The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica.

Abstract

Hypovirulent strain EP713 of the chestnut blight fungus Cryphonectria (Endothia) parasitica harbors a family of viral encoded double‐stranded (ds) RNAs thought to be responsible for the hypovirulence phenotype. These include L‐dsRNA, described in the accompanying paper (Shapira et al., 1991); several prominent species in the estimated size range of 8 to 10 kb, referred to here as M‐dsRNAs; and several smaller species designated S‐dsRNAs which range in size from approximately 0.6 to 1.7 kb. The characterization of the M‐ and S‐dsRNA species is the subject of this report. Results from polymerase chain reaction mapping and molecular hybridization analysis indicate that the M‐ and S‐dsRNA species are internally deleted forms of L‐dsRNA. Three different S‐dsRNA species were cloned and sequenced. Each species contained a single deletion breakpoint and retained either 149, 155 or 156 bp of the terminus corresponding to the 5′‐end of the coding strand and 440, 447 or 449 bp of the other terminus. Two of the S‐dsRNA species contained, within the boundaries of the breakpoint, additional sequence information consisting of 42 bp or 95 bp that appeared to be unrelated to the L‐dsRNA sequence. These results demonstrate that defective RNAs contribute significantly to the complexity of dsRNA populations found in hypovirulent strains of C. parasitica and provide a first approximation of the location of cis‐acting signals involved in their replication.

A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus

“…1A) decreased the level of substrate for vsRNA biogenesis from the regions deleted in the DI RNA. This prediction was based on our previous report that the 8-to 10-kb DI RNA species associated with CHV1-EP713 infection retained Ϸ3.5 kb of each terminus and thus would lack all or portions of the regions of the viral genomic RNA that were underrepresented in the cloned vsRNA population (11). Extensive polymerase chain reaction (PCR) amplification and nucleotide sequence analysis was used to further define the structure of CHV1-EP713-associated DI RNA species.…”

“…Rapid recombination also appears to be an underlying cause of the instability and deletion of foreign, nonviral sequences from recombinant viral RNA vectors (6; reviewed in 7). Genome instability presents one of the most important obstacles to the use of recombinant RNA viruses as gene expression vectors for various practical applications, including gene therapy (8-10).Single-strand, positive sense mycoviruses in the family Hypoviridae, hypoviruses that persistently infect and attenuate virulence of the chestnut blight fungus, Cryphonectria parastica, generate internally deleted DI RNAs at a very high frequency (11,12). Efforts to use recombinant hypoviruses to express foreign genes also have encountered the limitation of instable nonviral nucleotide sequences (13).…”

“…Single-strand, positive sense mycoviruses in the family Hypoviridae, hypoviruses that persistently infect and attenuate virulence of the chestnut blight fungus, Cryphonectria parastica, generate internally deleted DI RNAs at a very high frequency (11,12). Efforts to use recombinant hypoviruses to express foreign genes also have encountered the limitation of instable nonviral nucleotide sequences (13).…”

Hypovirus Papain-Like Protease p29 Functions in trans To Enhance Viral Double-Stranded RNA Accumulation and Vertical Transmission

“…As observed for ⌬p29-infected colonies, pigment production was significantly increased in ⌬p40-infected fungal strains relative to CHV1-EP713-infected strains (44). The activity domain of p40 responsible for efficient viral replication was mapped to the N-terminal domain Thr 40 -Arg 64 by a gain-of-function analysis with p40 deletion mutant viruses. Moreover, restoration of symptoms directly correlated with increased accumulation of viral RNA.…”

ICTV Virus Taxonomy Profile: Hypoviridae

“…2), plays a critical role [4]. Many hypoviruses have shorter-than-fulllength, internally-deleted, defective interfering and defective replicative form dsRNA molecules; others have replicative forms of satellite-like RNAs [5,6]. The host RNA silencing pathway has been reported to promote defective interfering RNA production [7].…”