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Animal: RNA Viruses |
Istituto di Ricerche di Biologia Molecolare ‘P. Angeletti’, via Pontina Km 30600, 00040-Pomezia (Roma), Italy1
Author for correspondence: Licia Tomei. Fax +39 06 91093225. e-mail Tomei{at}IRBM.it
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Introduction |
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; Kuo et al., 1989 ). Chronic infection with HCV has also been linked to the development of liver cirrhosis and hepatocellular carcinoma (Chien et al., 1992 ). It has been estimated that about 1% of the world’s population is affected by the disease (Alter, 1995 ) but, so far, no efficient therapy and no vaccine exist.
The RNA-dependent RNA polymerase (RdRp) activity of HCV is provided by the viral protein NS5B (Behrens et al., 1996 ), located at the extreme C terminus of the HCV polyprotein. Generation of the mature RdRp relies on the activity of the viral NS3/NS4A serine protease complex (reviewed by De Francesco et al., 1998 ; Kwong et al., 1998 ).
Full-length NS5B has been purified previously as a non-fusion protein from insect cells infected with a recombinant baculovirus (Behrens et al., 1996 ; De Francesco et al., 1996 ) or as a tagged protein from both insect cells (Lohmann et al., 1997 ) and E. coli (Yuan et al., 1997 ). In vitro, the RdRp activity of recombinant NS5B is dependent on an RNA template and requires RNA or DNA as a primer (Behrens et al., 1996 ; Lohmann et al., 1997 ). On RNA templates of heteropolymeric sequence, the 3'-OH of the template is used as a primer and elongation proceeds via a ‘snap-back’ mechanism, thus leading to a double-stranded molecule in which template and product RNA are covalently linked (Behrens et al., 1996 ). The same mechanism has been shown to be used by purified NS5B for the transcription of the entire HCV genome in vitro (Lohmann et al., 1998 ). Despite the fact that purified NS5B can use HCV RNA as a template, no specificity has been observed for RNA templates containing HCV-derived sequences (Behrens et al., 1996 ; Lohmann et al., 1997 ). On the basis of analogy with other (+)-strand RNA viruses, it is generally assumed that NS5B corresponds to the elongation factor that, in association with other viral and/or cellular proteins, is part of the virus replication complex (Lai, 1998 ). HCV replication complexes are thought to assemble on intracellular membranes. This hypothesis is supported by the observation that all HCV non-structural proteins are membrane-associated when expressed in heterologous cell systems (Tanji et al., 1995 ). NS5B has been described to be membrane-associated and localized in the perinuclear region (Hwang et al., 1997 ). Recently, a potential NS5B membrane-anchoring domain has been identified tentatively at the C terminus of the NS5B protein. The C-terminal 21 residues correspond to a very hydrophobic sequence, the deletion of which, in a GST-fused NS5B, prevented perinuclear localization of the protein (Yamashita et al., 1998 ).
Purified full-length NS5B has very poor catalytic activity in vitro compared with the poliovirus RdRp 3Dpol (Lohmann et al., 1998 ; L. Tomei & S. Altamura, unpublished observations) or other well studied RNA- or DNA-dependent polymerases. We thought that this might not be an intrinsic property of HCV RdRp but, instead, could indicate that expression of full-length NS5B in heterologous systems does not allow authentic folding of the protein. We reasoned that the presence of the C-terminal hydrophobic domain might disturb protein folding or promote aggregation when expressed in the absence of membranes or extracted by the use of detergents. This is in line with recently published observations indicating that deletion of the 21 C-terminal residues does not interfere with NS5B activity (Lohmann et al., 1997 ) but increases the solubility of the protein expressed in E. coli (Yamashita et al., 1998 ; Ferrari et al., 1999 ).
