Initiation of viral RNA-dependent RNA polymerization

Alberdina A. van Dijk, Eugene V. Makeyev{dagger} and Dennis H. Bamford

Institute of Biotechnology and Faculty of Biosciences, PO Box 56, Viikinkaari 5, FIN-00014 University of Helsinki, Finland

Correspondence
Dennis H. Bamford
dennis.bamford{at}helsinki.fi


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
INSIGHTS FROM THE RdRP...
ROLE OF DIVALENT CATIONS
TEMPERATURE DEPENDENCE
REFERENCES
 
This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3' terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.

{dagger}Present address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
INSIGHTS FROM THE RdRP...
ROLE OF DIVALENT CATIONS
TEMPERATURE DEPENDENCE
REFERENCES
 
RNA-dependent RNA polymerases (RdRPs) are central components in the life cycle of RNA viruses. Correct initiation of RNA synthesis is essential for the integrity of the viral genome. Other virus- and cell-encoded components are also often needed in viral RNA synthesis to ensure RdRP activity through the formation of RNA–protein complexes similar to DNA-dependent RNA transcription complexes (Buck, 1996; Lai, 1998, and references therein; Patton et al., 1997). As RNA viruses demonstrate a variety of mechanisms for the initiation of RNA synthesis (Fields et al., 1996), it is expected that RdRPs from different sources will possess distinct molecular adaptations facilitating precise and efficient initiation.

The structures of the RdRPs solved to date show that they all have a basic right hand-like structure with fingers, palm and thumb subdomains (Fig. 1), similar to the DNA-dependent RNA polymerases (DdRPs), reverse transcriptases (RTs) and DNA-dependent DNA polymerases (DdDPs) (Cheetham & Steitz, 2000; Doublie et al., 1999; Ollis et al., 1985).



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Fig. 1. Basic right hand structure of the bacteriophage {pi}6 polymerase. Red, fingers; green, palm; blue, thumb; and yellow, priming domain. (Courtesy of J. M. Grimes & D. I. Stuart.)

 
Several conserved RdRP motifs have been identified (Koonin, 1991; Poch et al., 1989). Most of these motifs are in the palm subdomain, with the motifs A, B and C being most prominent (Ollis et al., 1985; Poch et al., 1989; Hansen et al., 1997). Based on structural similarities and the presence of conserved motifs, it was proposed that all polymerases utilize a common two-metal mechanism of catalysis (Fig. 2). This involves two conserved aspartic acid residues from the A (AspA) and C (AspC) motifs, respectively, and two divalent metal ions for the formation of the phosphodiester bonds (Joyce & Steitz, 1995; Steitz, 1998). The carboxylates anchor a pair of divalent metal ions, which play the major role in catalysis; one divalent metal ion (Mg2+ ion 1) promotes the deprotonation of the 3' hydroxyl of the nascent strand, while the other (Mg2+ ion 2) facilitates the formation of the pentacovalent transition state at the {alpha}-phosphate of the dNTP and the exit of the inorganic pyrophosphate group (PPi).



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Fig. 2. Catalytic mechanism of nucleotidyl transfer. [Modified from Steitz (1998) with permission.]

 
Here, we will point out that, in addition to the obvious similarities, viral RdRPs have a number of unique features which are important for efficient and accurate initiation of RNA-dependent RNA synthesis in the different viruses.

Diverse terminology has been used in the literature to describe components of the polymerase initiation complex. In this review, the term ‘RdRP’ will be used to refer to the catalytic subunit of RNA virus polymerases. The template nucleotides at the 3' end of the template (which is used to initiate de novo RNA synthesis) will be called T1, T2,..., in the 3'->5' direction. Nucleotides of the daughter strand are denoted D1, D2,..., in the 5'->3' order, such that D1 base pairs with T1, D2 with T2, and so on. In the literature, D1 is sometimes called the initiation nucleotide (NTPi) and D2 is called NTPi+1. With regard to the polymerization reaction, we differentiate between replication (synthesis of genomic RNA) and transcription (synthesis of viral mRNAs).

Initiation mechanisms
Although diverse RNA viruses use an amazing variety of replication scenarios, there are only two principally different mechanisms by which RNA synthesis can be initiated (Fig. 3): de novo and primer-dependent initiation (reviewed by Kao et al., 2001, and references therein; Ranjith-Kumar et al., 2002b; Paul et al., 1998).



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Fig. 3. Schematic diagram of initiation mechanisms of viral RNA-dependent RNA polymerases. (A) De novo initiation from the 3' terminus of the viral genome (i) or internal to the template during subgenomic synthesis (ii). (B) Primer-dependent initiation where the primer can be a protein primer, a snatched cap or an oligonucleotide (iii) or the 3' terminus of the template that folds back mimicking a template–primer structure (iv).

