Satellite and defective RNAs of Cryphonectria hypovirus 3-Grand Haven 2, avirus species in the family Hypoviridae with a single open reading frame

Cryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most r ecently characterized member of the Hypoviridae family of viruses associate d with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 co ntain either three or four double-stranded (ds) RNAs that are visible on et hidium bromide-stained agarose or polyacrylamide gels. Only the largest dsR NA appears to be required for virus infectivity, and was characterized prev iously (C. D. Smart et al., 1999, Virology 265, 66-73). In this study, we r eport the cloning, sequencing, and analysis of the other three dsRNAs. Size s of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 k b (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). Al l three accessory dsRNA species are polyadenylated on the 9'-end of one str and, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, w ith the 5'-terminal 1.4 kb derived from the 5'-end of dsRNA I and the 3'-te rminal 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 transla tion 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 dsRN A 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 t he 5'-terminus and would initiate the largest ORF on dsRNA 4, with the codi ng 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 i dentical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant then 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, (C) 2000 Academic Pre ss.