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The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.
Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the template-dependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science.
Several model systems for biochemical study of viral RNA-dependent RNA synthesis exist (4, 19, 20, 25, 37, 42). Well-characterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47-49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60).
RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed conformation, even in the absence of template, in contrast to DNA-dependent RNA polymerases, which transition from open to closed complexes upon template recognition (13). The closed form of the phage 6 RdRp has been proposed to allow specific recognition of the single-stranded viral RNA (7). The template channel formed by the closed structure, however, is too narrow to accommodate the partially duplexed RNA that forms during RNA synthesis, and hence, the closed conformation needs to undergo significant rearrangements in the ternary complex. Biswal et al. (3) have captured an X-ray crystallographic structure of a partially open conformation of the HCV RdRp. Bovine viral diarrhea virus (BVDV) RdRp was also shown to exist in a partially open conformation (11). Ranjith-Kumar and Kao (49) demonstrated that the HCV RdRp could initiate RNA synthesis from a circular RNA template, and thus, the threading of a single-stranded RNA into the template channel is not required for de novo-initiated RNA synthesis. Altogether, these results raise the possibility that the HCV RdRp can undergo rearrangements from the closed conformation seen in the crystal structure prior to de novo initiation.
A secondary structure that extends from the finger to the thumb subdomains, named the Δ1 loop, has been proposed to serve as a gate to cover the template channel and regulate the switch from de novo initiation to elongation (5, 10). Mutations that affect the interaction between the Δ1 loop and the mostly hydrophobic residues that it contacts have resulted in polymerases that are defective for de novo initiation but can bind to partially duplexed RNA and can extend from the 3′ terminus of an RNA primer (10).
Two general models for RNA synthesis by the HCV RdRp can be proposed (Fig. (Fig.1).1). The first posits that the HCV RdRp functions as a monomer at least during de novo initiation because the closed template channel is needed for specific recognition of the template (5, 7, 10). It was presumed that the Δ1 loop and thumb domain interaction in the HCV RdRp is stable and mutations that disrupted this interaction would render the enzyme catalytically inactive (5, 24). However, a deletion of five residues in the tip of the Δ1 loop did not prevent RNA synthesis from a primed template by the polymerase (10). Furthermore, a genotype 2a RdRp was crystallized in a form with altered interaction between the Δ1 loop and thumb domain in comparison to the 1b RdRp (3). Interestingly, a low-affinity GTP binding site exists on the thumb domain close to the base of the Δ1 loop binding pocket. GTP binding at this site has been proposed to stabilize the Δ1 loop and thumb domain interactions, favoring the closed monomer model (6). A second model is based on the reports that HCV RdRp can oligomerize and that oligomerization increases its activity (12, 16, 46, 54). The dimer could be active due to either the second subunit increasing the stability of the Δ1 loop and thumb interactions in the first subunit to increase de novo initiation or the two subunits forming a common template-binding domain (Fig. (Fig.1).1). Here we have attempted to determine whether monomers or oligomers of the HCV RdRp can better perform de novo initiation using biochemical and biophysical analyses.
The prototype HCV RdRp used in the study is derived from HCV 1b isolate strain BK. The hydrophobic C-terminal tail was truncated 21 and 51 residues from the C terminus to increase solubility of the proteins. All proteins were expressed in Escherichia coli with the 6×-His tag at the C terminus of the protein. The culture was shaken at 37°C until the optical density at 600 nm (OD600) reached 0.8 to 1.0, and then the temperature was changed to 16°C and isopropyl-β-d-thiogalactopyranoside (IPTG) was added to 0.5 mM. After a 12- to 16-h period, the cells were pelleted at 8,000 × g for 15 min, and the pellets were frozen at −70°C until needed.
RdRp purification used cells suspended in lysis buffer (20 mM Tris, pH 7.5, 300 mM NaCl, 10% glycerol, 10 mM imidazole, a protease inhibitor cocktail at the concentration suggested by the manufacturer [Sigma Inc.], 5 mM beta-mercaptoethanol [BME], and 0.1 mg/ml lysozyme). The suspension was sonicated for 2 min and clarified by centrifugation at 16,000 × g for 30 min. The supernatant was loaded onto nickel-nitrilotriacetic acid (Ni-NTA) columns equilibrated with 20 mM Tris, pH 7.5, 300 mM NaCl, 10% glycerol, 10 mM imidazole, and 5 mM BME. The columns were washed with the same buffer containing 20 mM imidazole, followed by elution of the bound proteins with lysis buffer containing 500 mM imidazole. Δ51 and some preparations of Δ21 along with the I432V mutant (used in differential scanning fluorimetry [DSF] assays) were further purified on a poly(U) agarose column as described previously for Δ21 (10).
