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

Abstract

Defective interfering (DI) RNAs, helper virus-dependent deletion mutant RNAs derived from the parental viral genomic RNA during replication, have been described for most RNA virus taxonomic groups. We now report that DI RNA production in the chestnut blight fungus, Cryphonectria parasitica, persistently infected by virulence-attenuating positive sense RNA hypoviruses, depends on one of two host dicer genes, dcl-2. We further report that nonviral sequences that are rapidly deleted from recombinant hypovirus RNA virus vectors in wild-type and dicer gene dcl-1 deletion mutant strains are stably maintained and expressed in the ⌬dcl-2 mutant strain. These results establish a requirement for dcl-2, the C. parasitica dicer gene responsible for antiviral defense and generation of virus-derived small interfering RNAs, in DI RNA production and recombinant virus vector RNA instability.defective interfering RNA ͉ hypovirus ͉ RNA silencing ͉ RNA recombination V irus RNA recombination is an important component of virus evolution that contributes to the emergence of new viruses (reviewed in 1) and the generation of internally deleted mutant RNAs, termed defective interfering (DI) RNAs, that are derived from, and are dependent on, the parental viral genomic RNA (2). The presence of DI RNAs can suppress parental virus RNA accumulation, leading to attenuation of symptoms (3) and persistent virus infections (4, 5). 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).We recently reported that disruption of one of two C. parasitica dicer genes, dcl-2, results in increased susceptibility to mycovirus infection (14). We subsequently showed that dcl-2 functions to process mycovirus RNAs into virus-derived small interfering RNAs (vsRNAs) as part of an inducible RNA silencing antiviral response (15). Cloning and characterization of vsRNAs generated from hypovirus CHV1-EP713 revealed a nonrandom distribution of vsRNAs along the 12.7-kb genome RNA. Conspicuous was the very low representation of vsRNA mapping to an internal portion of the 12.7-kb genome RNA that encodes the viral polymerase and helicase domains, from map position 7500 to 11000 (15). It has been postulated that the propensity of hypoviruses to generate internally deleted DI RNAs results in a lower level of substrate for vsRNA biogenesis from this region (15). Here ...

A Novel Victorivirus from a Phytopathogenic Fungus, Rosellinia necatrix, Is Infectious as Particles and Targeted by RNA Silencing

“…Nuss and coworkers demonstrated that the RNA silencing components DCL-2 and AGL-2 are required for the generation of CHV1 DI-RNA species (27,28), and our previous study showed that the existing DI-RNA in EP155 rarely moved to the ⌬dcl-2 recipient side along with helper CHV1 in the lateral transmission assay (17). In the present study, we confirmed this with a virus mutant, CHV1-⌬p69, during RnVV1 coinfection assay in which DI-RNAs appeared only in the EP155 host backgrounds (see the asterisked DI-RNAs in Fig.…”

“…The RNA silencing pathway is able to suppress replication of hypoviruses, mycoreoviruses (26,27), and also a heterologous partitivi-rus (family Partitiviridae) (17). Furthermore, such RNA silencing is required for the generation and maintenance of defective interfering (DI)-RNAs of the prototype hypovirus, Cryphonectria hypovirus 1/EP713 (CHV1-EP713) (27,28).…”

Effects of Defective Interfering RNA on Symptom Induction by, and Replication of, a Novel Partitivirus from a Phytopathogenic Fungus, Rosellinia necatrix

“…As another model, DI-RNAs efficiently trigger, but are not targeted by, RNA silencing that degrades helper viral mRNAs as an antiviral defense mechanism in plants, leading to symptom attenuation (4,5). Another interesting link between RNA silencing and DI-RNAs has been noted by Nuss and coworkers (6): RNA silencing is associated with the generation of DI-RNA in fungal cells infected with a hypovirus with a (ϩ) singlestranded RNA (ssRNA) genome. They hypothesized that hypovirus RNAs fragmented by the host RNA silencing pathway increase the rate of, and provide substrate for, template switching to produce DI-RNAs.…”

“…In the present study, applying a transfection technique with purified virus particles that had recently been developed for a partitivirus (22), we were able to obtain a DI-free strain of RnPV2 in an RNA silencing-defective mutant of a heterologous fungus, C. parasitica (6). We report here the molecular and biological properties of RnPV2, focusing on the effects of DI-dsRNA on replication and symptom induction using both natural and experimental host fungi.…”

A single Argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination

“…A similar induction and repression of Dicer gene expression has not yet been reported for infected plants or animals. A surprising role for C. parasitica DCL2 in viral RNA recombination, the production of internally deleted, defective interfering (DI) RNAs and the instability of foreign gene sequences in recombinant RNA virus vectors, was recently demonstrated (17).…”