Several reports have characterized the enzymatic activity of HCV NS5B in various degrees of detail (2
). However, further characterization of the reaction pathway of NS5B-catalyzed nucleotide incorporation has been hindered in part due to the lack of template/primer pairs capable of efficiently assembling catalytically competent complexes with the enzyme. In this work, we demonstrate that stable, preannealed dsRNAs are poor substrates for HCV NS5B. Instead, the HCV polymerase utilizes more efficiently short oligonucleotides, 2 or 3 nt in length, to prime nucleotide incorporation which can be followed by extension of radiolabeled RNAs. We further demonstrate that initiation of RNA synthesis preferentially occurs from the 3′ terminus of the template RNA, suggesting that the replicase assembles at the 3′ terminus of viral RNA. Consistent with this possibility was the finding that 3′ termini of both HCV positive-strand and negative-strand RNAs can serve as templates for dinucleotide-initiated RNA synthesis.
Recent structural studies revealed that HCV NS5B has a fully encircled active site with a relatively rigid interdomain structure, resembling the nucleic acid-bound conformation of several other polymerases (1
). The encircled overall structure of this enzyme is the result of the extensive interactions between the fingers and thumb subdomains which are likely to be unique to viral RdRps. In addition, an HCV-specific β-hairpin structure located in the thumb subdomain, absent in PV 3Dpol
and HIV RT, protrudes toward the active site and may impose a steric barrier to prevent binding to dsRNA molecules (6
). A highly conserved RNA binding groove bordered by the fingers subdomain and the interdomain loops provides a positively charged molecular surface to be occupied by the 5′ overhang of the template (6
). Upon template/primer binding, NS5B is expected to undergo local conformational changes including those proposed for the β hairpin and the thumb subdomain (6
) (Fig. ). No large-scale domain movements, such as those observed in other polymerases upon nucleic acid binding, are expected. At present, it is not clear how such conformational changes, in particular those required for accommodation of the nascent double-stranded RNA, can be induced.
FIG. 8 Quaternary complex model for HCV NS5B. The crystal structure containing HIV-1 RT, template/primer DNA pair, and an incoming dNTP (PDB code 1RTD) (11) was used to guide the construction of a model for the analogous complex of HCV NS5B with RNAs and an (more ...)
In the absence of a liganded structure, a model for the HCV NS5B quarternary complex was created (Fig. ), based on the complex structure of HIV-1 RT containing the enzyme, template/primer DNA pair, and an incoming dNTP (11
). A single-stranded RNA template and dinucleotide primer were modeled into the complex with few structural overlaps. The single-stranded template occupies the RNA binding groove with its 3′ terminus stacked against a tyrosine residue (Y448) at the tip of the β hairpin. The template region in direct contact with the RNA binding groove consists of 5 nt (T+1 to T+5), which is consistent with our observation that the minimal template length is 5 nt (Fig. A). We propose that the unique β hairpin in the thumb subdomain may play an important role in positioning the 3′ terminus of the template for proper initiation of RNA synthesis. The side chain of the strictly conserved isoleucine residue (I160) in motif F (17
) packed against the T+3 template base and stabilized the base pairing between T+3 and the incoming NTP. Within the space above the active site at the base of the palm subdomain, the dinucleotide primer (P+1 to P+2) forms a short duplex with the terminal template bases (T+1 and T+2). The 5′ phosphate group of the dinucleotide is in close proximity of motif E and a highly conserved arginine (R386) which may play a role in stabilizing the short initiating nucleotide primer. The size of the NS5B active site in this model can accommodate only a short duplex RNA with up to 3 bp, including the one between the incoming nucleotide and the T+3 base. This structural feature is reminiscent of the quaternary complex structure of the bacteriophage T7 RNA polymerase (7
) in which a trinucleotide primer base pairs with the single-stranded template DNA and the incoming NTP base pairs with the T+4 template base. Based on the HCV NS5B model, a second nucleotide incorporation (base pairing with the T+4 base) would require that the template translocate towards the β hairpin so that the T+4 ribose will be within the distance to the active site for catalysis to occur. How this template translocation is accomplished requires additional studies. However, this may explain our hypothesis of a rapid dissociation after incorporation of the first nucleotide (Fig. B), perhaps a result of steric hindrance imposed by the β hairpin toward the passage of the duplexed template/primer beyond the β-hairpin structure.
Based on the unliganded NS5B structure, the β-hairpin loop is located within a space similar to that of the N-terminal domain of T7 RNA polymerase (6
). In the quaternary complex of T7 RNA polymerase, this N-terminal domain functions as a wedge to separate the nascent RNA strand from the DNA template (7
). The β hairpin in NS5B may serve a similar function and will be the focus of future studies.
So far, two forms of in vitro RNA synthesis activities have been demonstrated for HCV NS5B. One is from a preannealed primer (2
); the other initiates de novo (21
). It is generally believed that de novo initiation is the mode of HCV RNA replication in vivo since this mode ensures the faithful replication of the entire viral genome by initiating RNA synthesis from the exact 3′ terminus of the template RNA. However, it is not clear what mechanisms are involved in the initial priming steps of the replication process. We propose the following model for initiation of HCV RNA replication: HCV NS5B binds viral RNA containing a 3′ single-stranded overhang free of secondary structures (Fig. ). Although this model appears to contradict the prediction that the 3′ terminus of the HCV genome is occluded (14
), we believe that the HCV 3′ X region represents a preinitiation structure. With the help of NS3 helicase or other factor(s), the 3′ stem-loop could be melted or unwound to allow initiation as proposed in this study.
Following RNA binding, the initiating nucleotide (ATP for negative-strand synthesis and GTP for positive-strand synthesis) enters the active site through the conserved NTP channel (6
) and base pairs with the 3′-terminal template base of the viral RNA. This step has been shown to be sensitive to NTP concentration and is thus rate limiting (21
). Subsequently, the polymerase will add one or more nucleotides to the initiating nucleotide to produce short RNA transcripts. These short RNA transcripts may dissociate from the polymerase/template complexes, a process known as abortive initiation/cycling observed in both prokaryotic and eukaryotic RNA polymerases (22
). These abortive transcripts, predominantly di- or trinucleotides, can then be used by the enzyme to initiate new rounds of RNA synthesis in a fashion as described in this study. The dinucleotide may significantly accelerate the RNA synthesis by circumventing the first nucleotidyl transfer reaction from the initiating nucleotide which is rate limiting. Similar effects have been reported for T7 RNA polymerase in that the abortive product, a dinucleotide tetraphosphate (pppGpA), is much more efficient in initiating RNA synthesis (13
). The biological role of abortive initiation is not clear, although it is proposed that these abortive RNA transcripts may serve as primers for DNA replication (22
). An intracellular pool of di- or trinucleotides may exist as a result of abortive cellular RNA transcription. Whether or not these di- or trinucleotides prime HCV RNA replication in vivo can be addressed only in the context of a viral infection. It is conceivable that the initiating dinucleotide is a product of de novo synthesis and allows efficient RNA synthesis to a level suitable for in vitro kinetic analysis.
The single-nucleotide initiation may reflect the first step of replication initiation but lacks the efficiency for in vitro characterization. Identification of the optimal template and primer requirements for HCV NS5B RdRp will facilitate the mechanistic characterization of nucleotide incorporation catalyzed by this enzyme.