Δ21 purified from the Ni-NTA column was dialyzed into buffer consisting of 20 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, and 10 mM BME using a desalting column and then loaded onto a MonoS column (HR 100/10; GE Healthcare) which was preequilibrated with desalting buffer. Δ21 was eluted with a gradient of 100 to 600 mM NaCl in desalting buffer. The proteins generated from these two columns were highly pure and did not contain other bands in Coomassie blue-stained gels.
A version of Δ21 that could have the C-terminal histidine tag removed was engineered with a TEV protease cleavage site (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) immediately N-terminal to the 6-His tag sequence. After initial purification through a Ni-NTA column and later on a MonoS column, the protein was exchanged into a buffer containing 100 mM NaCl and 20 mM imidazole and then incubated with the TEV protease for 48 h. The cleaved Δ21 was passed through a second Ni-NTA column, and the flowthrough was collected, concentrated, and buffer exchanged before use for biochemical analysis.
Approximately 250 ng of proteins were mixed with the loading dye supplied by the manufacturer (Invitrogen Inc.) and electrophoresed in 3 to 12% native PAGE gels (Invitrogen Inc.) with a Coomassie blue-containing cathode buffer at 4°C for 2 h according to the manufacturer's instructions. The gel was stained with silver using a kit and protocols from the manufacturer (Invitrogen Inc.).
LE19 is a template typically used to measure several RNA synthesis activities in one reaction (47): de novo initiation, primer extension, template switch, and nontemplated nucleotide addition. Standard RdRp reaction mixtures contained 50 nM RNA, 0.1 mM ATP and UTP, 33 nM (α-32P)CTP and 0.01 mM GTP (unless stated otherwise), and the amount of NS5B as specified. The assays also contained 20 mM sodium glutamate (pH 8.2), 4 mM MgCl2, 12.5 mM dithiothreitol, 0.5% (vol/vol) Triton X-100, 20 mM NaCl, and 1 mM MnCl2. The reactions were incubated for 1 h at 25°C before extracting the products with a 1:1 mixture of phenol-chloroform and precipitated with ethanol in the presence of 0.3 M NaOAc. The RNA was electrophoresed on 20% urea denaturing polyacrylamide gels, and the gels were wrapped in plastic and exposed to phosphor screens prior to scanning by a PhosphorImager (Molecular Dynamics).
Differential scanning fluorimetry (DSF) was carried out in a Stratagene MX3005P PCR machine with filter sets for excitation at 492 nm and emission at 610 nm. Each sample of 25 μl contained a 2 μM final concentration of protein in 300 mM Tris (pH 7.5), 80 mM NaCl, and SYPRO orange (Molecular Probes) at a 2.5× final concentration (the actual concentration cannot be revealed by Molecular Probes). The samples were heated at a rate of 0.5°C/min, from 25 to 75°C, and the MxPro software program (Stratagene) was used to calculate the Tmapp by plotting the negative derivative of the observed fluorescence change as a function of temperature.
Dynamic light scatter (DLS) measurements were made with Zetasizer-NanoS instrument (Malvern Instruments, United Kingdom) in a buffer containing 300 mM Tris (pH 7.5) and a 1 μM final protein concentration. The buffer components were filtered through a 0.2-μm-pore filter (Nalgene) to avoid suspended particles. The measurements were made at 10-s intervals, with the data points collected after a 1-s scan. At least three measurements were made per sample, and the average intensity for the corresponding peaks was plotted.
Analytical gel filtration used a Superdex-200 HR column (GE Healthcare) equilibrated with the running buffer (50 mM Tris [pH 7.5], 100 mM KCl, 5% glycerol, and 1 mM BME) at a flow rate of 0.25 ml/min. About 100 μg of the respective proteins were either mixed with 20 mM GTP or not and were injected into the 24-ml Superdex 200 column before the run. This high concentration of GTP was used to maintain the NS5B:GTP ratio used in the RdRp assays. One-ml fractions were collected, and the fractions obtained were analyzed by SDS-PAGE. The column was calibrated with molecular mass standards (Bio-Rad Inc.): bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B12 (1.35 kDa).
