Rapid de novo generation of defective interfering RNA by cucumber necrosis virus mutants that do not express the 20-kDa nonstructural protein.

Abstract

It is generally believed that serial passage at high multiplicity of infection (moi) is required for the generation of defective interfering (DI) particles. High levels of DI RNAs are found associated with persistent infections initiated with laboratory cultures of cucumber necrosis virus (CNV). Two synthetic CNV transcripts that were derived through site-directed mutagenesis of a highly infectious CNV cDNA clone and that do not express the CNV 20-kDa nonstructural protein were found to generate high levels of symptomattenuating DI RNAs de novo without serial high-moi passage in transcript-inoculated plants. Such de novo generation of DI RNAs did not occur in infections initiated with wild-type transcript until at least eight serial high-moi passages. The observation that a CNV nonstructural protein mutant rapidly generates DI RNA de novo may provide insight into mechanisms that underly DI particle formation in RNA viruses in general.Defective interfering (DI) particles are found in association with most animal viruses (1, 2) and appear in cell culture as a consequence of several serial passages of virus inoculum at high multiplicity of infection (moi). DI RNAs are deletion mutants of the helper virus, usually replicate at the expense ofthe helper virus in doubly infected cells, and require helper virus for their multiplication in host cells (for reviews see refs. 3-5). It has been suggested that DI particles may act as modulators of disease symptoms in animals; however, this has not been firmly established (6). In contrast to the ubiquity of DI particles in animal viruses, DI particles have only been well documented in two related groups of plant viruses, the tombusviruses and the carmoviruses (7-11). Recently, DI particles have also been found to be associated with two other plant virus groups, the geminiviruses (12) and the potexviruses (13). In the cherry strain of tomato bushy stunt virus (TBSV-Ch), a tombusvirus, DI RNAs were found to be colinear deletion mutants of genomic RNA that replicated to high levels in plants at the expense of virus accumulation. TBSV-Ch DI particles require the presence of the helper virus genome and are correlated with a dramatic attenuation of the severe necrosis typical of the helper virus alone in solanaceous hosts (7). In addition, TBSV-Ch DI RNA interferes with the replication of TBSV genomic RNA in protoplasts (14). More recently, it has been shown that TBSV-Ch DIs arise spontaneously upon serial high-moi passage in planta and that the de novo appearance ofDI RNAs is closely associated with attenuation of symptoms induced by the virus (15). De novo generation of DI RNA by synthetic RNA inocula derived from full-length cDNA clones of the carmovirus turnip crinkle virus (9) and a tombusvirus cymbidium ringspot virus (CyRSV) (16) has also been demonstrated.Cucumber necrosis virus (CNV), a biologically distinct tombusvirus (17), is an isometric virus containing a monopartite, positive-polarity RNA genome. The sequence of the CNV genome predicts the synthesis of five di...

Sequence and structure of defective interfering RNAs associated with cucumber necrosis virus infections

“…1 b). This pattern of retained CNV genomic sequences has been observed in CNV DI RNAs recently sequenced in another laboratory (Y.-C. Chang, T. J. Morris & A. Jackson, personal communication) and those generated from the 20K mutant of CNV (Rochon, 1991) as well as DI RNAs from the related tombusviruses TBSV-Ch (Hillman et aL, 1987;Knorr et al, 1991) and CyRSV (Burgyan et al, 1989(Burgyan et al, , 1991Rubino et al, 1990). At their 5' ends, all clones maintained sequences up to and immediately upstream of the CNV 33K start codon and seven of the nine clones retained the actual start codon.…”

“…In addition, plants infected with CNV-Lc displayed attenuated symptoms in comparison with plants inoculated with synthetic wild-type (wt) CNV transcripts (Johnston & Rochon, 1990). These observations taken in conjunction with the fact that synthetic CNV mutants that do not express the 20K protein rapidly generate DI RNAs de novo (Rochon, 1991) suggested that the 400 nucleotide RNAs associated with CNV-Lc infection may be DI RNAs and further that the wt CNV genome, like the 20K mutant genome, is capable of generating DI RNAs de novo. In this paper we demonstrate that the 400 nucleotide RNAs are in fact DI RNAs and are likely to be responsible for the symptom attenuation associated with CNV-Lc.…”

“…The 21K and 20K proteins are expressed from extensively overlapping but distinct ORFs on a single, bifunctional 0"9 kb subgenomic mRNA (Rochon & Johnston, 1991). The functions of these two proteins are not known, but the 20K protein is suspected to play a role in CNV RNA replication based on the observation that a CNV mutant that does not express this protein rapidly generates defective interfering (DI) RNAs (Rochon, 1991).…”

“…DI RNAs generated de novo from wt transcripts DI RNAs from CNV-Lc (Burgyan et al, 1991), TCV (Li et al, 1989) and CNV mutants lacking expression of the 20K protein (Rochon, 1991). It was previously reported that RNA extracts of plants infected with a laboratory culture of CNV (CNV-Lc) contained high levels of a low M r (approximately 400 nucleotides) virus-related RNA species.…”

“…Serial high m.o.i. passages were carried out as described previously (Rochon, 1991). After 14 high m.o.i, passages, large amounts of low M r RNAs were detected in two out of six plants examined.…”