Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
Correspondence
Richard Kormelink
richard.kormelink{at}wur.nl
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ABSTRACT |
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MAIN TEXT |
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More recently, an in vitro assay has been developed in which purified TSWV particles were shown to support either virus transcription or replication, depending on the presence or absence of rabbit reticulocyte lysate, respectively (Van Knippenberg et al., 2002; see also Fig. 1
A). In its presence, virus transcription was observed, as demonstrated by de novo synthesis of subgenomic RNA molecules (Fig. 1A
, lane 3) that hybridized to strand-specific probes for the N and NSs genes and co-migrated with these mRNAs as found in total RNA from infected plants (Van Knippenberg et al., 2002
). Moreover, evidence for genuine virus transcription initiation in vitro was obtained by RT-PCR cloning of these mRNAs, revealing the presence of non-templated leader sequences at the 5' ends. These RNA leader sequences were derived either from globin mRNAs present in the reticulocyte lysate or from exogenously added AMV RNA.
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As a first step, TSWV transcription in vitro (i.e. in the presence of rabbit reticulocyte lysate) was performed as described previously (Van Knippenberg et al., 2002) in the absence or presence of edeine and cycloheximide. In brief, 1015 µg purified virus was incubated for 1·5 h at 30 °C in 25 µl reactions containing 4 mM magnesium acetate, 1 mM each NTP, 0·1 % NP-40, 0·8 U RNasin µl1, 2·5 µl AP-Biotech translation mix and 10 µl AP-Biotech rabbit reticulocyte lysate. For visualization of the RNA products, 2 µl [
-32P]CTP (800 Ci mmol1) was added instead of CTP. No exogenous (AMV RNA) cap donor was added to the transcription assays, since the endogenous globin mRNAs present in the reticulocyte lysate have previously been shown to be used as cap donors (Van Knippenberg et al., 2002
). After extraction and precipitation, radiolabelled RNA products were resolved by electrophoresis on a 1·5 % agarose gel, followed by downward Northern blotting in 10x SSC for 1·5 h. The RNA profile on this Northern blot was analysed by autoradiography.
The translation inhibitor cycloheximide stalls the ribosomes on the transcript by blocking elongation (peptidyl transferase) (Godchaux et al., 1967; Jaye et al., 1982
). Edeine prevents association of the 60S ribosomal subunit to the 40S subunit but, unlike cycloheximide, still allows scanning of the nascent transcript by the 40S subunit (Kozak & Shatkin, 1978
). When either of these inhibitors was added to the TSWV in vitro transcription reaction, no change in the RNA product profile was observed (Fig. 1B
, lanes 3 and 6) compared with a profile of the standard reaction (Fig. 1B
, lane 2). Increasing concentrations of the inhibitors, even levels far exceeding those reportedly necessary for complete inhibition of protein synthesis (Bellocq et al., 1987
; Vialat & Bouloy, 1992
), did not affect TSWV transcription (Fig. 1B
, lanes 4, 5, 7 and 8). These findings indicated that virus transcription does not depend on reticulocyte lysate for translation of a viral protein, nor for ribosome scanning of the nascent transcript.
To verify that cycloheximide and edeine were indeed functional in blocking in vitro translation, protein synthesis from control RNA templates (supplied with the rabbit reticulocyte lysate kit and performed according to the manufacturer's recommendations) was analysed by 12 % SDS-PAGE. In addition, protein synthesis in in vitro TSWV transcription reactions was analysed by supplying 35S-labelled methionine to the assay, instead of 32P-labelled CTP. Strikingly, although the control RNA was properly translated (Fig. 2, lane 1), no detectable protein synthesis was observed in the 35S-labelled TSWV transcription reaction (Fig. 2
, lane 3). In the presence of translation inhibitors, translation of control RNA was already effectively blocked at the lowest concentration used (results not shown). These data indicated that cycloheximide and edeine were indeed fully effective in blocking translation.
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To substantiate further the evidence that translation inhibitors did not affect TSWV transcription in vitro, viral RNA synthesized in vitro in the presence of cycloheximide or edeine was analysed by RT-PCR using a viral N gene-specific primer in combination with a primer corresponding to the 5'-leader sequence of globin mRNAs. Using this approach, viral mRNAs can be specifically amplified and discriminated from (anti)genomic RNA molecules, as previously shown by Duijsings et al. (1999, 2001)
and Van Knippenberg et al. (2002)
. PCR fragments of expected sizes were obtained (Fig. 3
A, lanes 2 and 3) and subsequent cloning and sequence analyses (Fig. 3B
) confirmed their identity as N gene transcripts with globin mRNA leader sequences. These results indicated that TSWV transcription initiation was not affected by inhibition of translation.
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Recently, TSWV in vitro transcription was also demonstrated to require the addition of reticulocyte lysate (Van Knippenberg et al., 2002). The experiments described here have demonstrated that addition of translation inhibitors had no effect on either elongation or initiation of transcription. Moreover, whereas the in vitro transcription reactions of La Crosse and Germiston virus are really coupled transcriptiontranslation reactions, no visible protein synthesis could be detected for TSWV in vitro transcription. These results seem to indicate that the lysate-dependence of TSWV in vitro transcription is similar to that of Germiston and La Crosse viruses in that no actual viral protein synthesis is required, yet differs from it with respect to exactly which factor of the lysate confers stimulation of transcription.
The only other segmented ambisense RNA plant virus for which an in vitro transcription assay has been established is the tenuivirus rice hoja blanca virus (Nguyen et al., 1997). This virus, although a member of a genus sharing many characteristics with the Bunyaviridae, does not share the requirement for reticulocyte lysate for in vitro transcription. In the past, cellular factors (e.g. tubulin) have been demonstrated to be required for transcription of negative-strand RNA viruses such as Sendai virus, vesicular stomatitis virus, human parainfluenza virus type 3 and measles virus (Moyer et al., 1986
; Hill et al., 1986
; De et al., 1990
; Ray & Fujinami, 1987
). The identity of the factor required for TSWV transcription remains to be determined.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 October 2003;
accepted 7 January 2004.
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