Cryphonectria hypovirus 3, a Virus Species in the Family Hypoviridae with a Single Open Reading Frame

Abstract

Isolate Grand Haven (GH) 2 is a naturally occurring isolate of the chestnut blight fungus, Cryphonectria parasitica, that is greatly reduced in virulence due to the presence of a double-stranded RNA virus. Unlike many other virus-infected, hypovirulent isolates, GH2 is not substantially reduced in pigmentation, conidiation, or laccase expression compared to its virus-free counterpart. The dsRNA genome of the GH2 virus was cloned, sequenced, and compared to hypovirulence-associated viruses of the family Hypoviridae. GH2 dsRNA is considerably smaller than previously characterized members of the family, 9.8 kb compared to 12.5-12.7 kb for other members. The genome organization of GH2 dsRNA reflected the substantial difference in genome size. Like other members of the family, one strand contained a poly(A)(+) tail at the 3' end and a long sequence with several minicistrons at the 5' end of the same strand. Only a single open reading frame (ORF) of 8622 nucleotides was predicted from deduced translations of the poly(A)(+)-containing strand, however. This contrasts with the two-ORF structures of previously characterized members. Analysis of the deduced ORF of GH2 dsRNA revealed putative proteinase, RNA polymerase, and helicase domains similar to those previously identified in confirmed members of the virus family Hypoviridae. GH2 dsRNA was more distantly related to Cryphonectria hypovirus (CHV) 1-EP713 and CHV2-NB58 than the latter two were to each other but has features in common with each of those viruses. We propose that the GH2 virus be included in this taxon as a member of the genus Hypovirus, representing a strain of a new species, CHV3.

Novel Predicted Peptidases with a Potential Role in the Ubiquitin Signaling Pathway

“…41 CHV3 encodes a single polyprotein containing a HC-Pro like protease, whereas CHV4 is similar in genome organization and size to CHV3 but does not have the HC-Pro-like protease. 42 Thus, the PPPDE peptidase detected in CHV3 and CHV4 is likely to be the main protease mediating polyprotein processing in the latter and an accessory protease (or, perhaps, a protease that cleaves host proteins) in the former. The presence of multiple proteases involved in polyprotein cleavage is characteristic of several other families of positive-strand RNA viruses.…”

Contribution of Protein p40 to Hypovirus-Mediated Modulation of Fungal Host Phenotype and Viral RNA Accumulation

“…Conidia were liberated from 1-month-old colonies with a glass rod in 15 ml of 0.15% Tween 80, filtered through three layers of Miracloth to remove mycelial fragments, and quantified with the aid of a hemacytometer. Laccase activity produced by virus-free and transfected fungal strains was measured by the method of Smart et al (30). Colonies were cultured for 1 week on Bavendamm's (4) solid media containing 0.5% tannic acid (pH 4.5), 1.5% malt extract, and 2% Bacto-agar (Difco).…”

“…Infection-related phenotypic changes can include reduced pigment production, suppressed asexual sporulation, altered colony morphology, loss of female fertility, and modified expression of specific host genes (1,12,(22)(23)(24)(25). Efforts to identify hypovirus symptom determinants have benefited from the complete nucleotide sequence determinations of four members of the family (7,18,28,30) and construction of full-length infectious cDNA clones for two family members: the prototypic severe hypovirus isolate CHV1-EP713 (9) and the mild isolate CHV1-Euro7 (7).Using the robust transformation protocol available for C. parasitica (10), Choi and Nuss (8) showed that the 5Ј-proximal CHV1-EP713 coding domain, open reading frame (ORF) A, when expressed in C. parasitica transformants in the absence of virus infection, caused a subset of phenotypic changes exhibited by CHV1-EP713-infected strains, e.g., a white phenotype (reduction in orange pigmentation), reduced asexual sporulation, and a slight reduction in the production of fungal laccase activity. ORF A encodes a 69-kDa polyprotein (p69) that is autocatalytically processed into p29 and p40 by the action of a papain-like cysteine protease domain located in the p29 coding region.…”

“…Infection-related phenotypic changes can include reduced pigment production, suppressed asexual sporulation, altered colony morphology, loss of female fertility, and modified expression of specific host genes (1,12,(22)(23)(24)(25). Efforts to identify hypovirus symptom determinants have benefited from the complete nucleotide sequence determinations of four members of the family (7,18,28,30) and construction of full-length infectious cDNA clones for two family members: the prototypic severe hypovirus isolate CHV1-EP713 (9) and the mild isolate CHV1-Euro7 (7).…”

“…The deduced p40 amino acid sequence is very basic (pK a ϭ 11.96), indicative of a possible role in viral RNA binding or replication. Moreover, this protein is conserved between two major hypovirus species, CHV1 and CHV2 (30). Thus, the observation that p40 is not required for CHV1-EP713 replication (32) was surprising.…”