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Short Communication |
1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninsky Gory 1, Laboratory Building A, Moscow 119992, Russia
2 Department of Virology, Moscow State University, Leninsky Gory 1, Laboratory Building A, Moscow 119992, Russia
Correspondence
J. G. Atabekov
atabekov{at}genebee.msu.su
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Only these two replicase proteins are essential for viral RNA replication (Ishikawa et al., 1991; Lewandowski & Dawson, 2000; Meshi et al., 1987). The 30 kDa movement protein (MP) and 17.4 kDa coat protein (CP) are expressed via individual 3'-co-terminal subgenomic (sg) RNAs (Deom et al., 1987; Hunter et al., 1976; Meshi et al., 1987). The dicistronic intermediate-length RNA-2 called sgRNA I2 is translated to produce the MP (Bruening et al., 1976; Higgins et al., 1976; Beachy & Zaitlin, 1977; Goelet & Karn, 1982), whereas the 3'-proximal CP gene of I2 RNA is translationally silent. This gene is expressed from a monocistronic sgRNA called low molecular component, LMC (Beachy & Zaitlin, 1977). The 75 nt leader sequence of TMV U1 sgRNA I2, called internal ribosome entry site (IRES)MP,75U1, promotes translation of the downstream ORF in dicistronic reporter constructs (Skulachev et al., 1999). A similar element called IRESMP,75CR has been detected in the RNA of crucifer-infecting tobamovirus (crTMV) (Dorokhov et al., 1993, 1994; Skulachev et al., 1999). The intracellular transport of a movement-deficient TMV U1-KK6 mutant (Lehto et al., 1990) lacking IRESMP,75U1 was largely restored by the insertion of IRESMP,75CR, which was apparently due to the translation-enhancing ability of IRESMP,75CR (Zvereva et al., 2004). Furthermore, the 148 nt IRES (IRESCP,148CR) is located upstream of the crTMV-CP gene (Ivanov et al., 1997).
Previously, we suggested that, concurrently with conventional TMV CP gene expression via cap-dependent translation of LMC, the IRESCP,148CR allows cap-independent translation of the CP gene from full-length genomic RNA and/or from RNA I2 of crTMV via an internal ribosome entry mechanism (Ivanov et al., 1997; Dorokhov et al., 2002). It has been shown that cap-independent translation activity mediated by IRESCP,148CR in cell-free systems from plant, animal and yeast cells was higher than that of a widely used IRES from encephalomyocarditis virus RNA (Dorokhov et al., 2002). Analysis of IRESCP,148CR sequence and structure revealed a bulged stem–loop (SL) structure flanked by two polypurine (A)-rich sequences (PARS), crucial for IRES activity (Dorokhov et al., 2002; Ivanov et al., 1997). Remarkably, the equivalent 148 nt sequence from TMV U1 RNA (U1CP,148SP) was incapable of mediating internal initiation of in vitro translation (Dorokhov et al., 2002; Ivanov et al., 1997).
Here, we have examined the contribution of IRESCP,148CR to CP production under conditions when the functional activity of the CP gene sg promoter (SGP) was abolished. Binary vectors containing a tobamovirus genome were delivered to plant cells by the agroinjection technique (Dorokhov et al., 2004), which is known to infect at least 94 % of the cells of injected leaves (Marillonnet et al., 2005). cDNA copies of TMV U1 and crTMV containing GFP fused with the N-terminal part of their CP genes (TMV U1-CP–GFP and crTMV-CP–GFP) were constructed (Fig. 1a and b, respectively). TMV U1-CP–GFP and crTMV-CP–GFP vectors contain viral cDNA that is fused to the transcription start site of the actin 2 promoter of Arabidopsis thaliana and the nos transcription terminator. Most of the CP gene was substituted by GFP, using the additional BamHI, ApaI and XbaI sites introduced into the CP sequence and in front of the 3'-non-translated region, respectively. The whole cassette was inserted into the binary vector pBin19 between KpnI and SalI (U1) or HindIII sites. To allow comparisons, the size of the remaining CP gene sequence (25 codons) was similar for both viral vectors. It should be noted that: (i) an enhancer element is located between nt +25 and +55 with respect to the TMV U1-CP translation start site (Man & Epel, 2004) and (ii) the crTMV-CP gene overlaps the MP gene by 75 nt (Dorokhov et al., 1994). In order to prevent CP–GFP synthesis in agrobacteria, we inserted a small synthetic intron into the GFP ORF.