Here, we describe the biochemical properties of a truncated NS5B lacking the C-terminal 21 residues (NS5B-
C21) expressed in E. coli. We decided to rely on a non-fusion protein in order to obtain an enzyme as close as possible to the native form. The biochemical properties of the deleted NS5B protein have been compared with those of the full-length enzyme (NS5B-FL) obtained with the baculovirus expression system. We show that, besides improving solubility, deletion of the C-terminal hydrophobic tail also confers enhanced catalytic efficiency on NS5B.
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Methods |
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Recombinant plasmids. ). pT7NS5B-FL contains a cDNA fragment encoding amino acids 2420–3010 of the HCV-BK polyprotein cloned between the NdeI and SmaI sites of the pT7-7 expression vector. pT7NS5B-C21 contains a 3'-terminal deletion of 63 bp. pGEMDCoh-NC contains the DraI–MscI fragment from pT7DCoH inserted into the SmaI site of pGEM-1 vector (Promega).
Expression and purification of the NS5B proteins.
Construction of a recombinant baculovirus carrying the full-length NS5B sequence and infection of Sf9 cells have already been described (Behrens et al., 1996 ). Purification of the protein was carried out at 4 °C, essentially as reported previously (Behrens et al., 1996 ; De Francesco et al., 1996 ) with the following modifications. The peak fractions from the heparin–Sepharose chromatography were applied to a Resource-S column and eluted with a 0·15–0·8 M NaCl gradient in sodium phosphate buffer, pH 8·0. The NS5B fractions were diluted to 0·15 M NaCl, loaded on a poly(U)–Sepharose column and eluted with a 0·15–0·85 M NaCl gradient in a buffer containing 20 mM Tris–HCl (pH 8·0) instead of sodium phosphate.
Expression in bacteria of NS5B-FL and the C21 mutant was obtained by transforming the pT7-7 derivatives into E. coli BL21 (DE3) (Studier et al., 1990 ). Bacteria were grown at 37 °C in standard LB medium up to an OD600 of 0·8. The temperature of the culture was then lowered to 18 °C and expression was induced with 0·4 mM IPTG for 23 h. NS5B-FL was purified as described for the protein expressed in Sf9 cells, using Triton X-100 as the detergent. For NS5B-C21, Triton X-100 was replaced by n-octyl 
-D-glucopyranoside (NOG) in both the lysis buffer (20 mM HEPES–NaOH, pH 8·0, 1 mM EDTA, 0·5 M NaCl, 50% glycerol, 10 mM DTT, 0·2% NOG, Complete protease inhibitor cocktail) and the chromatographic buffer (20 mM HEPES–NaOH, pH 8·0, 1 mM EDTA, 20% glycerol, 3 mM DTT, 0·2% NOG). The purification steps were essentially those followed for NS5B-FL with some modifications. Briefly, the flow-through of DEAE-Sepharose FF was loaded on a heparin HyperD column (BioSepra) and eluted with a 0·3–0·85 M NaCl gradient. The NS5B-C21 fractions were brought to 0·15 M NaCl and loaded on a Resource-S column and eluted with a 0·15–0·8 M NaCl gradient. The protein was further purified on a HiLoad 26/60 Superdex 75 column equilibrated at 0·5 M NaCl. Pure NS5B-C21 was concentrated on an HR 5/5 Mono-S column equilibrated at 0·15 M NaCl and eluted at 0·8 M NaCl.
Polymerase assay.
Reactions were carried out in 20 µl buffer containing 20 mM Tris–HCl (pH 7·5), 0·05% Triton X-100, 2% glycerol, 50 mM NaCl, 1 mM DTT, 0·1 µg/µl BSA, 0·25 units/µl RNasin, 5 mM MgCl2 and 40 nM purified NS5B. Poly(rA)–oligo(rU)18 and poly(rC)–oligo(G)18 template–primer couples were present at a final concentration of 60 µM AMP–0·5 µM oligo(rU)18 and 12 µM CMP–0·12 µM oligo(rG)18, respectively. The poly(rA)–oligo(rU)18 and poly(rC)–oligo(rG)18 concentrations reported in the figure legends refer to the primer concentration. UTP and 0·1 µCi [3H]UTP (Amersham, 47 Ci/mmol) per µM cold UTP or GTP and 0·03 µCi [
-32P]GTP (NEN Dupont, 3000 Ci/mmol) per µM cold GTP were added at the concentrations specified below. Heteropolymeric RNA templates were used at a final concentration of 40 nM and 100 µM ATP, CTP and UTP, 1 µM GTP and 2 µCi [-32P]GTP were present.