 
De novo initiation
De novo initiation, also known as primer-independent initiation, requires interactions of at least the following four components: (i) the RdRP; (ii) the RNA template with a virus-specific initiation nucleotide; (iii) the initiation nucleoside triphosphate (D1); and (iv) a second NTP (D2). The first phosphodiester bond is formed between D1 and D2. The initiation nucleotide (essentially a one-nucleotide primer) provides the 3'-hydroxyl for the addition of the next nucleotide. The advantage of de novo RNA synthesis for viral RNA replication is that no genetic information is lost during replication and no additional enzymes are needed to generate the primer or to cleave the region between template and newly synthesized RNA. In most cases, the productive de novo initiation event is immediately followed by elongation. However, in some instances de novo initiation leads to the formation of abortive RNA products (abortive initiation) or gives rise to short RNA oligonucleotides that are subsequently used as primers (prime and realign mechanism).

Abortive initiation.
The de novo synthesis of short abortive RNAs (two to five nucleotides) has been reported for T7 bacteriophage, Escherichia coli and eukaryotic DdRPs (Martin et al., 1988; Carpousis & Gralla, 1980; Ackerman et al., 1983) and viral RdRPs such as those of turnip crinkle carmovirus (Nagy et al., 1997), reovirus (Yamakawa et al., 1981) and rotavirus (Chen & Patton, 2000). Several RdRPs that initiate de novo can also use these oligonucleotides to replace D1 in vitro (Downing et al., 1971; Garcin & Kolakofsky, 1992; Honda et al., 1986; Kao & Sun, 1996; Nagy et al., 1997).

Prime and realign.
With some negative-strand RNA viruses such as Bunya-, Arena- and Nairoviruses, the 5' ends of the genomic and antigenomic RNAs contain non-templated nucleotides (Garcin & Kolakofsky, 1990, 1992; Garcin et al., 1995; Jin & Elliott, 1993). Arenavirus RNA initiation is hypothesized to take place from an internal templated cytidylate. After the synthesis of a few nucleotides, the daughter RNA is shifted to the position 3' of T1 so that the initiation GTP overhangs the end of the template for genomic and antigenomic RNA synthesis. The prime and realign mechanism explains the extra guanylate present at the 5' ends of the genomes and antigenomes and may be selected because it allows de novo initiation to take place from a protected internal nucleotide without losing genetic information (Kao et al., 2001).

Primer-dependent initiation
Several viruses initiate RNA synthesis using either an oligonucleotide or protein primer: (i) Oligonucleotides cleaved from the 5' end of a capped cellular mRNA (cap-snatching) are used by many segmented negative-strand RNA viruses for transcription (Hagen et al., 1995). (ii) A short leader RNA (two to five nucleotides) synthesized by viral RdRPs during abortive cycling (described above) can also serve as primer (McClure, 1985). (iii) With template-primed initiation (also known as loop-back, copy-back, turn-around or back-priming synthesis), the 3' end of the template RNA loops back on itself to serve as a primer (Behrens et al., 1996; Zhong et al., 1998, 2000b; Luo et al., 2000; Laurila et al., 2002). (iv) With protein-primed initiation, an amino acid provides the hydroxyl group for the formation of a phoshodiester bond with the first nucleotide. This mechanism is used by Picornaviridae (Paul et al., 1998) and DNA viruses such as adenoviruses and bacteriophages {pi}29 and PRD1 (Salas, 1991).

RNA viruses can use either one or sometimes both of these mechanisms for initiation of RNA synthesis. De novo initiation is used by viruses with positive, negative, double-stranded (dsRNA) and ambisense RNA genomes (Kao et al., 2001). Specific examples include the dsRNA viruses such as the Cystoviridae (Makeyev & Bamford, 2000a, b; Yang et al., 2003a) and rotavirus (Chen & Patton, 2000) and negative-strand RNA viruses such as vesicular stomatitis virus (VSV) (Testa & Banerjee, 1979). De novo initiation is widely used by positive-strand RNA viruses; examples include plant alphavirus-like viruses (Strauss & Strauss, 1994; Goldbach et al., 1991), members of the family Flaviviridae, namely hepatitis C virus (HCV) (Kao et al., 1999, 2001; Luo et al., 2000; Oh et al., 1999; Zhong et al., 2000a), Kunjin (Guyatt et al., 2001) and dengue 2 (Ackermann & Padmanabhan, 2001), as well as bacteriophage Q{beta} (Blumenthal, 1980). Poliovirus exclusively uses primer-dependent initiation (Paul et al., 1998), while influenza virus employs a combination of the two mechanisms with the choice being determined by the type of RNA to be synthesized (Honda et al., 1986). Back priming, however, appears to be an artefact of in vitro reactions. Although RdRPs from HCV and bovine viral diarrhoea virus (BVDV) preferentially utilize back priming in vitro, they most likely use de novo initiation in vivo (Ranjith-Kumar et al., 2002a, and references therein; Kao et al., 2001; Gong et al., 1998a, b).