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). Deletions of 39 or 59 nt caused unfolding of the stem in this putative structure. Activity of the sgRNA promoter decreased significantly when the length of base-paired sequence in the stem was shortened (Grdzelishvili et al., 2000). In order to inactivate TMV U1-CP–GFP-SGP, we inserted, by overlapping PCRs, 3 nt substitutions into the SL as indicated in the resulting construct TMV U1-CP–GFP/CP-mut (Fig. 1a
). Although there is low similarity (18 %) between the sequences of CP sgRNA promoters of different tobamoviruses, most of them could be folded into similar SL structures (Grdzelishvili et al., 2000). Our computer-predicted folding of the putative crTMV-CP-SGP also predicted an SL structure. We substituted 4 nt in the stem and, in addition, changed a putative start nucleotide of crTMV-CP–GFP sgRNA to obtain crTMV-CP–GFP/SL-mut (Fig. 1b).
TMV U1-CP–GFP, crTMV-CP–GFP and their CP-SGP mutants were agroinjected into four locations of the same Nicotiana benthamiana leaf (Fig. 1c). Efficient fluorescence could be detected under UV illumination in sites injected with TMV U1-CP–GFP and crTMV-CP–GFP. TMV U1-CP–GFP/SL-mut did not produce any fluorescence, whereas moderate but readily detectable fluorescence was observed after agroinjection with crTMV-CP–GFP/SL-mut. Approximately 5 µg total nucleic acid isolated from inoculated spots of the leaf was denatured, separated in 1.5 % agarose gels containing 10 % formaldehyde in MOPS buffer, pH 7.0 and transferred to a nylon membrane (Hybond-N+; Amersham). Membranes were incubated in a prehybridization solution containing 6x SSC, 0.5 % SDS, 5x Denhardt's reagent and 200 µg tRNA ml–1 for 2 h at 65 °C and probed with a denatured DNA fragment containing the GFP gene. Probes were labelled with [
32P]dATP (3000 Ci mmol–1) in a PCR. Fig. 2 shows that a large amount of CP–GFP sgRNA was present in crTMV-CP–GFP- (lanes 1 and 3) and TMV U1-CP–GFP- (lanes 5 and 7) injected zones. Only small amounts of genomic RNA were revealed by Northern blotting, which is apparently due to RNA degradation in the absence of the CP. In separate experiments, we showed that joint agroinjection of viral vectors and the CP gene led to a significant increase of genomic RNA accumulation (data not shown). This is consistent with the conclusion (Asurmendi et al., 2004) that the replication of genomic RNA is much more efficient in the presence of the CP.
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), suppressing genomic RNA accumulation. In our experiments with another CP-lacking crTMV-based vector, crTMV–GFP (Dorokhov et al., 2004), accumulation of genomic and sgRNA was low (Fig. 2, lane 10), but co-expression of potato virus Y Hc-Pro suppressor of RNA silencing drastically stimulated genomic RNA accumulation (Fig. 2, lane 11).
Importantly, the nucleotide substitutions in CP-SGP abolished the synthesis of CP–GFP sgRNA by crTMV-CP–GFP/SL-mut (Fig. 2, lanes 2 and 4) and TMV U1-CP–GFP/SL-mut (Fig. 2, lanes 6 and 8), indicating that SGPs were completely inactivated. In accordance with this observation, no CP–GFP production by TMV U1-CP–GFP/SL-mut vector virus could be detected by Western blot analysis (Fig. 3, lanes 4 and 6). It was particularly noteworthy that under the same conditions the CP–GFP fusion protein was detected in leaves agroinjected with crTMV-CP–GFP/SL-mut that contained IRESCP,148CR upstream of the CP gene. Fig. 3 shows that protein samples without dilution (lane 3) or after fivefold dilution (lane 5) displayed the CP–GFP band. Our calculations suggest that the level of CP–GFP synthesis in the leaves agroinjected with IRESCP,148CR-containing crTMV-CP–GFP/SL-mut reached a level of 2–4 % of control leaves injected with crTMV-CP–GFP. In controls (Fig. 3, lanes 1 and 2) CP–GFP production was abundant, corresponding to 1 and 2 g per 1 kg leaf material 10 days after injection with TMV U1-CP–GFP and crTMV-CP–GFP, respectively. Taking into account that the CP-SGP of crTMV-CP–GFP/SL-mut was inactivated, it is reasonable to suggest that the CP–GFP production by crTMV-CP–GFP/SL-mut was mediated by IRESCP,148CR. Experiments on in vitro translation proved that nucleotide substitutions in crTMV-CP-SGP did not affect the activity of IRESCP,148CR in expression of the 3'-proximal gene of the bicistronic transcript (data not shown).
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). It could be speculated that early in the viral replicative cycle production of the CP is needed for crucifer-infecting capacity of crTMV. The different strategies of crTMV and TMV U1 in the expression of CP gene might be explained by the location of ORF6, which is not present in the corresponding region of crTMV (Canto et al., 2004).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Received 3 April 2006;
accepted 5 May 2006.
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