A Novel Alcohol Oxidase/RNA-binding Protein with Affinity for Mycovirus Double-stranded RNA from the Filamentous FungusHelminthosporium (Cochliobolus)victoriae

Abstract

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FADdependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10-to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding ␤-␣-␤ fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the filamentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein. victoriae 190S virus (Hv190SV) has been extensively studied (2-9), and is classified as a definitive member of the genus Totivirus in the family Totiviridae (10, 11). The Hv145SV, on the other hand, has only been subjected to limited biochemical characterization (2). Because of similarity in size and number of dsRNA segments between the Hv145SV and viruses in the genus Chrysovirus in the family Partitiviridae, the Hv145SV was tentatively classified as a member of the genus Chrysovirus (12). We have recently (13) isolated a cellular protein, Hv-p68, that copurifies with viral dsRNA from H. victoriae isolates infected with both Hv190SV and Hv145SV. Additionally, Hv-p68 was demonstrated to be present as a minor component in the viral capsids (13). Our initial biochemical characterization studies of Hv-p68 indicated that it occurs in vivo as an octamer and that it is consistently present in higher levels in virus-infected H. victoriae isolates than in virus-free ones. We have also demonstrated by a gel retardation assay that Hv-p68 has RNA binding activity (13).In the present study, we report the isolation and complete nucleotide sequence of a cDNA clone of Hv-p68. We present evidence that Hv-p68 belongs to the FAD-dependent GMC family of oxidoreductases and that it has high sequence similarity to the alcohol oxidases of methylotrophic yeasts. Furthermore, we show that overexpression of Hv-p68 is associated with virus infection and that...

Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H 2 O 2 in Brown Rot Decay of Wood

“…Genes encoding AOX have thus far been identified, and cDNAs have been cloned from several yeasts (38,44,51) and mold fungi (27,44,48,49). Here we present for the first time the primary structure of AOX from a basidiomycete fungus that compared to the yeast and/or mold enzymes exhibited some unique features.…”

RNA Sequence Determinants of a Coupled Termination-Reinitiation Strategy for Downstream Open Reading Frame Translation in Helminthosporium victoriae Virus 190S and Other Victoriviruses (Family Totiviridae)

“…Total RNA was extracted from virusinfected or vector-transformed fungal mycelia using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The full-length plus-stranded RNA transcript of HvV190S (ϳ5.2 kb) was synthesized in vitro using T7 RNA polymerase and plasmid pT7HvV190S containing full-length HvV190S cDNA as template (23,46). RNA samples (15 g) were then fractionated on a denaturing agarose gel.…”

Characterization of alcohol oxidase from Aspergillus ochraceus AIU 031

“…In the production of AODs by filamentous fungi, Ko et al have recently reported that Thermoascus aurantiacus NBRC 31963 inductively produced two AODs by incubation with the pectin medium, and characteristics of both enzymes have been revealed by his group (Ko et al, 2005). The genes showing a high homology to AODs from methylotrophic yeasts have also been isolated from a few filamentous fungi (Holzmann et al, 2002;Segers et al, 2001;Soldevila and Ghabrial, 2001), but their proteins have not been purified.…”