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The structural basis for nucleotide incorporation fidelity remains an open question for all nucleic acid polymerases. Addressing this question for the viral RNA-dependent RNA polymerase (RdRp) is of particular, practical significance because it is a determinant of sensitivity to antiviral nucleosides and may be a determinant of viral virulence. All polymerases are thought to employ the same catalytic mechanism, but the rate of nucleotide incorporation can vary substantially. Here we review some of the recent work with the RdRp that leads us to suggest that structure provides only a partial understanding of RdRp function and dynamics may be the missing link.
RNA viruses represent the largest class of existing and emerging human pathogens. Few vaccines have been licensed to prevent RNA virus infections, and only one broad-spectrum antiviral (ribavirin) has been licensed to treat RNA virus infections in humans. Unfortunately, this single antiviral agent is not effective against all RNA viruses . Replication of the RNA virus genome is catalyzed by the virus-encoded RNA-dependent RNA polymerase . Like other viral polymerases, the RdRp is an important target for development of antiviral therapeutics. Rational design of RdRp-specific, nucleotide-based inhibitors and/or substrates would benefit from knowledge of how catalytic efficiency and nucleotide specificity are engineered into the active site of the enzyme. Addressing these very fundamental issues regarding polymerase function is among the most active areas of polymerase enzymology. Herein we review some of the most recent studies of RdRp structure and function. We conclude that existing gaps in our ability to connect RdRp structure to RdRp function may require investigations into RdRp dynamics.
RdRps have a semi-classical structural organization that is often compared to a right hand (Fig. 1a). The enzyme has the fingers, palm and thumb subdomains found in other classes of nucleic acid polymerases but also has a fingertips subdomain that leads to formation of a completely encircled active site. Seven conserved structural elements, termed motifs A-G, exist. Motifs A-E are located in the palm subdomain and contribute to aspects of nucleotide and nucleic acid binding, as well as catalysis. Motif C is the most well known motif as it contains the GDD sequence that is a hallmark of viral RdRps. The function of motif D is only now becoming clear and will be discussed below. Motifs F and G are located in the fingertips and fingers subdomains, respectively. Motif F contains basic amino acid residues that appear to be responsible for ground-state binding of the triphosphate moiety of the incoming nucleotide . The function of motif G is not absolutely clear, although substitutions of residues in this motif inhibit polymerase activity .
RdRps have often been characterized as error-prone polymerases. However, it is now clear that the low fidelity is primarily due to the absence of proofreading exonuclease activity . RNA viruses with genomes longer than 10,000 nt, coronaviruses for example, may encode a proofreading activity [5••]. Transition mutations do occur with a frequency of 1 in 10,000, primarily due to GMP misincorporation opposite uridine (Fig. 1b). RdRps do not distinguish correct nucleotides from incorrect nucleotides or nucleotide analogues, such as ribavirin triphosphate (RTP), at the binding step as the apparent dissociation constants (Kd,app) for all nucleotides are quite similar (Fig. 1b), consistent with motif F interactions with the triphosphate governing nucleotide binding . The primary fidelity checkpoint for the RdRp occurs after binding and represents a conformational change that has been attributed to reorientation of the triphosphate into a position for efficient catalysis [4,6]. Changes in the equilibrium constant for this conformational-change step manifest as a change in the maximal rate constant for nucleotide addition (kpol) (Fig. 1b).
One of the most exciting advances in the field of RdRp enzymology recently was the solution of structures for the RdRp from foot-and-mouth disease virus (FMDV) in complex with primed template and various correct nucleotides and RTP [7,8••]. Because the fingertips of the RdRp constrain movement of the fingers subdomain relative to the thumb subdomain, reorganization of these subdomains did not occur as observed in polymerases that lack a fingertips subdomain [8••]. This observation suggests that subdomain movements are not responsible for rate-limiting, conformational-change steps observed by kinetic analysis of the single-nucleotide-addition cycle. The most substantial difference between a complex with a correct nucleotide (UTP in Fig. 1c) relative to a complex with an inefficiently incorporated nucleotide analogue (RTP in Fig. 1c) was the position of residues, such as Lys-369, on motif D. The complexes are essentially superimposable when atoms involved in catalysis are interrogated (Fig. 1d). As discussed below, Lys-369 (Lys-359 in poliovirus (PV) RdRp) of motif D is a dynamic element that influences the rate of catalysis. Stuctural studies may fail to identify the basis of fidelity because they are unable to capture the trajectory of motif D during a catalytic cycle.