Most viruses initiate both replication and transcription at the very terminal end of the genome. However, some may employ internal initiation of RNA synthesis for transcription (reviewed by Miller & Koev, 2000, and references therein). Examples include VSV, brome mosaic virus (BMV) and cucumber necrosis virus (CNV). VSV transcription initiates at different positions in vitro and in vivo. In vitro, VSV initiates transcription at the genomic 3' end (Emerson, 1982), but in vivo transcription initiates internally directly at the first gene-start sequence (Whelan & Wertz, 2002). RNA-dependent RNA replicases of the plant viruses CNV and BMV have distinct modes of initiation site recognition for initiation at the 3' terminus and internal initiation (Panavas et al., 2002; Ranjith-Kumar et al., 2003). Enhancer-like activity of a viral RNA promoter seems to be important in this differentiation.


   INSIGHTS FROM THE RdRP STRUCTURES
Top
ABSTRACT
INTRODUCTION
INSIGHTS FROM THE RdRP...
ROLE OF DIVALENT CATIONS
TEMPERATURE DEPENDENCE
REFERENCES
 
To date, the structures of six viral RdRPs have been determined (Table 1), namely those of poliovirus (PV) 3Dpol (Hansen et al., 1997; Hobson et al., 2001), HCV NS5B of the BK strain and HCV NS5B of the J4 strain (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999; O'Farrell et al., 2003), bacteriophage {pi}6 protein P2 (Butcher et al., 2001; Salgado et al., 2004), rabbit haemorrhagic disease virus (RHDV) 3Dpol (Ng et al., 2002) and reovirus protein {lambda}3 (Tao et al., 2002). The structures of initiation complexes have been solved for bacteriophage {pi}6 P2 (Butcher et al., 2001; Salgado et al., 2004), reovirus {lambda}3 (Tao et al., 2002) and HCV HC-J4 NS5B (O'Farrell et al., 2003).


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Table 1. Information on known structures of viral RNA-dependent RNA polymerases

 
The new structural data confirm the proposal that all nucleic acid polymerases share similarities in structure and the mechanism of catalysis (Joyce & Steitz, 1995). The basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for catalysis. However, RdRPs also have specific features that distinguish them from other polymerases. The structural characteristics of viral RdRPs that have been determined closely resemble each other (Hansen et al., 1997; Bressanelli et al., 2002; Butcher et al., 2001; Doublie et al., 1999; Huang et al., 1998; Tao et al., 2002; O'Farrell et al., 2003). One structural attribute that distinguishes most RdRPs from other polymerases is their ‘closed hand’ conformation, as opposed to the ‘open hand’ shape of the other known polymerases. The closed conformation is accomplished by interconnecting the fingers and thumb domains with several loops (fingertips) protruding from the fingers. The closed structure creates a well-defined template channel, which might regulate the recognition of the initiation site (Bressanelli et al., 2002; Butcher et al., 2001). The template channel and additional structures near the active site ensure that initiation of minus-strand RNA synthesis takes place at or near the end of the 3' termini of the HCV and bacteriophage {pi}6 RNAs (Hong et al., 2001; Laurila et al., 2002). The NTP (substrate) tunnel is another common structural RdRP element. The hypothesis is that negatively charged incoming NTPs interact sequentially with positively charged amino acids in the tunnel to reach the active site. DdRPs also have a well-defined channel for nucleotide diffusion (Gnatt et al., 2001; Murakami et al., 2002). Co-crystallization with nucleoside triphosphates and/or with oligonucleotides has mapped substrate-binding sites, while the binding of Mg2+ and/or Mn2+ has mapped the active site of the enzymes.

The overall structural similarity and the conservation of secondary and tertiary structure elements in the palm and thumb domains of polymerases of the families Picorna-, Flavi-, Cysto- and Retroviridae has led to speculation that they may have evolved from a common ancestor (Butcher et al., 2001; O'Farrell et al., 2003). These structural similarities are not predictable by comparative sequence analyses alone (Koonin, 1991; Iyer et al., 2003). The general significance of structural conservation in the context of virus evolution has been discussed elsewhere (Bamford et al., 2002; Bamford, 2003).

Model for the initiation of RNA synthesis of bacteriophage {pi}6
The structures of the initiation complexes of the RdRPs of reovirus (Tao et al., 2002) and bacteriophage {pi}6 (Butcher et al., 2001; Salgado et al., 2004) allow one to elaborate on their initiation mechanisms. Here we will discuss the proposed model for initiation of viral RNA synthesis for bacteriophage {pi}6 RdRP (Butcher et al., 2001). The bacteriophage {pi}6 RdRP is a compact, spherical molecule (Butcher et al., 2001). Its shape is attributed to two elaborations of the basic right hand architecture, a polypeptide chain that connects the fingertips and thumb similar to the chain identified for the HCV RdRP and a C-terminal elaboration, known as the initiation or priming platform, resembling the C-terminal {beta}-hairpin of the HCV RdRP (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999).

The following structures have been solved for the {pi}6 RdRP: the RdRP complexed with a DNA (Butcher et al., 2001) or RNA oligonucleotide representing the 3' end of the negative-sense strand of the viral genome (Salgado et al., 2004); the RdRP complex with DNA and two GTPs (Butcher et al., 2001); and that of a dead-end initiation complex (Salgado et al., 2004). The latter structure demonstrates that the polymerase is active in the crystal form. The structural details of these complexes complement and extend the biochemical and genetic information on the initiation mechanisms.