The NS5B protein was preincubated with template RNA in a 15 µl volume in which all the components except NTPs were present at 1·33 times the final concentration. After 20 min incubation at 23 °C, the reaction was started by the addition of 5 µl NTP mixture and incubated at 37 °C for the time specified. The activity was measured as the radioactivity present in the acid-insoluble material.
Km for nucleotides and kcat values were determined from non-linear least-squares fits of the Michaelis–Menten equation to the experimental data. The assays were performed under initial rate conditions as described.
Heteropolymeric RNA templates were prepared as described previously (Behrens et al., 1996 ). For product analysis under single processive cycle synthesis, the elongation reaction was started by the addition of cold and -32P-labelled nucleotides and 50 ng/µl heparin. With poly(rA)–oligo(rU)18 template–primer, 2·5 µl of the reaction mixture was withdrawn at successive time-points, diluted with 5 µl 95% formamide–20 mM EDTA and loaded on a 6% acrylamide–urea sequencing gel. With heteropolymeric RNA templates, 20 µl aliquots were withdrawn at successive time-points and the reaction was stopped by the addition of an equal volume of 2x proteinase K (PK) buffer (150 mM Tris–HCl, pH 8·0, 50 mM EDTA, 2% SDS). Samples were incubated for 30 min at 30 °C with 20 µg PK. Next, 100 µl 4 M guanidium isothiocyanate was added and the RNA was precipitated with 500 µl isopropanol and 10 µg carrier tRNA. Samples were analysed on 5% acrylamide–7 M urea gels.
Gel-retardation experiments.
Binding reactions were carried out in 20 µl with the buffer used for the polymerase assay with the amount of purified NS5B specified in the figure. The enzyme was incubated with 20000 Cerenkov counts of labelled RNA probe (D-RNA-NC, 180 nt) for 15 min at 23 °C. At the end of the incubation, 5 µl 20% Ficoll was added and samples were analysed by 6% PAGE with 0·25x TBE.
Labelled D-RNA-NC probe was obtained by using T7 polymerase with the linearized plasmid pGEMDCoh-NC as the DNA template and the MEGAscript T7 kit (Ambion).
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Results |
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C21 mutant ; De Francesco et al., 1996 ) with some minor modifications (see Methods; Fig. 1). As already reported, solubilization of the protein in the total cell extract required high salt and glycerol concentrations and the presence of 2% Triton X-100 in the lysis buffer. In this way, more than 90% of the NS5B protein was recovered in the soluble fraction (Table 1).
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), as judged by Coomassie blue-stained SDS–PAGE (not shown). High glycerol and salt concentrations, together with 2% Triton X-100, were needed to bring NS5B-FL into solution. Moreover, the protein eluted from all the columns used as very broad peaks that did not allow sufficient purification and also determined that the yield of purified protein was rather modest (Table 1). In addition, its activity was lower than that of the NS5B-FL enzyme purified from Sf9 cells, suggesting that a large part of the protein was not functional (not shown).