Several surprises regarding the mechanistic basis for RdRp fidelity came from studies of poliovirus (PV) that were aimed at identifying a high-fidelity polymerase by selecting for PV mutants that exhibit reduced sensitivity to ribavirin [9,10]. The first surprise was that a site remote from the catalytic center controlled fidelity. Gly-64 of the PV RdRp, a residue in the fingers subdomain, was changed to Ser. Gly-64 has an indirect connection to motif A in the active site; this motif contributes to interactions in the ribose-binding site and coordination of Mg2+ ions required for catalysis . A second surprise was that this same mutant was isolated independently by two different laboratories, suggesting that few solutions may exist to development of resistance to this class of antiviral nucleosides, a distinct contrast to other classes of nucleoside-based RdRp inhibitors .
Mechanistic studies of the PV G64S polymerase revealed that this substitution decreased the equilibrium constant for the conformational-change step (K2) preceding chemistry (Fig. 2a) . The more difficult it is for the enzyme to achieve the catalytically competent conformation, the more faithful the nucleotide-addition cycle will be. Structural studies of the G64S polymerase in complex with substrates have not been completed, but the structure of the unliganded enzyme provided no insight into changes in the organization of motif A as might have been predicted (Fig. 2b). Because conformational changes appear to be altered by the G64S substitution, analysis of the dynamics during the nucleotide-addition cycle may be most insightful.
Biological studies of PV encoding the G64S substitution have shown that the fidelity of the PV RdRp appears to be important for pathogenesis. The ability for G64S PV to cause disease was attenuated in a very permissive mouse model [12•,13•]. The attenuated phenotype was shown to be a reflection of the reduced population diversity produced by a high-fidelity polymerase by using chemical mutagenesis to restore virulence to the G64S PV population [13•]. Importantly, G64S PV and related mutants confer long-term protection of mice from a lethal challenge with wild-type PV [14••]. Therefore, an understanding of how fidelity is engineered into the RdRp structure has practical implication for development of vaccines.
Nucleic acid polymerases employ a two-metal-ion mechanism for catalysis . One metal (B in Fig. 3a) enters the active site with the nucleotide; the other (A in Fig. 3a) enters after the nucleotide. Metal B likely influences organization of the triphosphate in the active site. However, metal A lowers the pKa value for the 3′-OH of the primer and contributes to charge neutralization that forms during the transition state. The acceptor for the proton from the primer 3′-OH is not known. Recent studies of the nucleotidyl transfer reaction catalyzed by the RdRp and other polymerases have made the unexpected observation that two protons are transferred during the reaction, not just one proton . Moreover, it is now clear that the second proton derives from a basic amino acid (general acid) of the polymerase and is transferred to the pyrophosphate leaving group (Fig. 3a) [17••]. Protonation of the pyrophosphate leaving group is not absolutely essential but contributes 50- to 1000-fold to the rate constant for nucleotide addition. The general acid is located on motif D of RdRps and reverse transcriptases (RTs), on helix O of A-family polymerases (e.g. T7 RNA polymerase), helix P of B-family polymerases (e.g. RB69 DNA polymerase) and the trigger loop of multi-subunit RNA polymerases (Fig. 3a) [17••].
The general acid is Lys-359 in PV RdRp, Lys-369 in FMDV RdRp, and Lys-220 in the RT from human immunodeficiency virus (HIV). This residue is absolutely conserved in all RdRps and RTs. Motif D is the most dynamic structural element of RdRps and RTs (Fig. 3b). Surprisingly, none of the structures available for RdRps or RTs, presumably poised for catalysis, reveal a conformation that is consistent with the ability of this residue to function as a general acid. The general acid (Lys-374) of the RdRp from a Norovirus (NV) comes the closest (Fig. 3b).
It is not at all clear why the FMDV RdRp and HIV RT structural studies have not succeeded in capturing the general acid and motif D in its active conformation. Studies of the other polymerases have successfully captured the general acid in the active conformation (Fig. 3c).