The {pi}6 RdRP has two positively charged tunnels that, respectively, allow the access of the RNA template and NTP substrates to the active site. The template RNA enters the polymerase through the channel formed by the interaction between the thumb and the fingers, while NTPs enter the catalytic site through the opening formed primarily by the fingers and palm domains. The template tunnel is wide enough to accommodate single-stranded RNA (ssRNA) but not dsRNA. The distance from the surface to the active site can be spanned by a 5-mer single-stranded oligonucleotide.

Structural and biochemical studies indicate that the C-terminal domain and initiation platform of the {pi}6 RdRP have three functions (Butcher et al., 2001; Laurila et al., 2002). They (i) stabilize the initiation complex by interacting with the initiation nucleotides; (ii) prevent back priming; and (iii) serve as a physical barrier to block the exit end of the template tunnel during initiation.

From the structure of the bacteriophage {pi}6 RdRP–DNA complex, Butcher et al. (2001) proposed a sequence of events that could result in the formation of the initiation complex as follows (Fig. 4). Steps I–IV: the template enters the tunnel and interactions with the specificity pocket, S, lock it in place; NTPs occupy site I, presumably in rapid exchange. Step V: the D1 GTP binds to the initiation platform (P), stabilized by three hydrogen bonds to the cytidine of the template and stacking interactions with Tyr-630 of the initiation platform in the C-terminal domain. Step VI: the template ratchets back, facilitated by electrostatic attraction to Arg-268 and Arg-270, freeing T1 from the specificity pocket. Step VII: a second GTP, D2, enters the P site to lock the initiation complex into its active form; catalysis releases pyrophosphate, freeing the nucleotide from Arg-268 and Arg-270 so that it can ratchet down, displacing the C-terminal domain of the protein (step VIII). This may be facilitated by attraction of the phosphates of the GTP in site P to the Mn2+ ion. The next NTP slips into the catalytic site, C, from site I, which sets the ratchet for the chain elongation to start.



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Fig. 4. Model for initiation and chain elongation of the bacteriophage {pi}6 RdRP, illustrating key points in the reaction mechanism. Red boxes highlight experimental results. (I) Apo structure with bound Mn2+. Binding sites are identified in black letters. (II) NTP bound in site I. (III) Template bound. (IV) Template bound and NTP non-productively bound at site I. (V) Initial productive binding at site P. (VI) Template ratchets back. (VII) Second GTP bound at site P. Polymerization can occur. (VIII) Polymerization has occurred, releasing nascent duplex from ordered binding at the active site C. The C-terminal domain moves allowing the duplex to ratchet forward, out of the active site. [From Butcher et al. (2001), with permission.]

 
When RNA oligonucleotides bind in the RdRP template tunnel, the orientation of the bases varies resulting in different oligonucleotide–RdRP interactions (Salgado et al., 2004). The most important difference is in the specificity pocket, where the strictly conserved T1 cytidine is rotated approximately 180°. The T2 RNA nucleotide is stabilized by base stacking with the T3 uracil, an effect not observed with DNA. Overall, the presence of an extra hydroxyl group in RNA changes the sugar conformation of the nucleotides and slightly rearranges the side-chains of interacting polymerase residues, leading to the formation of extra hydrogen bonds between the protein and oligonucleotide, further stabilizing the RNA complex compared with the DNA complex (Fig. 5). This might be the basis for the preference of the bacteriophage {pi}6 RdRP for RNA templates.



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Fig. 5. Superposition of an RNA oligonucleotide and DNA oligonucleotide binding in the template tunnel of bacteriophage {pi}6. In general, RNA (red) and DNA (blue) bind to the template tunnel in a similar way, although they exhibit different base and sugar orientations, particularly in the 3' and 5' ends. The presence of the hydroxyl group in the sugar causes rearrangements of the nucleotides and the surrounding residues (not shown). The most important difference observed is in position 1, the conserved cytidine in the specificity pocket (indicated by black arrows). There is a movement of roughly 180° of the base, accompanied by a change in sugar orientation. (Courtesy of P. S. Salgado, J. M. Grimes & D. I. Stuart.)

 
Structural features facilitating initiation of RNA synthesis
Structural elements that ensure de novo initiation have been identified. The initiation mechanism for bacteriophage {pi}6, where the two initiation nucleotides stack against each other and against a specialized priming platform followed by repositioning of this element to allow egress of the product, may also apply to the RdRPs of HCV (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999) and reovirus (Tao et al., 2002). It may even be used as an initiation paradigm in many RNA polymerases. Structural comparisons of unrelated RNA polymerases have recently revealed general features of initiation (Murakami & Darst, 2003). Similar to the {pi}6 C-terminal domain, the N-terminal domain in the bacteriophage T7 DdRP initiation complex blocks the path of the elongating RNA transcript and also requires conformational changes to facilitate the transition from initiation to elongation. However, the finger domains differ significantly among viral RNA polymerases. This may be a result of specific adaptations to structurally diverse substrates (Hobson et al., 2001). Structural details of individual RdRPs are as follows.

