1 Department of Virology, Moscow State University, Vorobiovy Gory Moscow 119899, Russia
2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobiovy Gory Moscow 119899, 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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Recently, we reported that TMV U1 and a crucifer-infecting tobamovirus (crTMV) contain internal ribosome entry sites (IRESs) upstream of the MP gene (IRESU1MP,75 and IRESCRMP,75, respectively) (Skulachev et al., 1999; Dorokhov et al., 2002
). It has been suggested that the IRESCRMP,75 sequence is functionally active when located at or within the 5'UTR of the I2 sgRNA, but not in the full-length genomic RNA context. This could be due to the extensive secondary structure and inaccessibility of this region to ribosomes in full-length tobamovirus RNA. It is still unclear whether the TMV I2 sgRNA is capped, although the primer extension data of Grdzelishvili et al. (2000)
presented indirect evidence for the capped nature of this RNA. Consequently, the question has arisen as to whether the 5'-proximal MP gene of the I2 sgRNA is translated by a ribosome-scanning mechanism or whether its 5'-terminal IRESMP,75 sequence mediates internal ribosome binding. It is possible that both mechanisms operate concurrently (Skulachev et al., 1999
). Our recent data show that the IRESCRMP,75 sequence acts as a translational enhancer, being located at the 5' position of monocistronic mRNAs (M. V. Skulachev and others, unpublished observations). The expression patterns of the MP and CP sgRNAs in TMV-infected cells are markedly different. The TMV MP is produced transiently, early in infection (Watanabe et al., 1984
; Lehto et al., 1990
), and the level of MP accumulation is relatively low (Ooshika et al., 1984
). By contrast, the CP gene is expressed at later stages of TMV replication (Siegel et al., 1978
). Little is known concerning the regulation of MP gene expression (for a review, see Lehto & Dawson, 1990
).
Lehto et al. (1990) constructed the movement-deficient TMV mutant (KK6) by insertion of an additional subgenomic promoter (SGP) (CP SGP-2 in Fig. 1
A) upstream of the MP gene. This promoter was capable of mediating I2 sgRNA synthesis with the 24 nt 5'UTR transcribed from the new transcription start site. Consequently, KK6-produced I2 sgRNA was lacking the IRESU1MP,75 sequence of wild-type (wt) TMV304 (Fig. 1A
). Remarkably, the KK6 mutant was movement deficient, which correlated with the fact that production of the MP in KK6-infected cells was delayed in comparison with wt strain TMV304. This feature of KK6 could be due to reduction of the MP-coding I2 sgRNA accumulation transcribed from CP SGP-2 or to reduction of this RNA translational efficiency caused by modification and shortening of its 5'UTR (Fig. 1A
).
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Plasmid KK6, which is a derivative of wt TMV304 (Fig. 1A), was kindly provided by K. Lehto (University of Turku, Finland). The TMV MP gene was amplified by PCR to introduce the NcoI site including the initiation codon at the 5' terminus of the MP gene. To insert the IRESCRMP,75 just upstream of the MP gene (Fig. 1A
), the KK6 plasmid vector was digested with XhoI/PstI and ligated together with three inserts: the IRESCRMP,75 (Skulachev et al., 1999
; XhoI/NcoI), the MP fragment (NcoI/HindIII) and the 3'-terminal part of the TMV genome (MPCP3'UTR; HindIII/PstI). Plasmid KS4 was used as a negative control and contained a non-physiological polylinker-derived nucleotide sequence (PL80) upstream of the MP gene referred to as a control sequence (CS; Fig. 1A
). The plasmid construction was verified by sequencing.
Comparison of the time-course of symptom development on Nicotiana tabacum cv. Samsun plants infected with TMV304, KK6 and K86 RNA transcripts showed that all the viruses induced systemic infection of tobacco plants, whereas KS4 infection was symptomless. However, systemic symptoms (yellow spots) produced by KK6 could not be detected earlier than 1517 days post-inoculation (p.i.), while systemic symptoms of K86 and wt TMV304 (yellow spots and mild mosaic) were induced 10 and 57 days p.i., respectively (data not shown).