The protein in a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 5% glycerol, and 1 mM BMEΕ was adjusted to ~5 ng/μl, adsorbed to freshly glow-discharged carbon-coated copper grids (EMS), and stained with uranyl acetate (1% [wt/vol] aqueous solution). Electron microscopy was performed using a transmission electron microscope operated at 80 kV (Jeol 1010). Dimeric particles of the HCV RdRp were selected using the BOXER routine in the EMAN software program (29), filtered, and centered. Class averages were generated with these filtered and centered particles without imposed symmetry. Centered particles (about 4,000) were subjected to analysis using the Multirefine software program to sort the particles into an initial model. A molecular mass of 130 kDa was used for the surface-rendering threshold of the three-dimensional (3D) structures of the dimers. 3D reconstructions were visualized using the UCSF Chimera software package (43).
Blue-native gel electrophoresis (BN-PAGE) was used to examine the oligomeric and/or conformational state of the HCV Δ21 protein. The proteins were coated with Coomassie blue and separated in an acrylamide gel based on a combination of sizes, shapes, and/or oligomeric states (33, 57). We examined Δ21 (Fig. (Fig.2A)2A) along with several mutant derivatives that are affected for oligomerization (E18R mutant ), de novo initiation (m26-30 and I432V mutants [10, 27]), or in the GTP-binding allosteric site (P495/V499A mutant, named PV ) (Fig. (Fig.2B).2B). While Δ21 migrated at the expected molecular mass of 65 kDa in SDS-PAGE, it existed as multiple bands of different intensities in a BN gel (Fig. (Fig.2B).2B). m26-30, with a five-amino-acid deletion in the Δ1 loop which renders it defective for de novo initiation but not primer extension (10), had a different banding pattern. The E18R and PV mutants deviated significantly from the banding pattern of Δ21 and exhibited some poorly resolved high-molecular-weight complexes (Fig. (Fig.2B).2B). The I432V mutant had bands in a position similar to that of Δ21. The variety of bands exhibited by Δ21 and mutant RdRps in the BN-PAGE suggests that each of the RdRps exists in complex arrays of oligomeric states and/or conformations.
Dynamic light scatter spectroscopy (DLS) was used to measure the hydrodynamic diameter of Δ21 to estimate the dimensions of the protein. In a Tris buffer containing 40 mM NaCl, Δ21 existed in three peaks (Fig. (Fig.2C).2C). The first has a mean diameter of more than 8 nm and represents the majority of the protein based on volume distribution of the peak. This hydrodynamic diameter was higher than expected based on the X-ray structure of the monomeric HCV RdRp, indicating polydispersity (25%). The other two peaks are likely to be high-molecular-weight oligomers. Higher salt concentrations slightly increased the abundance of peak I and altered the abundance and positions of the other two peaks in DLS (Fig. (Fig.2C).2C). This is consistent with the idea that the three peaks are in equilibrium in a manner that can be affected by the monovalent salt concentration, as reported previously (12). Finally, Δ21 eluted as three distinct peaks in a Superdex-200 gel filtration column when in a buffer containing 100 mM monovalent salt. However, it showed a significantly delayed elution pattern, with the most abundant peak eluting at a mass smaller than 65 kDa, compared with molecular mass markers (see below). Similar observations were made by Gu et al. (16).
Next, we visualized Δ21 by electron microscopy after staining with uranyl acetate. Δ21 existed in particles of different sizes that are likely monomers (~6-nm average diameter), dimers, and higher-order complexes, with the longest diameters being up to 20 nm (Fig. 2E and F). In several independent experiments, we did not observe particles whose average diameters were larger than 20 nm. The diameters of ~450 Δ21 particles that could be selected from randomly picked sections of five micrographs are plotted in Fig. Fig.2G.2G. The distribution of Δ21 particles on electron microscopy (EM) grids matched with the polydisperse nature of peak 1 observed in DLS (compare Fig. 2D and G). We have previously reported that Δ21 is primarily a monomer in solution based on results from analytical centrifugation (10), while others have shown using the same technique that the Δ21 apoenzyme is a monomer that can form dimers and tetramers in the presence of an RNA that can direct de novo initiation (24). The reason for the discrepancy is unclear.