HCV.
The fingers domain of the HCV RdRP has a long binding groove, which constitutes a template tunnel (O'Farrell et al., 2003). Other specific structural features of the RdRP include a loop that connects the fingers with the thumb (the {Lambda} or A1-loop) and a {beta}-hairpin protruding from the thumb domain toward the active site at the base of the palm domain (Bressanelli et al., 1999; Lesburg et al., 1999). The A1-loop may be responsible for the closed conformation of the RdRP and the ‘clamping’ motion of the enzyme (Labonte et al., 2002). Mutation of Leu-30 (which has a strong hydrophobic interaction with the thumb domain) to polar residues was detrimental to RdRP activity, confirming that it is a critical element for enzyme activity (Labonte et al., 2002). The {beta}-hairpin seems to be important in positioning the 3' terminus of the viral genome for correct initiation of replication. It allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site and may function as a gate preventing the 3' terminus of the template RNA from slipping through the active site, thus ensuring terminal initiation of replication (Hong et al., 2001). Recently, a regulatory motif in the C-terminal non-catalytic region of the HCV RdRP has been identified upstream of the membrane anchor domain. It comprises a unique conserved hydrophobic pocket, which protrudes into the RNA-binding cavity. Several functions have been proposed for this motif. One is that, together with the {beta}-hairpin, it forms a rigid bulky loop at the active site that serves as an initiation platform similar to that of bacteriophage {pi}6 (Butcher et al., 2001). Other possible functions are that it could play a role in initiation in ensuring correct RNA replication by preventing primer-dependent and back-primed initiation and recognition of the correct secondary structure at the 3'-terminal end of the HCV genome (Leveque et al., 2003).

Crystal structures of the HCV RdRP (genotype 1b, strain J4) complexed with metal ions and NTPs revealed that the fingers domain forms a positively charged NTP tunnel (O'Farrell et al., 2003). The HCV RdRP–rNTP complexes indicated how incoming NTPs might access the metals by moving along the funnel and how, subsequently, pyrophosphate exits the active site without necessitating large structural changes. The J4 RdRP has a single NTP molecule in the polymerase active site (O'Farrell et al., 2003), while NTP complexes of the RdRP of the HCV BK {Delta}55 strain accommodated two NTPs (Bressanelli et al., 1999). The difference has been attributed to the deletion of the C terminus in the {Delta}55 RdRP, which improves solubility (Yamashita et al., 1998; Tomei et al., 2000) but may change its properties. Structurally this deletion causes a shift in the positions of the {beta}-flap backbone atoms, which increases the active site volume and may allow the binding of an extra triphosphate (Bressanelli et al., 1999). Structure-guided site-directed mutagenesis elucidated the role of some basic amino acids in NTP trafficking. The last positively charged amino acid (Arg-222) in close proximity to the active site is critical in delivering NTPs to the active site. Substitution of Lys-151 at the beginning of the NTP tunnel with glutamic acid increased enzyme activity compared with the wild-type due to the formation of more productive pre-initiation complexes (Labonte et al., 2002). Arg-518 in the BVDV RdRP and its HCV RdRP equivalent, Arg-386, were important for de novo RNA synthesis (Lai et al., 1999; Hong et al., 2001). HCV Arg-386 proved to be important for dinucleotide-initiated RNA synthesis. These observations support the hypothesis that Arg-386 plays a critical role in stabilizing the interaction between the 3' terminus of the RNA template and the initiating nucleotide(s) (Hong et al., 2001).

The crystal structure of the HCV RdRP indicates that there is not enough space underneath the {beta}-hairpin to allow the passage of a dsRNA product. Therefore, the {beta}-hairpin should be flexible to give way to the nascent dsRNA (Hong et al., 2001). It was hypothesized that a change in conformation of the thumb domain takes place upon template binding to allow efficient de novo initiation of RNA synthesis (Bressanelli et al., 2002).

Reovirus.
Structural features of the reovirus RdRP that facilitate initiation of RNA synthesis include a cube-like cage structure, tunnels and a priming loop. The reovirus RdRP polypeptide chain folds into a compact unit with a central cavity. It has three domains: a central polymerase domain, which contains the fingers, palm and thumb subdomains, an N-terminal domain, which bridges the fingers and thumb on one side of the catalytic cleft, and a C-terminal bracelet domain, which covers the catalytic cleft on the other side (Tao et al., 2002). The catalytic site, enclosed in the centre, is accessible to solvent, substrate or template/product through four channels of various sizes at the ‘front’, ‘left’, ‘rear’ and ‘bottom’. The front channel is the bracelet opening and is large enough to accommodate dsRNA. The left channel is at the interface of the polymerase and bracelet domains and the rear channel is at the interface of the polymerase and N-terminal domains. All three domains border the bottom channel. Tao et al. (2002) suggested that the roles of the channels are as follows. The front channel is for the exit of newly formed dsRNA produced during replication. The left channel is for template entry and the rear channel is the substrate tunnel. The bottom channel in the reovirus RdRP is a structural feature not yet observed in other polymerases. It is most likely the exit route for mRNA produced during transcription.

