viruses of woody plants, including Grapevine leafroll-associated virus-3 (56).…”

The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast

“…This is somewhat surprising, since it is known that several viruses depend on or even actively influence cellular iron homeostasis to facilitate viral infections. For example, several plant viruses carry genes that encode proteins with the AlkB domain that require iron for activity, and these proteins might be involved in maintaining the integrity of the viral genomic RNA (76). Also, several human viruses, such as hepatitis C virus, can also actively influence iron homeostasis.…”