Satellite and Defective RNAs of Cryphonectria hypovirus 3-Grand Haven 2, a Virus Species in the Family Hypoviridae with a Single Open Reading Frame

Abstract Cryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most recently characterized member of the Hypoviridae family of viruses associated with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 contain either three or four double-stranded (ds) RNAs that are visible on ethidium bromide-stained agarose or polyacrylamide gels. Only the largest dsRNA appears to be required for virus infectivity, and was characterized previously (C. D. Smart et al., 1999, Virology 265, 66–73). In this study, we report the cloning, sequencing, and analysis of the other three dsRNAs. Sizes of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 kb (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). All three accessory dsRNA species are polyadenylated on the 3′-end of one strand, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, with the 5′-terminal 1.4 kb derived from the 5′-end of dsRNA 1 and the 3′-terminal 2.2 kb from the 3′-end of dsRNA 1. A single major open reading frame (ORF) is evident from deduced translations of dsRNA 2. The deduced translation product is a 91-kDa protein that represents a fusion consisting of the entire N-terminal protease and the entire putative helicase domain. DsRNAs 3 and 4 represent satellite RNAs that share very little sequence with dsRNA 1 and 2. DsRNA 4 is 937 nucleotides, excluding the poly(A) + . The first AUG of the polyadenylated strand of dsRNA 4 occurs eight residues in from the 5′-terminus and would initiate the largest ORF on dsRNA 4, with the coding capacity for a 9.4-kDa protein. Within the deduced ORF and approximately 100 nucleotides from the 5′-end of dsRNA 4 is a 22-base sequence that is identical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant than dsRNA 4. DsRNA 3 was found to represent a head-to-tail dimer of dsRNA 4 linked by a poly(A)/(U) stretch of 40–70 residues.