The C-terminal 21 residues of NS5B correspond to a very hydrophobic region of the protein. It has been reported recently that their deletion increases the solubility of tagged NS5B expressed in E. coli (Yamashita et al., 1998 ; Ferrari et al., 1999 ). Therefore, we tried to produce an NS5B mutant lacking the C-terminal 21 residues (NS5B-C21) as a non-fusion protein in E. coli. As for NS5B-FL expression, we found that performing the IPTG induction at 18 °C and disrupting the cells in a buffer containing high salt and glycerol concentrations maximized the recovery of soluble protein. In this way, more than 90% of the NS5B-C21 protein was present in the soluble fraction (Table 1 and Fig. 1). Purification was performed by following essentially the same scheme used for NS5B-FL, except that the poly(U)–Sepharose affinity column was replaced by a gel filtration step. The purified protein was homogeneous, as judged by Coomassie blue-stained SDS–PAGE (Fig. 1), and about 6 mg pure protein was obtained per litre of bacterial culture (Table 1). Although detergents could be omitted during extraction and purification, they were important in avoiding precipitation of the pure protein during long-term storage. For the same reason, glycerol must be maintained at 5–10% and the salt concentration must be higher than 0·15 M.
Activity of NS5B-FL and NS5B-C21 on homopolymeric and heteropolymeric RNA templates
A steady-state kinetic analysis of the two forms of the enzyme was performed in order to compare their activities on different RNA templates. On homopolymeric RNA templates, the activity of the NS5B RdRp has been shown to be strictly primer-dependent (Behrens et al., 1996 ; Lohmann et al., 1997 ). Both DNA and RNA oligonucleotides are accepted, but the highest activity is obtained if RNA primers are annealed to the homopolymeric templates. First, we decided to use poly(rA)–oligo(rU)18 and poly(rC)–oligo(rG)18 couples to compare the NS5B-FL and NS5B-C21 enzymes. The assay conditions used were found to be optimal for both enzyme forms in terms of pH and NaCl and Mg2+ ion concentrations. Mn2+ at 0·5 mM could substitute for 5 mM Mg2+ without affecting the polymerase activity significantly (not shown). The optimal template–primer ratio was determined to be one molecule of oligo(rU)18 primer every 120 bases of the poly(rA) template and one molecule of oligo(rG)18 primer every 100 bases of the poly(rC) template. Both full-length and NS5B-C21 RdRps showed maximum activity at 37 °C and the efficiency of the reaction started to decrease above this temperature (not shown).
The kinetic parameters calculated for NS5B-FL and NS5B-C21 on the homopolymeric templates are reported in Table 2. While 1·5–2-fold higher kcat values were measured for NS5B-C21, the Km for UTP and GTP were essentially the same for the two enzymes. For this reason, the observed differences in the kcat/Km ratios mainly reflected differences in the kcat values.
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). The kcat/Km ratios measured for the two enzymes on the D-RNA template indicated that NS5B-C21 was about 4-fold more active than NS5B-FL (Table 2). The higher activity observed also reflected differences in only the kcat values in this case, since the Km for GTP was unchanged. Time-courses of elongation (Fig. 2) revealed that the plateau of the reaction by NS5B-FL was reached after only 10 min, while for NS5B-C21, incorporation continued for at least 60 min. Equivalent results were also obtained on RNA templates derived from the HCV genome (not shown), indicating that the enhanced activity of NS5B-C21 did not depend on the sequence of the heteropolymeric RNA template. As was the case for NS5B-FL, no specificity for HCV genomic sequences could be observed with NS5B-C21 (not shown).
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C21 polymerasesC21, we preincubated NS5B-FL and NS5B-C21 in the absence or presence of poly(rA)–oligo(rU)18 at 23 and 37 °C before the addition of UTP. Activities were compared with those of samples that were not subjected to preincubation, the activity of which was arbitrarily set at 100%. As shown in Fig. 3(A), preincubation in the absence of RNA greatly diminished polymerase activity. In the case of NS5B-FL, up to 90% of the catalytic efficiency was lost upon preincubation at 23 °C and almost no activity remained after preincubation at 37 °C. A similar effect was observed on NS5B-C21, but the extent of the phenomenon was less dramatic, suggesting that NS5B-FL is less stable than the deletion mutant. However, preincubation in the presence of the RNA template greatly enhanced the stability of both enzymes (Fig. 3B). The activity of both was doubled by preincubation at 23 °C, while at 37 °C the efficiency of the elongation reaction was comparable to that of control samples. The enhancement of activity upon preincubation with RNA before NTP addition might be explained by the need for a stable polymerase–template complex to be formed before an efficient elongation reaction can take place. This suggests that association of NS5B with the RNA template may be one of the rate-limiting steps of the reaction. The lower activity after preincubation at 37 °C could be due to denaturation of the enzymes or to a faster dissociation of productive polymerase–RNA complexes. For this reason, the polymerase enzymes were preincubated at 23 °C in the presence of RNA templates in all of the kinetic experiments. Since the behaviour of the two enzymes was comparable, different stability in the presence of RNA cannot be the reason for the different activity observed.