The reovirus RdRP also has a special priming loop that supports the stacking of the priming NTP, D1. This loop is a unique insertion within a strand present in the palms of all other polymerases. In the fully active polymerase complex, this loop retracts towards the palm with respect to its position in the apo-enzyme and in the initiation complex to fit into the minor groove of the product duplex (Tao et al., 2002).

RHDV and poliovirus.
These viruses have a virus-encoded VPg covalently linked to the 5' terminus of their genome. In vitro, the RHDV RdRP uses back priming from the 3'-hydroxyl end of the genome for minus-strand synthesis (López Vázquez et al., 2001). However, Paul et al. (1998) reported that poliovirus VPg facilitates protein-primed RNA synthesis and predicted that this mechanism should function in most plus-strand RNA viruses with protein-linked genomes. The thumb domain of the RHDV RdRP seems to play an important role in RNA synthesis. Comparisons among the structures of the alternative conformational states of RHDV RdRP and RdRPs from HCV and poliovirus suggest novel structure–function relationships (Ng et al., 2002). The RHDV thumb domain can adopt two conformations. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important for enzymic function in RdRPs. The most dramatic difference between the RHDV and poliovirus RdRPs and that of HCV occurs in the loop connecting the third and fourth helices in poliovirus and RHDV RdRPs. In the HCV RdRP, this loop is replaced by a long {beta}-hairpin insert that occludes the active site cleft. The shorter loops seen in the poliovirus and RHDV enzymes are consistent with their ability to utilize dsRNA as templates in vitro (Arnold & Cameron, 2000; López Vázquez et al., 2001; Hong et al., 2001) and with the fact that the poliovirus RdRP does not have a structural feature equivalent to the HCV RdRP {beta}-hairpin or the bacteriophage {pi}6 RdRP initiation platform and uses protein-primed initiation for genome replication (Paul et al., 1998).

For bacteriophage {pi}6 and HCV RdRPs, the initiation mechanism of RNA synthesis could be changed from de novo to primer-dependent initiation by reducing their initiation platforms. For the {pi}6 RdRP, YKW (aa 630–632) was changed to GSG (Laurila et al., 2002) and the HCV {beta}-hairpin (aa 443–454) from LDCQIYGACYSI to LGGI (Hong et al., 2001).

Specialized features that facilitate dsRNA strand separation
Specialized features have been proposed to facilitate dsRNA strand separation and provide the basis for the mechanism that ensures feeding of the correct strand to the catalytic site for initiation of RNA synthesis. These include the reovirus 5'-cap binding site, which was identified on the surface of the RdRP between the template entrance and exit channels (Tao et al., 2002), and the bacteriophage {pi}6 plough, which is adjacent to a positively charged groove over the polymerase surface (Butcher et al., 2001). Structural comparisons and bioinformatic analysis of the HCV, RHDV and bacteriophage {pi}6 RdRPs also identified the N-terminal region of motif F as possibly being involved in unwinding of dsRNA for transcription (Bruenn, 2003). Whereas replication is a single event for dsRNA viruses, transcription involves multiple rounds of initiation. The mechanism of reinitiation on dsRNA templates is presently unknown. According to some models, it may be facilitated by a helicase suitably positioned in relation to the polymerase to displace the nascent chain from the transcription complex (Butcher et al., 2001).

Active and inactive polymerase conformations
The repeated observations of an open conformation (not to be confused with the general ‘open hand’ structure of polymerases other than the RdRPs) in three structures of apo forms of RdRPs (RHDV, PV and HCV) suggest that these enzymes may prefer to adopt a catalytically inactive state, which can be transformed to a catalytically competent state by the binding of a primer, template, divalent metal ions and nucleotide triphosphates (Ng et al., 2002).

RHDV.
The thumb domain of the RHDV RdRP can adopt either an open or closed conformation (Ng et al., 2002). Under physiological ionic strength conditions, divalent metal ions are bound to the enzyme and the active site cleft is open to the binding of RNA primer–template duplexes. Comparisons between the RHDV RdRP structure and the structures of PV and HCV RdRPs indicate that conformational changes similar to those seen in DNA polymerases may be important to the catalytic mechanism of RdRPs. The closed conformation is probably the active form of the RdRP because the ion coordination closely matches that seen in active enzyme–NTP–primer–template complexes formed by related DdDPs and the human immunodeficiency virus 1 RT (Doublie et al., 1999; Huang et al., 1998).