Interestingly, all of the structural elements that contain the general acid undergo substantial changes in conformation after nucleotide binding (Fig. 3c). This conformational change may be related to the rate-limiting step (K2) that is used for fidelity of nucleotide addition. Consistent with this possibility, amino acid substitutions in helix O of A-family polymerases and the trigger loop of multisubunit RNA polymerases contribute to fidelity [18–23•]. If this is also the case for motif D of the RdRp, then this motif may represent an important target to alter fidelity of the viral RdRp for viral attenuation and vaccine development as done for the G64S PV RdRp. Motif D may represent a universal target for control of viral fidelity and viral attenuation in all RNA viruses. Also worth noting is the observation that one of the attenuating mutations in the Sabin vaccine strain for PV type 1 converts Thr-362 of the RdRp to Ile . Given its proximity to Lys-359, the Thr-to-Ile substitution at position 362 may impact Lys-359 function. In conclusion, dynamics of motif D will clearly impact function. We suggest that motif D dynamics may hold the key to our understanding of RdRp fidelity and our ability to tune RdRp fidelity for applications such as vaccine development.
Picornaviruses produce a single polyprotein that is post-translationally processed by viral proteases to produce a variety of incompletely and terminally processed products. For example, in addition to the 3C protease and 3D RdRp, a 3C-3D fusion protein (3CD) is also made. Like 3C, 3CD has protease activity, but unlike 3D, 3CD lacks RdRp activity. The molecular basis for this difference has been of great interest to picornavirologists. A recent suggestion was that the fingers subdomain of 3D was derived from a “chameleon” sequence . As long as the amino terminus of 3D is free to bury itself into the fingers subdomain, then the fingers subdomain would fold into conformation suitable for RdRp function . When the amino terminus of 3D is fused to 3C, however, a new conformation that is inconsistent with RdRp function would be realized. The first structure of a picornaviral 3CD protein was solved recently [26•]. As shown in Fig. 4, 3CD is a composite of 3C and 3D. The only hypothesis that could be put forward to explain the absence of RdRp activity in 3CD was that the dynamics of the 3D domain of 3CD may be negatively impacted [26•].
Processing intermediates have the ability to expand the viral proteome, potentially creating new functions or, as described here, inactivating some functions. Moreover, a single amino acid change of a residue that influences protein dynamics can be sufficient to alter the global dynamics of a protein and potentially its function . If this is the case, then the combination of a viral quasispecies and variable extents of polyprotein processing creates unlimited possibilities for the acquisition and evolution of function by a virus with limited coding capacity.
In spite of all of the structural information now available for the RdRp (see Supplementary Table 1), many gaps exist in our understanding of RdRp function. We would like to suggest that dynamics may represent the missing link to our complete understanding of RdRp function (Fig. 5). Given the numerous high-resolution structures available for the RdRp in the same and different families, it should be possible to use molecular dynamics simulations (MD) to determine the extent to which dynamics co-evolves with structure and function. In addition, the impact of mutations or fusions on dynamics should be tractable by using MD. Indeed, MD has become extraordinarily powerful . A recent study on HIV RT reported the use of a 360 ns simulation to explain the impact of a non-nucleoside reverse transcriptase inhibitor on HIV RT dynamics [29•]. It should also be possible to use hydrogen-deuterium exchange mass spectrometry (HDXMS) to evaluate RdRp dynamics. HDXMS should permit analysis of RdRp dynamics in complex with substrates, as well as when fused to other proteins. Again, HDXMS studies of HIV RT have recently been used to identify regions of RT that change conformation in the absence of substrates [30•]. Finally, the molecular weight of most viral RdRps is in the 50–65 kDa range. Enzymes in this size range can now be studied by using nuclear magnetic resonance spectroscopy, which offers an empirical approach to the evaluation of dynamics on the widest range of timescales.
RdRp fidelity may be a tractable target for viral attenuation and vaccine development. Our current understanding of the basis of RdRp fidelity is incomplete even with the availability of multiple crystal structures. Computational and empirical studies of RdRp dynamics may complete our understanding of RdRp function.
Our work on RdRp mechanism has been supported by grant AI045818 from NIAID/NIH.
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