The complete nucleotide sequence of the largest double‐stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3′‐poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496–498 and extending to nucleotide positions 11 859–11 861. The junction between ORF A and ORF B consisted of the sequence 5′‐UAAUG‐3′, where UAA served as the termination codon for ORF A and AUG was the 5′‐proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino‐terminal portion of the ORF B‐encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence‐associated dsRNAs.

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Computer-assisted analysis of the putative polypeptide products encoded by the two open reading frames present in a large virus-like double-stranded RNA, L-dsRNA, associated with hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, revealed five distinct domains with significant sequence similarity to previously described conserved domains within plant potyvirus-encoded polyproteins.These included the putative RNA-dependent RNA polymerase, RNA helicase, two papain-like cysteine proteases related to the potyvirus helper-component protease, and a cysteine-rich domain of unknown function similar to the N-terminal portion of the potyvirus helper-component protein. Phylogenetic trees derived from the alignment of the polymerase domains of L-dsRNA, a subset of positive-stranded RNA viruses, and double-stranded RNA viruses, using three independent algorithms, suggested that the hypovirulence-associated dsRNA and potyvirus genomes share a common ancestry. However, comparison of the organization of the conserved domains within the encoded polyproteins of the respective viruses indicated that the proposed subsequent evolution involved extensive genome rearrangement.The phenomenon of transmissible hypovirulence represents a natural form of biological control in which the virulence of Cryphonectria parasitica, the chestnut blight fungus, is modulated by the presence of virus-like genetic elements composed of double-stranded RNA (dsRNA) (reviewed in refs. 1 to 4). Efforts to understand the molecular mechanisms responsible for transmissible hypovirulence have provided an emerging view of the structural and functional properties of hypovirulence-associated dsRNA genetic elements (reviewed in ref. 5). The largest dsRNA present in C. parasitica strain Ep713, L-dsRNA (12,712 base pairs), was shown recently to contain two contiguous coding domains, designated open reading frame (ORF) A and ORF B, consisting of 622 and 3165 codons, respectively (6). Both ORFs encode polyproteins that undergo autocatalytic processing during or immediately after translation (6, 7). On the basis of the similarity of the L-dsRNA genetic organization and expression strategy to those of several viral genomes, it was suggested that L-dsRNA should be considered the equivalent of a viral genome or replicative form and the descriptive term hypovirulence-associated virus (HAV) was proposed (6).Similarities between one of the HAV-encoded proteases, p29, and the potyvirus-encoded protease, HC-Pro, were noted previously (7). In addition, computer-assisted analysis of the C-terminal portion of the ORF B-encoded polyprotein revealed a domain that was clearly related to the RNA helicase of potyviruses (6). We now extend these observations by demonstrating that three additional domains are conserved between the gene products of HAV and those of potyviruses, including the putative RNA polymerase. A detailed analysis of the sequence and organization of the conserved domains within the HAV-encoded and potyvirusencoded polyproteins suggests tha...

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The physicochemical interaction of pediocin PA-1 with target membranes was characterized using lipid vesicles made from the total lipids extracted from Listeria monocytogenes. Pediocin PA-1 caused the time-and concentration-dependent release of entrapped carboxyfluorescein (CF) from the vesicles. The pediocin-induced CF efflux rates were higher under acidic conditions than under neutral and alkaline conditions and were dependent on both pediocin and lipid concentrations. A binding isotherm constructed on the basis of the Langmuir isotherm gave an apparent binding constant of 1.4 ؋ 10 7 M ؊1 at pH 6.0. The imposition of a transmembrane potential (inside negative) increased the CF efflux rate by 88%. Pediocin PA-1 also permeablized synthetic vesicles composed only of phosphatidylcholine. Sequence alignments and secondary-structure predictions for the N terminus of pediocin PA-1 and other class IIa bacteriocins predicted that pediocin PA-1 contained two ␤-sheets maintained in a hairpin conformation stabilized by a disulfide bridge. The structural model also revealed patches of positively charged residues, consistent with the argument that electrostatic interactions play an important role in the binding of pediocin PA-1 to the lipid vesicles. This study demonstrates that pediocin PA-1 can function in the absence of a protein receptor and provides a structural model consistent with these results.