Accumulation of genomic and sgRNAs in total RNA extracts from N. tabacum cv. Samsun plants inoculated with in vitro-produced T7 RNA transcripts of TMV304, KK6 and K86 was analysed by a Northern blot hybridization assay using a probe containing sequences of the CP gene, as described previously (Dorokhov et al., 2002). Only negligible amounts of viral RNA could be detected in KS4-inoculated leaves (Fig. 1B
), due to a very low level of infection produced by this mutant. Fig. 1(B)
shows that 7 days after inoculation, the wt TMV304 and K86 produced similar amounts of RNA, whereas the level of viral RNA production by the KK6 mutant was significantly reduced. This reduction reflected the delay in the spread of the KK6 mutant throughout the infected plant.
This suggestion was consistent with time-course Western blot analysis of CP (Fig. 2A) and MP (Fig. 2B
) accumulation in tobacco leaves inoculated with wt TMV304 and with the KK6 and K86 mutants. The amount of CP and MP produced by wt virus increased from 3 to 10 days p.i. As shown by Lehto et al. (1990)
, production of MP by KK6 was considerably delayed. Relatively low amounts of KK6 CP and MP could be detected only at 7 and 10 days p.i. The efficiency and time course of CP and MP production by the K86 mutant were intermediate between the wt virus and the movement-deficient KK6 (Fig. 2A and B
). This observation suggested that insertion of the IRESCRMP,75 element into the 5'UTR of the KK6 genome resulted in a significant restoration of K86 mutant cell-to-cell movement ability. To examine this effect more closely, we compared the size of the local lesions produced by the wt and the mutant viruses in non-transformed N. tabacum cv. Xanthi nc plants (Fig. 2C
) and in N. tabacum cv. Xanthi nc, line 2005, which is transgenic for the TMV MP gene (Fig. 2D
), kindly provided by R. Beachy (Deom et al., 1991
). Our data indicated that the size of local lesions produced by the K86 mutant in non-transgenic plants was intermediate between that of wt virus and the KK6 mutant of Lehto et al. (1990)
. It was particularly noteworthy that the difference in the size of lesion produced by all three viruses in MP transgenics was not significant: the minor differences were within the limits of standard error (Fig. 2D
). This indicated that the movement deficiency could either be restored partially by insertion of the IRESCRMP,75 element into the 5'UTR of the KK6 genome or restored completely by trans-complementation in MP-producing transgenics.
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It is reasonable to suggest that partial restoration of the K86 movement function was due to a peculiar feature of the IRESCRMP,75 sequence mentioned above, i.e. its ability to serve as a translational enhancer when inserted into the 5'UTR of mRNA (M. V. Skulachev and others, unpublished observations). To test this suggestion, in vivo model experiments were undertaken using agroinjection of N. benthamiana leaves with 35S RNA-based monocistronic cDNA constructs containing the U1, KK6, K86 and KS4 I2 sgRNA-derived leaders cloned upstream of the reporter green fluorescent protein (GFP) gene. The GFP gene contained a normal intron in its coding region (GFP-int) to prevent GFP production in Agrobacterium tumefaciens cells (Fig. 3A). The intron sequence was taken from the actin 2 gene of Arabidopsis thaliana (cv. Columbia). The natural intron was modified to get the optimal context for donoracceptor splicing sites and to remove some restriction sites. Specific primers were designed for both the intron and gene sequences. The 77 nt synthetic intron was inserted into the 5'-proximal region of the GFP gene using a PCR that started from the internal part of the gene and amplified the whole plasmid.
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Thus, the results of in vivo experiments showed that insertion of the IRESCRMP,75 sequence into the 5'UTR of KK6 I2 sgRNA resulted in significant translational enhancement of the reporter GFP gene expression in agroinjected leaves. Taken together, our data indicate that insertion of the IRESCRMP,75 sequence corresponding to the 5'-untranslated leader of I2 sgRNA into the movement-deficient TMV KK6 genome conferred an increased efficiency of MP production and cell-to-cell movement in the resulting K86 mutant. We suggest that the contribution of the IRESCRMP,75 sequence to TMV movement is due to its translation-enhancing function.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 November 2003;
accepted 19 December 2003.
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