Multiple mitochondrial viruses in an isolate of the Dutch elm disease fungus Ophiostoma novo-ulmi

nucleotide sequences of three mitochondrial virus double-stranded (ds) RNAs , RNA-4 (2599 nucleotides), RNA-5 (2474 nucleotides), and RNA-B (2343 nucle otides), in a diseased isolate Log1/3-8d(2) (Ld) of the Dutch elm disease f ungus Ophiostoma novo-ulmi have been determined. All these RNAs are A-U-ric h (71-73% A + U residues). Using the fungal mitochondrial genetic code in w hich UGA codes for tryptophan, the positive-strand of each of RNAs 4, 5, an d 6 contains a single open reading frame (ORF) with the potential to encode a protein of 783, 729, and 695 amino acids, respectively, all of which con tain conserved motifs characteristic of RNA-dependent RNA polymerases (RdRp s). Sequence comparisons showed that these RNAs are related to each other a nd to a previously characterized RNA, RMA-3a, from the same O. novo-ulmi is olate, especially within the RdRp-like motifs. However, the overall RNA nuc leotide and RdRp amino acid sequence identities were relatively low (43-55% and 20-32%, respectively). The 5'- and 3'-terminal sequences of these RNAs are different, but they can all be folded into potentially stable stem-loo p structures. Those of RNA-4 and RNA-6 have inverted complementarity, poten tially forming panhandle structures. Their molecular and biological propert ies indicate that RNAs 3a, 4, 5, and 6 are the genomes of four different vi ruses, which replicate independently in the same cell. These four viruses a re also related to a mitochondrial RNA virus from another fungus, Cryphonec tria parasitica, recently designated the type species of the Mitovirus genu s of the Narnaviridae family, and to a virus from the fungus Rhizoctonia so lani. It is proposed that the four O. novo-ulmi mitochondrial viruses are a ssigned to the Mitovirus genus and designated O, novo-ulmi mitovirus (OnuMV ) 3a-Ld, 4-Ld, 5-Ld, and 6-Ld, respectively. Northern blot analysis indicat ed that O. novo-ulmi Ld nucleic acid extracts contain more single-stranded (ss, positive-stranded) RNA than dsRNA for all three newly described mitovi ruses. O. novo-ulmi RNA-7, previously believed to be a satellite-like RNA i s shown to be a defective RNA, derived from OnuMV4-Ld RNA by multiple inter nal deletions. OnuMV4-Ld is therefore the helper virus for the replication of both RNA-7 and another defective RNA, RNA-10. Sequence comparisons indic ate that RNA-10 could be derived from RNA-7, as previously suggested, or de rived directly from RNA-4. (C) 1999 Academic Press.