Since Δ21 can be present as an oligomer, we wanted to examine whether oligomerization can affect a particular mode of RNA synthesis. For this analysis, we used a template, LE19, that can report on both de novo initiation (producing a 19-mer RNA) or primer extension products (producing a 32-mer RNA, Fig. Fig.3A).3A). The reaction mixtures had a 10 μM concentration of the initiating nucleotide GTP, 50 nM template RNA, and from 20 to 60 nM Δ21. Over this concentration range, we observed a geometric increase in de novo initiation but a linear increase in primer extension and template switch products (Fig. 3B and C). This suggested that conditions that favor higher-order RdRp complexes favored de novo initiation.
To confirm the results without the complex template switch products, we used LE19P, which can direct de novo initiation but not primer extension due to a puromycin being attached to the 3′-most hydroxyl of the template. A second RNA, named PE46, which can only direct primer extension, was also present to allow comparative analysis of the two modes of RNA synthesis (Fig. (Fig.3A).3A). Both RNAs were at a 25 nM final concentration. Again, Δ21 concentrations from 20 to 120 nM yielded a geometric increase in de novo-initiated RNAs, while the primer extension product was inhibited at the highest concentration tested (Fig. (Fig.3C).3C). These results show that the mode of RNA synthesis by Δ21 is dependent on the Δ21 concentration. The divalent cation manganese stimulated de novo initiation in all of these reactions, while primer extension was inhibited at higher enzyme concentrations in a divalent metal-independent manner (not shown). This is consistent with our previous findings that manganese has a selective effect on de novo initiation (47).
To examine the relationship between oligomerization and de novo-initiated RNA synthesis, we needed a mutant RdRp that was defective for de novo initiation but could retain interaction with Δ21. The protein m26-30, which had been previously characterized (10), met these criteria and was selected for this activity.
First, we examined whether highly purified m26-30 (Fig. (Fig.4A)4A) was altered in conformation/oligomerization compared to Δ21 using the differential scanning fluorimetry (DSF) assay. The assay uses the dye SYPRO orange, which fluoresces when bound to hydrophobic regions of the protein, to report on its denaturation profile (38). A clear difference was observed between the melting profiles of Δ21 and m26-30 (Fig. 4C and D). Δ21 had a relatively symmetric melting curve with a maximum Tmapp of 45.5°C; m26-30 had a more complex melting profile and a maximum Tmapp of 41.5°C.
We next compared RNA synthesis by Δ21 and m26-30 over a range of protein concentrations. While de novo initiation increased by 69-fold over the range of concentrations tested, m26-30 produced only a small increase in de novo-initiated products over the same concentration range tested (Fig. (Fig.4C).4C). Next, Δ21 at a constant 20 nM (a concentration that produces only a faint amount of de novo-initiated products) was preincubated with increasing amounts of m26-30 before the enzyme mix was used in RdRp assays. m26-30 was found to cause a concentration-dependent increase in the de novo products when it was present with Δ21 (Fig. (Fig.4D).4D). These results suggest that the intermolecular interaction between m26-30 and Δ21 led to increased de novo initiation by Δ21 or by m26-30. Due to the deletion in m26-30, we consider rescue of de novo initiation in m26-30 to be less likely.
To test whether the interaction is specific or could be due to a molecular crowding effect, recombinant green fluorescent protein (GFP) and E. coli TraM proteins were added to Δ21 at the same concentrations as m26-30. Both had a more modest effect on de novo initiation by Δ21 than m26-30 assayed in the same reaction (Fig. (Fig.4E).4E). These results suggest that while molecular crowding did contribute to higher de novo initiation by Δ21, m26-30 had a larger effect.