Reovirus.
Crystalline reovirus RdRP is catalytically active. Structures of complexes have been obtained of the enzyme stalled at the initiation complex and of a fully active enzyme performing polymerization (Tao et al., 2002). The structure of the apo form of the RdRP shows that only internal adjustments are required to accommodate substrates. Upon the formation of the first phosphodiester bond, the reovirus priming loop that supports the priming nucleotide D1 through base stacking retracts towards the palm with respect to its position in the apo-enzyme and in the initiation complexes to fit into the minor groove of the product duplex. This allows the newly synthesized RNA to exit the polymerase and facilitates the transition between the initiation and elongation stages of RNA synthesis (Tao et al., 2002).

HCV.
The orientation of the nucleotides in the active site of HCV RdRP was identified by superpositioning HCV structures on to the initiation complex of the bacteriophage {pi}6 RdRP (Bressanelli et al., 2002). Density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent. A network of triphosphate densities was detected that superimposed on the corresponding nucleotide moieties seen in the {pi}6 RdRP initiation complex, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. Three HCV RdRP amino acids that bind the triphosphate moiety of the nucleotide at the priming site, Arg-158 in the fingers and Ser-367 and Arg-386 in the thumb, are conserved among HCV with different genotypes and in most Flaviviruses. A large solvent cavity was noticed in the region where the priming nucleoside moiety should lie, suggesting that a different conformation of the HCV RdRP thumb is needed for efficient initiation, in which the protein, through an element similar to the protein platform provided by residue Tyr-630 of the bacteriophage {pi}6 RdRP, would hold the D1 base in place to make Watson–Crick interactions with the 3' base (T1) of the template.

Analysis of complexes of the HCV RdRP with NTPs and divalent metal ions revealed a specific GTP-binding site in a shallow pocket at the molecular surface of the enzyme at the interface between fingers and thumb. The position of this site suggested a possible role of GTP either in triggering the conformational change or in stabilizing the active conformation for efficient initiation (Bressanelli et al., 2002). Since no conformational change was observed in the crystal of the enzyme with bound GTP, it was speculated that the presence of both template in the RNA-binding groove and GTP at the surface site might be necessary for such a conformational change. Alternatively, the surface site may provide an oligomerization surface for RdRP, and the oligomeric form could adopt the correct conformation required for efficient initiation. Recently, Qin et al. (2002) reported a possible dimerization of the HCV RdRP and identified two surface amino acids as being essential for dimerization and activity.

Template requirements for initiation
Specific template recognition by the replicase is essential for faithful genome replication. In most viral systems, RdRP–RNA interactions account only partially for the overall template specificity. Factors that usually contribute to the template specificity of RNA viruses include: (i) RdRP template preferences determined by the primary sequence and secondary structure at the 3' end or internal initiation site; and (ii) specific template interactions of other cell- and virus-encoded proteins. For example, the Q{beta} replicase ensures specificity as a heterotetramer consisting of the viral RdRP and three host proteins (see, for example, Brown & Gold, 1996). In dsRNA viruses such as bacteriophage {pi}6, template specificity is achieved by exclusive packaging of virus-specific RNAs in a polymerase complex comprised of several structural proteins (Mindich, 1999). For some viruses, template specificity may be coupled with other processes, such as translation. Here, we will discuss the significance of the interaction between RdRPs and their cognate templates.

The initiation nucleotide
Initiation efficiency can be affected by the affinity of the polymerase for the initiation nucleotide of the template and the initiation NTPs (D1 and D2). Our structural data show that the specificity pocket of bacteriophage {pi}6 RdRP is designed for 3'-terminal template cytidylates (Butcher et al., 2001; Salgado et al., 2004). Biochemical analysis shows that for the BVDV RdRP the N3- and C4-amino group of the initiation template cytidylate are essential for RNA synthesis (Kao et al., 1999; Kim et al., 2000). Consistently, GTP is the preferred D1 nucleotide for RdRPs of BMV, Q{beta}, BVDV, HCV, GB virus C (GBV-C) and members of the family Cystoviridae (Blumenthal, 1980; Jorgensen et al., 1969; Kao & Sun, 1996; Luo et al., 2000; Sivakumaran & Kao, 1999; Ranjith-Kumar et al., 2002b; Yang et al., 2001, 2003b).

Although they all use de novo initiation, the RdRPs of the flaviviruses HCV, BVDV and GBV-C have distinct initiation preferences (Ranjith-Kumar et al., 2002b). The BVDV RdRP prefers to initiate from the 3'-terminal cytidylate, but can also use a penultimate cytidylate. The BMV, turnip yellow mosaic virus and phage Q{beta} are known to use penultimate cytidylate for initiating RNA synthesis (Deiman et al., 1998; Singh & Dreher, 1998, Sivakumaran & Kao, 1999; Sun et al., 1996; Yoshinari & Dreher, 2000). The ability of RdRP to initiate from penultimate bases may be important for HCV and other RNA viruses whose polymerases can add extra nucleotides to the RNA 3' end in the process of a terminal transferase reaction (Ranjith-Kumar et al., 2002c).