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C21 were challenged with a labelled RNA molecule and the protein–RNA complexes were separated from unbound RNA by native PAGE. We chose a 180 nt RNA fragment that is part of the D-RNA template. This molecule was long enough to be utilized by NS5B as a template. At the same time, it was short enough to allow the electrophoretic separation of free and polymerase-bound RNA. As shown (Fig. 4), NS5B-FL bound the RNA probe efficiently. The protein–RNA complexes, however, were very large and did not enter the gel but accumulated in the loading wells. In contrast, distinct band-shifts were produced by NS5B-C21. In this case, at least three bands migrating slower than the free probe were distinguished clearly. The proportions varied during protein titration, the slower-migrating forms becoming more intense as the protein concentration increased. The formation of multiple C21–RNA complexes suggests that more than one protein molecule can associate with the RNA molecule. None of these complexes, however, was as large as those formed by NS5B-FL, since retention of radioactivity in the gel wells was never observed.
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C21C21 could reflect differences in the processivity of the two enzymes. Moreover, the results of the time-course experiments on the D-RNA template could be explained by enhanced template turnover of the NS5B-C21 molecules, while reinitiation events for NS5B-FL are limited. We decided, therefore, to compare the activity of the two proteins under continuous or single processive cycle synthesis in the presence of heparin. Heparin binds free enzyme as well as enzyme that dissociates from the template–primer during the course of the reaction. For this reason, the activity can be observed of only those polymerase molecules that were bound to the template RNA before the addition of the trapping agent, and reinitiation events are impeded. Therefore, the percentage of activity that is lost upon heparin addition is an indirect measure of the number of initiation events during continuous polymerization. On the other hand, nucleotide incorporation measured in the presence of heparin is the result of a single processive cycle of polymerization and is therefore an index of polymerase processivity. The results shown in Fig. 5 were obtained by adding increasing amounts of heparin together with NTPs, after preincubation of the enzymes with the D-RNA template. Under single processive cycle conditions, the activity of NS5B-C21 was slightly lower (about 0·06 nM NMP per nM RdRp) than that of NS5B-FL (about 0·1 nM NMP per nM RdRp). This result would suggest that NS5B-FL is only slightly more processive than NS5B-C21. However, more than 95% of the C21 activity was trapped by heparin (Fig. 5A), while that retained by NS5B-FL was around 50% of the catalytic efficiency observed under continuous polymerization conditions in the absence of heparin (Fig. 5B). We interpreted this latter observation as suggestive of an increased template turnover capability of NS5B-C21 with respect to the full-length enzyme.
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C21 in this case. However, only 40–50% of NS5B-FL activity was trapped by heparin, while more than 90% of the NS5B-C21 catalytic efficiency was lost upon addition of heparin.
Product distribution under single processive cycle conditions
We decided to compare the elongation rates under single processive cycle conditions of both NS5B-FL and NS5B-C21 by measuring the length of the RNA products synthesized on the poly(rA)–oligo(rU)18 template–primer. The experiments were performed in the presence of heparin as the trapping molecule, added together with [-32P]UTP. For both enzymes, the length of the RNA products synthesized by a single polymerase molecule after 0·5 min elongation was about 350–400 nt (Fig. 6A), suggesting an elongation rate of about 700 nt/min.