We wanted an additional de novo initiation-defective mutant to confirm that intermolecular interactions could stimulate de novo initiation by Δ21. The E18 residue was a good candidate, since it was previously claimed to be involved in a homomeric pair with H502 (46). E18 may also be needed for Δ1 loop and thumb interactions, since it formed a salt bridge with R401 in the thumb domain (Fig. (Fig.5A).5A). More than 200 sequences from the 1b HCV strains revealed that E18 and R401 (or E18-K401) were highly conserved (data not shown). When the purified E18R mutant protein was subjected to the DSF assay, it was found to exhibit a broader denaturation peak and also a lower Tmapp than Δ21 (Fig. (Fig.5B).5B). When tested for RNA synthesis with LE19P and PE46 templates, the E18R mutant was also defective for de novo initiation but not primer extension, except at very high concentrations of the protein (Fig. (Fig.5C).5C). When it was coincubated with a 20 nM Δ21 protein, increasing de novo initiation was observed (Fig. (Fig.5D),5D), thus representing a second set of interactions where the presence of an initiation-compromised derivative and Δ21 could increase de novo initiation, presumably by wild-type Δ21. An R401A mutant was not adversely affected for de novo initiation or primer extension activity from the LE19 template, confirming our previous results involving mutations in the template channel (10) (Fig. (Fig.5E).5E). These results suggest that the ionic interaction between R401 and E18 is not required during RNA synthesis by the polymerase.
GTP can stimulate de novo initiation while inhibiting primer extension (28, 48). However, it is not clear if GTP acts at the initiation site (NTPi site ), the allosteric site on the thumb domain, or both (6) (Fig. (Fig.6A).6A). The low-affinity GTP-binding pocket is at the base where the Δ1 loop interacts with the thumb domain, suggesting a relationship with the conformations needed for de novo initiation (Fig. (Fig.6A).6A). To examine whether the allosteric site has an effect on de novo initiation, we tested a double mutant, the P495/V499A mutant (referred to as PV), for RNA synthesis using LE9P and PE46 as templates. Both Δ21 and PV can extend from PE46 to similar levels, but the PV mutant synthesized only a fraction of the de novo-initiated product made by Δ21 while producing more of the primer-extended product (Fig. (Fig.6B6B).
The DSF assay (38) was used to examine whether PV exists in an altered conformation relative to Δ21 in the absence of GTP. PV has a broader melting curve than Δ21, and the Tmapp of PV was also 2°C lower than that of Δ21 (Fig. (Fig.7A).7A). Next, the effect of increasing GTP concentrations on the Tmapp of Δ21 and the PV mutant was determined. GTP increased the thermal stability of Δ21 by ~2.5°C in a concentration-dependent manner (Fig. 7A and B). A similar increase in stability was observed with UTP (not shown). These results likely represent the combination of the effects at both the allosteric site and the NTPi site. In contrast, the PV protein had a reduced response to either GTP or UTP at all concentrations tested (Fig. (Fig.7B7B and data not shown). Given that the PV mutant has a defect in the low-affinity site, the response to GTP likely represents an effect associated with GTP binding at the NTPi site.
Last, we examined whether increasing GTP concentrations can rescue de novo initiation by the PV mutant (Fig. (Fig.7C).7C). Given that the PV mutant was significantly reduced in de novo initiation relative to Δ21, we measured the amount of stimulation at a range of GTP concentrations and looked at the ratio of products at 200 versus 10 μM GTP. At 20 nM Δ21 or PV, the ratio of de novo initiation products synthesized at the two GTP concentrations was 2 for Δ21, but no products were made by the PV mutant at either GTP concentration. At 120 nM, both the Δ21 and PV proteins had an increase of de novo-initiated product as a function of the GTP concentration (Fig. (Fig.7C).7C). Altogether, these results suggest that the allosteric GTP binding site is required for optimal de novo initiation and that some rescue of de novo-initiated RNA synthesis by the PV mutant can take place under conditions that favor intermolecular interactions.
The Δ21 protein used in the present study contained a C-terminal histidine tag to aid in purification. To rule out the effect of the tag on the ability of NS5B to oligomerize, we expressed and purified a Δ21 protein with a TEV protease cleavage site immediately N-terminal to the histidine tag and subjected it to TEV protease treatment. The TEV protease-treated protein was passed through a Ni-NTA column, and the protein flowthrough was used for analysis. Western blot analysis using a monoclonal antibody specific to the histidine tag confirmed its removal (Fig. (Fig.8A).8A). The resulting protein was functional for the production of both the de novo-initiated and primer extension products (Fig. (Fig.8B).8B). Furthermore, a nonlinear increase in the amount of de novo-initiated product was evident as a function of the protein concentration. The protein lacking the histidine tag also had a Tmapp nearly identical to that of the protein with the tag when examined by DSF (data not shown). These results demonstrate that the C-terminal His tag did not affect de novo initiation by Δ21.
