Institut für Virologie der Universität zu Köln, Fürst-Pückler-Str. 56, 50935 Köln, Germany1
Author for correspondence: Marcus Klein. Fax +49 221 4783902. e-mail marcus.klein{at}medizin.uni-koeln.de
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Abstract |
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As part of the replication complex (Bienz et al., 1992 ), the 329-amino-acid protein 2C is involved in viral RNA replication. This was proven by the finding, among others, that relevant mutations for both resistance to or dependence on the picornaviral RNA replication inhibitors 2-(
-hydroxybenzyl)-benzimidazole (HBB) and guanidine map within its coding region (Hadaschik et al., 1999
; Klein et al., 2000
; Pincus & Wimmer, 1986
; Tolskaya et al., 1994
). Subsequently, sequential and functional analysis of 2C revealed the presence of three highly conserved NTP-binding subdomains (Gorbalenya et al., 1990
; Klein et al., 1999
; Mirzayan & Wimmer, 1992
). That 2C can hydrolyse ATP and GTP has already been proven (Klein et al., 1999
; Mirzayan & Wimmer, 1994
; Rodriguez & Carrasco, 1993
). In addition, 2C is considered to be a putative RNA helicase (Gorbalenya et al., 1988
; Kadaré & Haenni, 1997
), but the experimental evidence for this activity is still missing.
One further function of 2C has been reported for poliovirus: the ability to bind RNA, first demonstrated by Rodriguez & Carrasco (1993) . Experiments with truncated 2C revealed that two regions (aa 2145 and 312319) are involved in RNA binding (Rodriguez & Carrasco, 1995
). Recently, specific binding of poliovirus 2C to a distinct part of the negative-stranded 5'-nontranslated region (ntr) was reported (Banerjee et al., 1997
).
The aim of this study was to identify and characterize a putative RNA-binding function of 2C protein of echovirus-9 strain Barty. The protein was expressed in E. coli and purified as a histidine-tagged (His)-protein, according to earlier work (Klein et al., 2000 ). In order to control for induction of the protein, an SDS10% polyacrylamide gel was run with an equal amount of induced and uninduced bacteria (Fig. 1a
). Inclusive of the 6x His-tag and a short linker sequence of the expression vector, a total molecular mass of 40·9 kDa was calculated for His-2C. One clear and sharp protein band of the expected size was visible, confirming proper expression of full-length His-2C (Fig. 1a
, lane 2). Since we preferred to work with native and undenatured protein, His-2C was purified from the supernatant fraction of the lysed bacteria by Ni2+ affinity chromatography according to the manufacturers protocol (Qiagen). After step-wise washing and elution, His-2C-containing fractions were analysed by SDS12% PAGE. Most of the His-protein was eluted at 60 mM imidazole, possibly due to the His-tag not being completely exposed at the N terminus of 2C (Fig. 1b
, lane 3). To ensure the use of highly purified His-2C protein, only fractions eluted with 100, 150 and 200 mM imidazole were pooled (Fig. 1b
, lanes 57), concentrated with spin columns (Amicon), and dialysed against 20 mM HEPESKOH (pH 7·5), 100 mM KCl, 5 mM DTT and 25% glycerol.
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The binding reaction was performed with 50000 c.p.m. labelled RNA and His-2C of high purity in 10 mM HEPESKOH (pH 7·5), 50 mM potassium acetate, 3 mM magnesium acetate, 10 mM DTT, 2·5 mM ATP, 0·02% BSA, 0·5% Tween 20 and 5% glycerol. After incubation for 15 min at 37 °C, the reactions were subjected to a 3·5% electrophoretic mobility shift assay (EMSA) in 0·25x Trisborate buffer at 4 °C. The dried gels were autoradiographed or analysed by phosphorimager.
It was shown for the first time that echovirus-9 protein 2C binds to RNA (Fig. 2): 20 ng of 2C protein led to noticeable complex formation and 100 ng (approx. 200 nM) completely retained the inserted labelled RNA (Fig. 2a
). As a negative control, the same amounts of another viral His-tagged protein (human papillomavirus type 8 E2) were used, and no binding was observed (data not shown). However, the RNAHis-2C complex did not appear as an individual band with reduced mobility; instead, the emerging complexes failed to enter the gel, suggesting a very high molar ratio of protein to RNA (Fig. 2
). It has been reported that picornavirus 2C tends to oligo- or multimerize (Mirzayan & Wimmer, 1994
; Tolskaya et al., 1994
). We suggest that this effect probably strongly pertaining to echovirus-9 may be the reason for the abolished mobility of the RNA2C complexes. Similar findings have been reported for the P30 protein of tobacco mosaic virus, which binds to linear nucleic acids only if a minimum number of nucleotides is exceeded (Citovsky et al., 1992
).
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Although the influence of RNA length was not examined explicitly, a substantial criterion for binding by 2C seems to be the secondary structure of the target RNA. Obviously, the echovirus 2C protein ignores strongly folded RNA. Using up to 1 µg of tRNA, successful competition of the 2C/linear RNA interaction was not detectable (Fig. 2b). This is in accordance with further binding experiments performed using as RNA target the first 119 nucleotides of the 5'-ntr of echovirus-9 in both positive- and negative-stranded orientation, which are supposed to build up complex secondary structures. In these experiments, almost no binding of His-2C to this kind of RNA was detectable (Fig. 3
).
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For the first time, the interaction with RNA of the highly conserved echovirus 2C protein has been characterized at the molecular level. The finding that this obviously multifunctional protein binds preferably to single-stranded RNA fits well with current opinion concerning participation of 2C in RNA replication and/or RNA encapsidation.
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Acknowledgments |
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References |
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Received 28 April 2000;
accepted 21 June 2000.