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C21 with D-RNA, the elongation reaction was started by the addition of NTPs, including [-32P]GTP, and heparin. The same results were obtained with both enzyme forms, but only those for NS5B-C21 are reported (Fig. 6B). As shown, in the first 30 s of elongation in the presence of heparin (lanes 1–9), labelling of only the template RNA could be observed, and bands corresponding to dimer-sized products were clearly visible only after 2 min incubation. Starting from 1 min, many bands, probably corresponding to sites of pausing of transcription, began to be detectable. These bands gradually disappeared, probably being elongated to dimer-sized products by RdRp molecules that were still bound to the RNA. During the incubation, the intensity of the labelled template band also decreased as that of the full-length hairpin product increased. In contrast, in experiments performed in the absence of heparin (lanes 10–18), where reinitiation events were allowed, the intensity of the bands corresponding to template and pausing sites increased, although at a slower rate than those corresponding to the full-length, dimer-sized molecules. These results suggest that template labelling corresponded to the initiation step of the elongation reaction. The observation that labelling of only the template RNA could be observed in the first 30 s might indicate that initiation is one of the rate-limiting steps of the HCV polymerase reaction on heteropolymeric RNA template molecules in vitro. |
Discussion |
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; De Francesco et al., 1996 ). Attempts to purify an active NS5B-FL from E. coli failed due to the high insolubility of the protein expressed in this system, which is found mainly in inclusion bodies. As already reported by others (Yamashita et al., 1998 ; Ferrari et al., 1999 ), we found that deletion of the C-terminal 21 residues vastly improved the solubility of NS5B produced in E. coli, which could easily be extracted and purified, even without the aid of detergents.
We attempted to compare the activity of NS5B-C21 with that of NS5B-FL, reasoning that the increased solubility might be a result of better overall folding of the protein, which, in turn, might be reflected in the polymerase catalytic efficiency. We found, indeed, that the catalytic activity of NS5B-C21 was higher than that of NS5B-FL on all the RNA templates assayed, with the maximum difference being observed on RNA molecules of heteropolymeric sequence (about 4-fold after 10 min of reaction and more then 10-fold after 30 min; Fig. 2). Of the several possible explanations, we could exclude both significant variations of the Km values for the NTPs (Table 2) and different stability of the two enzymes, at least in the presence of RNA (Fig. 3).
Other possibilities have been investigated and will be discussed. Firstly, we found that, in contrast to NS5B-C21, NS5B-FL binds the RNA as a multimeric form (Fig. 4). Even though we cannot rule out that the full-length protein oligomerizes only in the presence of RNA, as in the case of the 3Dpol RdRp (Pata et al., 1995 ), we strongly suspect that the enzyme does exist in multimeric forms in solution. Gel-filtration experiments (not shown) indicate that purified NS5B-FL forms large aggregates composed of at least four NS5B molecules. On the other hand, NS5B-C21 exists in a monomeric form in solution, as shown by light-scattering measurements (not shown). Assuming that NS5B-FL oligomers do not retain full catalytic activity, but do retain full RNA-binding activity, their formation would diminish both the amount of active protein and the number of RNA template molecules available for polymerization.
Secondly, we found that the amount of RdRp activity that was trapped by heparin was larger in the case of NS5B-C21 (Fig. 5). This is suggestive of a higher reinitiation rate associated with the C21 protein compared with NS5B-FL. The same conclusion can be drawn from the observation that the time-course of the reaction performed on heteropolymeric RNA molecules with NS5B-FL reached a plateau after only 10 min (Fig. 2). By this time, only 0·2% of the limiting nucleotide has been incorporated and only 0·05% of the D-RNA templates have been converted into double-stranded template–product molecules. Even though the possibility exists that the higher activity of the C21 mutant might derive from a higher percentage of active enzyme in the preparation, this could not explain the higher turnover of this enzyme form on RNA templates. There are at least three ways to explain these results. (i) Formation of productive polymerase–RNA complexes is inefficient. The observation that incorporation efficiency increases upon preincubation of the polymerase with the template RNA before the addition of nucleotides might indicate that stable association of the enzyme with the template is one of the rate-limiting steps of the reaction (Fig. 3). Even though no differences in the behaviour of the two enzyme forms appear from the experiments reported, the association rates could be different. (ii) Dissociation rate constants of the enzyme–RNA complexes are low. It should be pointed out that we cannot discriminate between these two possibilities, since we have not yet calculated the relative kinetic constants. (iii) Stability of the two enzyme forms when not associated with the RNA template is different. As a matter of fact, we observed that, in the absence of RNA, the NS5B-FL enzyme is less stable than NS5B-C21 (Fig. 3). This could cause the full-length molecules that dissociate from the RNA after one round of processive polymerization to denature faster and thus prove incapable of re-associating with the template in order to perform successive rounds of replication.