To examine further whether the C-terminal tail contributed to de novo initiation resulting from intermolecular interactions, we used a version of NS5B that lacked the C-terminal 51 residues. The Δ51 enzyme was more active in both primer extension and de novo initiation than the Δ21 enzyme; however, a nonlinear increase in de novo initiation activity was observed as a function of higher enzyme concentrations (Fig. (Fig.8C)8C) even with this enzyme. These results confirm that the C-terminal portion of the HCV NS5B protein does not affect the stimulation of de novo initiation associated with higher enzyme concentrations. Interestingly, the inhibition of primer extension was not seen at higher Δ51 concentrations (Fig. (Fig.8C),8C), perhaps due to a larger template channel that can accommodate double-stranded RNA better.
The specificity of intermolecular interactions between Δ51 subunits was examined by keeping Δ51 at a constant 20 nM concentration while adding a 1 or 3 molar excess of several other proteins. The de novo initiation-defective m26-30 and PV stimulated de novo activity by Δ51 when complexed with Δ51. The other proteins (HCV NS3, NS5A [a gift from Craig Cameron], and proteins unrelated to HCV) either had no effect or had an inhibitory effect on de novo initiation by Δ51. The inhibitory effects could be due to binding to RNA or effects on nucleoside triphosphates (NTPs). These results are consistent with those seen with Δ21 (Fig. (Fig.4E)4E) and further imply that specific interactions between NS5B subunits contribute to a stimulation of de novo initiation activity.
The HCV genotype 2a JFH1 strain can establish cell culture infections, and its subgenomic replicon shows faster replication kinetics in cell culture than the replicon from genotype 1b (2). NS5B from strain JFH1 was recently demonstrated to be more competent for de novo initiation than primer extension due to polymorphisms in the thumb domain that make it a more closed structure than NS5B from the 1b HCV genotype (data not shown) (51). We examined whether de novo initiation by JFH1 Δ21 was affected by conditions that favored oligomerization. Consistent with the reports of Simister et al. (51), we observed that the 2a Δ21 protein produced more de novo initiation products than the primer extension reaction (data not shown). However, consistent with the effects of 1b Δ21, increasing the 2a Δ21 concentration resulted in a stimulation of de novo-initiated RNA synthesis without a comparable effect on primer extension.
All of the characterizations so far show that there are complex interactions and conformational changes associated with de novo initiation by the HCV RdRps. The dynamic nature of the RdRp will make it a challenge to define all of the changes necessary for RdRp function, especially by X-ray crystallography. However, 3D reconstruction from negatively stained particles obtained by electron microscopy (EM) can inform us about major changes in protein conformation. This approach has already been used to define conformations of larger molecules, such as ribosomes, GroEL, and innate immune receptors (30, 36, 52). With the immune receptors, we have determined that ligands can induce dramatically different conformations, including oigomerization (36, 52).
To capture the de novo initiation-competent structure, we collected more than 3,000 individually selected dimers/oligomers of Δ21 from negatively stained electron micrographs. However, the final models generated did not yield a good match for the class averages of the data set, and the Fourier transform over the eight rounds of refinement showed that convergence was suboptimal (data not shown). Therefore, we analyzed several other mutant RdRps and found that the I432V mutant was a good choice for reconstruction.
The I432V substitution was originally identified in a mutant HCV replicon that was resistant to cyclosporin A and subsequently was shown to be better at de novo initiation than the wild-type (WT) Δ21 protein, especially at higher enzyme concentrations (27) (Fig. 9A and B). Gel filtration chromatography profiles of the two proteins showed that the elution profile of the I432V mutant lacked a second peak seen with the WT Δ21 protein in the absence of GTP (Fig. (Fig.9C,9C, see asterisks) while the differences were negligible when the elution was performed with GTP (Fig. (Fig.9C).9C). In the DSF assay, noticeable differences in the thermostabilities of the Δ21 and I432V proteins were also observed as a function of increasing GTP concentrations (Fig. (Fig.9D).9D). Finally, the dimensions of the I432V particles were more homogenous than that for Δ21, especially measured in the absence of GTP (compare Fig. 9E and F). Together these results supported our contention that the Δ21 enzyme exists in a heterogeneous state and further suggest that the I432V protein has a lesser degree of heterogeneity. Given that the I432V mutant was competent for de novo initiation and less heterogenous than Δ21, it was selected for single-particle reconstruction.
