Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

Abstract

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer. INTRODUCTIONTombusviruses are small isometric plant viruses belonging to the genus Tombusvirus, family Tombusviridae. Their genome is a monopartite, single-stranded, positive-sense RNA of 4800 nt containing five ORFs. ORF1 and -2 encode the replicase proteins of 33 or 36 kDa (p33 or p36), depending on the species, and the corresponding readthrough products, p92 and p95. ORF3 encodes the coat protein (CP), and two nested ORFs (ORF4 and -5) encode two proteins involved in virus movement and pathogenesis Silhavy et al., 2002). Tombusvirus infections are often associated with small parasitic RNAs, which can be either defective interfering (DI) or satellite (sat) RNAs. DI RNAs are shortened forms of genomic RNA deprived of all viral genes required for replication, encapsidation and movement of the viral genome. They are generated by errors of the viral replicase, which bypasses local intramolecular base-paired regions of genomic RNA, thus leading to the synthesis of deletion mutants. All described tombusvirus DI RNA molecules contain segments of viral RNA, derived from the 59 leader sequence, the replicase and movement protein genes and the 39 noncoding sequence. Tombusviruses can support the replication of homologous or heterologous DI RNA in plant (Havelda et al., 1998) or yeast cells (Pantaleo et al., 2003(Pantaleo et al., , 2004Panavas & Nagy, 2003). Tombusvirus DI RNAs have been widely used for the analysis of cis-acting factors directing the replication of viral RNA both in vivo Chang et al., 1995;Ray & White, 1999White & Morris, 1994;Wu & White, 1998;Wu et al., 2001) and in vitro (Nagy & Pogany, 2000; Panavas et al., 2002a, b;Panavas & Nagy, 2003).The molecular biology of tombusvirus sat RNAs has been much less studied. So far, the best characterized sat RNA is the 621 nt sat RNA of Cymbidium ringspot virus (CymRSV) whose cis-acting sequence requirements essential for replication and the significance and ...

Cymbidium ringspot virus defective interfering RNA replication in yeast cells occurs on endoplasmic reticulum-derived membranes in the absence of peroxisomes

“…These proteins were expressed either by two separate plasmids (lane 4) or by only one plasmid expressing wt p92 (lane 3), whereas no progeny RNA was detected when p92 (lane 1) or p33 (lane 2) were omitted. As in previous experiments (Pantaleo et al, 2004;Rubino et al, 2004;Navarro et al, 2006), the progeny DI RNA was composed of monomers and dimers (Fig. 7a, lanes 3 and 4).…”

“…Only cells expressing both p33 and p92 would be able to replicate DI-3/MS2, switching the localization of GFP from the nucleus to the site of DI RNA replication. Transformed yeasts were grown as described previously (Pantaleo et al, 2003;Rubino et al, 2004) and divided in two aliquots, from one of which RNA was extracted and the other was fixed for immunofluorescence analysis. Northern blot analysis of RNA extracts showed that mutant DI RNA molecules containing the MS2 recognition sequence in both positive (Fig.…”

Properties of a novel satellite RNA associated with tomato bushy stunt virus infections

“…The primary structure of satRNA L was more similar to that of CymRSV satRNA than TBSV B1 and B10 satRNAs, particularly in the 59-terminal 60 nt and in the 39-terminal 4 nt, where the triplet CCC was preceded by an A instead of a G. The presence of a G at position 24 is an absolute requirement for the replication of tombusvirus genomic and DI RNAs and it may represent the position of initiation of minus-strand Characteristics of a TBSV satellite RNA synthesis (Dalmay et al, 1993b;Havelda & Burgyán, 1995;Pantaleo et al, 2003). By analogy with experimental evidence gained for CymRSV satRNA, a similar replication strategy for satRNA L can be suggested, where the concurrent replication of genomic RNA is required rather than the sole expression of the replicase proteins, which is sufficient to replicate DI RNAs (Kollar & Burgyán, 1994;Rubino et al, 2004).…”

Satellite RNAs and Satellite Viruses of Plants

“…Rubino et al [77] demonstrated that co-expressing the replication proteins of Carnation Italian ringspot virus (CIRV) in yeast cells could support the replication of several defective interfering RNAs derived from the genome of CIRV or the related CymRSV but not that of a satRNA originally associated with CymRSV. Taken together, the results suggest that satRNAs have adopted a system similar to but distinct from their cognate helper viruses for their replication and thus are not strong competitors of their helper viruses.…”