For processive nucleic acid synthesis, it can be shown that the steady state kcat value should equal the intrinsic polymerase elongation rate (Wilson et al., 1996 ). In the case of the HCV polymerase, the overall incorporation rates measured are still much lower than the elongation rates calculated under single processive cycle conditions, even for the more active C21 enzyme. The kcat value would, in fact, indicate that each polymerase molecule would be able to incorporate less then one nucleotide per minute. This strongly contradicts results derived from experiments performed in the presence of heparin as a trapping molecule, from which an elongation rate could be calculated of about 700 nt/min on the poly(rA)–oligo(rU)18 RNA (Fig. 6A). Recently, Lohmann et al. (1998) determined an elongation rate of about 150–200 nt/min on the full-length HCV genome. We also found that, on the 400 nt long D-RNA template, full-length product synthesis is completed by a single polymerase molecule after 2 min (Fig. 6B), and this would indicate an elongation rate of about 200 nt/min. The presence of elongation pausing sites impeded a correct estimate of the elongation rate, but suggests that its value is higher than 200 nt/min.
Several reasons could account for the apparent discrepancy between the overall incorporation rate and that calculated for a single polymerase molecule: (i) even though NS5B-C21 could be folded better, the active protein concentration could still be lower than the total protein concentration; (ii) the enzyme may be trapped in non-productive enzyme–template complexes; (iii) dissociation of the enzyme–template complex may be rate-limiting; (iv) initiation of polymerization may be rate-limiting. This latter possibility seems especially likely with heteropolymeric RNA templates, on which, in the absence of primer, the elongation reaction starts from 3'-OH ends that are transiently base-paired, forming unstable snapped-back duplexes containing the initiation site. We found that, at the very beginning of the elongation reaction by NS5B-C21 (Fig. 6B) as well as by NS5B-FL (not shown), labelling of only the template occurs. This suggests that initiation of the elongation reaction in vitro from the 3'-OH of heteropolymeric RNA molecules is a very inefficient step. In addition, the low velocity of the template-labelling reaction might greatly affect the overall rate of the reaction.
It should be taken into account that, in vitro, purified NS5B has no apparent template specificity by itself. Association with other cellular and/or viral factors is required to drive the polymerase specifically at the replication initiation site of the genome. Moreover, priming is often a complex reaction that does not rely solely on viral polymerases but also involves other functions (Salas, 1991 ). In the case of poliovirus, for example, the interaction of the RdRp with the viral precursor protein 3AB is thought to play an important role in virus replication. Indeed, besides increasing polymerase activity by improving initiation of elongation events (Plotch & Palant, 1995 ; Richards & Ehrenfeld, 1998 ) and being a likely candidate for the membrane tether of 3Dpol (Towner et al., 1996 ), 3AB is the direct precursor of VPg (3B), which probably functions as the replication-priming protein (Paul et al., 1998 ).
In the light of the pivotal role of the elongation reaction mediated by NS5B polymerase in the course of the HCV life-cycle, compounds able to interfere with the activity of this enzyme could be promising candidate drugs. The understanding of the enzymatic properties of the purified enzyme will be helpful for the development of first-generation inhibitors.
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Acknowledgments |
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References |
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Received 27 August 1999;
accepted 12 November 1999.
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