More than 4,000 dimer-like particles of the I432V mutant without GTP or with GTP were picked and sorted into class averages. The particles selected had a good coverage of the potential orientations, as determined by their distributions in the asymmetry triangle (Fig. 10A and E). The class averages generated are shown in Fig. 10B and F. Several initial models were selected and used for reconstructions without symmetry and with C2 symmetry, and the resulting models were congruous, but the model generated with C2 symmetry had higher resolution. They are shown in Fig. 10D. The final model for the complex was at 19-Å resolution without GTP and at 16 Å with GTP. The I432V molecule has the density set to the mass of 130 kDa (the mass of a Δ21 dimer) and reveals a central channel with a width of 16 Å that could accommodate RNA (Fig. 10D). The results are consistent with the model that two interacting RdRp subunits could form one template channel. However, the individual monomers were difficult to delineate in the models due to a possible combination of extensive interactions between the monomers and/or a low resolution. Interestingly, in the presence of GTP, the densities over the central channel closed and the central channel became more constricted (Fig. 10H), showing that a conformational change can reconfigure the central channel in the dimeric RdRp.
Many viral RdRps need to initiate genomic RNA synthesis de novo from the termini of their RNA genomes in order to avoid the loss of genomic information. This mechanism is fundamentally different from that of DNA-dependent polymerases and may ensure the specificity of synthesis from viral templates. In this report, we show that factors such as GTP and interactions between intermolecular subunits of a higher-order complex can modulate the conformations of the HCV RdRp and influence the mode of RNA synthesis.
The conformational changes in viral RdRps are just starting to be defined. However, it is important to put this in the context of RNA-dependent RNA synthesis. RNA synthesis by an RdRp in vitro can be divided into discrete steps: (i) formation of productive initiation complexes (de novo initiation or primer extension complexes), (ii) formation of the first or first few phosphodiester bonds, (iii) elongation of the stable primer along the template, and (iv) termination. The first two stages are rate limiting and are collectively called initiation events. The Kms for NTPs for initiation nucleotides are significantly lower for elongation than for initiation (15, 47), consistent with the idea of a conformational change in the RdRp between initiation and elongation in RNA synthesis.
The template channel in Δ21 seems to be a region whose dimensions can change depending on the enzyme concentration and the presence of NTPs. This will have a direct effect on the initiation events in RNA synthesis. A recent report has shown that the degree of “closedness” of the enzyme depends on the amino acid composition in the thumb domain (51). This implies that a weak Δ1 loop and thumb domain interaction will result in inefficient de novo initiation (51) but efficient primer extension, at least from short RNA templates (10). In this work, we propose that the interactions between RdRp subunits can promote the de novo initiation in a manner that involves the Δ1 loop and the thumb domain.
Given that de novo initiation by Δ21 and Δ51 can be induced by the presence of a de novo initiation-defective HCV RdRp (Fig. (Fig.4D4D and and8D),8D), the results suggest that the role of a second HCV RdRp subunit is to influence the conformation of the de novo-initiating subunit in the complex. Preliminary reconstruction of the de novo initiation-competent dimer of the I432V mutant also suggests that the two subunits may form a shared template channel that can be induced to close into separate entities in the monomers upon the addition of GTP (Fig. 10D and G). The changes in the template channel in the presence of GTP suggest that GTP binding at either the active site or the allosteric pocket can induce another significant conformational change in the complex that may activate the complex for template binding and RNA synthesis. A number of intermediate structures of the HCV RdRp thus remain to be better characterized in addition to the initially closed structure of the HCV RdRp monomer.
Our results supporting a dimer/oligomer of HCV RdRp being the active form for RNA synthesis (Fig. (Fig.1)1) are consistent with the results and interpretations of Qin et al. (46) and Wang et al. (54). Wang et al. (54) showed the presence of two broad interacting interfaces in the thumb region of one molecule and the finger region of another molecule. However, Cramer et al. (12) reported that a monomeric RdRp is active. This difference may be a matter of the sensitivity in detecting the de novo-initiated products, since we show that the HCV RdRp could exist in an array of conformations and/or oligomeric states, even at the nanomolar enzyme concentrations used in this study. Our results confirm and extend the results from the two reports of Qin et al. and Wang et al. (46, 54). Contact between the oligomerized polymerase subunits has been reported to increase the cooperativity in RNA synthesis in poliovirus 3Dpol (31, 40) and more recently for the Norovirus RdRp (18). Another proposal is that the interface contacts between poliovirus 3D polymerase monomers (thumb interface) and the poliovirus 3C protease could affect RNA synthesis through regulating protein priming (41). While the mechanism needs to be examined further for each species of RdRp, a higher-order structure of viral RdRps as a functional unit in RNA synthesis may be a shared property of the positive-strand RNA viruses (1, 45, 60). It should also be emphasized that the entire complex of replication machinery for viral RNA synthesis will likely take the form of highly ordered complexes that will involve specific targeting of viral and cellular proteins to specific sites on intracellular membranes (31, 34, 58). In this context, the association of the polymerase would also be facilitated by the cellular membrane environment.
The Δ1 loop and its interaction with the thumb domain could be interacting as part of an interface between two RdRp subunits. This would help to explain how mutations in the Δ1 loop, such as m26-30, and even the PV mutants were either incapable of or showed only a linear increase in de novo initiation activity with increasing enzyme concentrations. The residues in HCV NS5B identified to be critical for de novo initiation activity in this study, such as E18, L26-L30, and P495/V499, have all been documented to be needed for HCV replication in cells, underlying their biological significance (8, 10, 32). The interdomain interactions may also be needed for productive RNA binding when longer RNA templates are used. This is consistent with previous reports showing a cooperative effect of NS5B oligomerization on RNA synthesis using longer primer extension templates (54). Whether the elongation of RNA synthesis will require oligomeric contacts between subunits remains to be explored. However, our results indicate that in vitro, lower concentrations of RdRp (where the enzyme is not in excess of template concentrations) are capable of elongative RNA synthesis (Fig. (Fig.33).
The host-encoded prolyl isomerases, cyclophilins, could influence the rate of de novo initiation and, by extension, the level of HCV RNA replication through changes in RdRp conformation (50, 55). I432V, which confers cyclosporin A resistance to HCV (27, 50), is also more competent for de novo initiation at higher enzyme concentrations than at lower enzyme concentrations. This suggests that cyclophilins are likely to modulate conformational changes and possibly the oligomerized states of the RdRp. Chatterji et al. (9) have recently documented that the active site of cyclophylin A is required for HCV replication. NS5B phosphorylation in cells was reported to be needed for HCV replication (23), and phosphorylation may serve to “remodel” the oligomeric complexes of NS5B to assist in template loading.
Genotypic differences in HCV isolates could also impact the equilibrium between the different conformation/oligomeric states of the HCV RdRp and affect the rate of de novo initiation (56). The results reported by Simister et al. (51) demonstrating that the 2a RdRp is more active for de novo initiation are consistent with this model, although it should be considered that the increase in de novo initiation by the 2a RdRp could be by favoring a higher-order conformation rather than a more closed conformation.
Finally, it is interesting to note a difference between viruses that use a polyprotein processing mechanism (including HCV) and viruses that use different RNAs to express the replication proteins. The former class of viruses should, theoretically, have more comparable stoichiometries of all the viral proteins. In contrast, viruses that use distinct RNAs to express the replication proteins could have dramatically reduced expression of the RdRp without affecting the overall level of RNA synthesis (39, 59). These two strategies will certainly allow selection for additional activities of the RdRp (such as binding of the retinoblastoma protein ), as well as distinct ways to deal with the host's innate immunity mechanisms.
We thank C. T. Ranjith-Kumar and the Kao lab members for helpful discussions, Barry Stein for assistance in EM, and especially Laura Kao for editing the manuscript.
This work was funded by National Institute of Allergy and Infectious Diseases grant 1RO1AI073335 to C.C.K.
Published ahead of print on 7 April 2010.