Although the RdRP seems to contribute to the overall template selection of the replication/transcription machinery by selecting specific T1 or/and D1, it is important to remember that both T1 and D1 requirements may not be very stringent, especially in vitro. This may account for some controversy in the RdRP literature. For example, Zhong et al. (2000a) observed that BVDV and HCV initiate from a purine nucleotide in the template, while others found that the RdRPs have a specificity for initiation from pyrimidines (Luo et al., 2000; Oh et al., 2000; Sun et al., 2000). In contrast to the observations of Kao and co-workers (2000), Zhong et al. (2000a) and Kao et al. (1999) demonstrated that short templates containing a 2',3'-dideoxynucleotide could direct de novo initiation by the HCV RdRP and BVDV RdRP, respectively. Two possible explanations have been offered. One is that the two polymerases have different initiation requirements. Another is that the RdRPs are slightly different. Kao et al. (2000) used a full-length HCV RdRP, while others used proteins that lacked 20–55 C-terminal residues (Kao et al., 1999; Luo et al., 2000). The C-terminal tail of the HCV RdRP is present in the active site of the crystal structure and has been hypothesized to play a role in template discrimination (Ago et al., 1999).

Some RNA polymerases may prefer pyrimidines in the D1 position (purines in T1) or have a naturally relaxed preference to the initiation nucleotide. The minus-strand RNA of a bovine coronavirus contains a uridylate as the 5'-terminal nucleotide (Hofmann & Brian, 1991). In Semliki Forest virus, the 5' termini of the replication intermediates, but not the virion RNAs, are pyrimidines (Sawicki & Gomatos, 1976). The use of either purine or pyrimidine triphosphates as D1 has also been documented in DdRPs (Schibler & Perry, 1977; Reddy & Chatterji, 1994).

De novo initiation of viral RNA synthesis seems to involve a higher Km for D1 than for other NTPs (Kao & Sun, 1996; Gaal et al., 1997; Joyce, 1997; Kim et al., 2000; Luo et al., 2000; Testa & Banerjee, 1979; Laurila et al., 2002), indicating that initiation is the replication efficiency-limiting step that may be subject to additional regulation. Examples of viruses for which this seems to be the case include bacteriophage {pi}6 (Laurila et al., 2002), Q{beta} (Blumenthal, 1980), BMV (Kao & Sun, 1996), HCV and BVDV (Kao et al., 1999), and also cellular and viral DdRPs (Losick & Chamberlin, 1976). Increased stability of the ternary complex has been demonstrated in the presence of a high initiation substrate concentration (Gaal et al., 1997). For the BMV replicase, the presence of GTP at the initiation site resulted in the formation of a more stable initiation complex (Sun & Kao, 1997a, b).

Specific template requirements
Beyond the first nucleotide, a stretch of nucleotides proximal to the initiation site may also stimulate initiation by RdRPs in virus systems. The initiation site context serves as a major determinant of template specificity, e.g. in turnip yellow mosaic virus, turnip crinkle virus and Q{beta} (Deiman et al., 1998, 2000; Singh & Dreher, 1997, 1998; Yoshinari et al., 2000). While these RNA viruses require additional regulatory sequences for RNA replication in vivo, cis-acting elements adjacent to the initiation site may form generic RNA structures that are highly tolerant of change (Blumenthal, 1980; Chen et al., 2000; Singh & Dreher, 1998). Specific sites within the polypyrimidine tract of the 3'-untranslated region of HCV have recently been identified where RNA synthesis is initiated de novo under in vitro conditions (Pellerin et al., 2002). Highly specific cis-acting signals have been also observed in several other systems (Adkins et al., 1997; Kim et al., 2000; McKnight & Lemon, 1998; Osman et al., 2000; Siegel et al., 1998; Sit et al., 1998; You & Padmanabhan, 1999). An RNA composed of a stem–loop and a single-stranded sequence is a common feature in initiating viral RNA synthesis for replication (Van Belkum et al., 1985; Seal et al., 1994; Netolitzky et al., 2000; Osman et al., 2000; Lahser et al., 1993; Fechter et al., 2001). One function of these 3'-end secondary structures may be to prevent back priming. For transcription, however, dsRNA viruses need to initiate from dsRNA templates. In vivo, dsRNA viruses efficiently transcribe dsRNA templates, but in vitro initiation from dsRNA templates with RdRPs is much less efficient than with ssRNA templates (Makeyev & Bamford, 2000b; Yang et al., 2001; Laurila et al., 2002). This could be due to a lack of strand-separation ability. When the {pi}6 polymerase complex contains drastically reduced amounts of its normal protein P4 complement, it could perform replication but not initiate transcription (Pirttimaa et al., 2002). However, adding this virus-specific RNA-dependent packaging NTPase (P4) to the bacteriophage {pi}6 RdRP does not stimulate in vitro transcription. Therefore, it has been speculated that efficient initiation of transcription requires the structural design of the {pi}6 polymerase complex or that the presence of a complete dsRNA genome in the polymerase complex at completion of replication might induce a conformational change that switches the RdRP from a replicase to a transcriptase (Makeyev & Grimes, 2004). The latter suggestion is supported by biochemical data showing that the large genome segment of bacteriophage {pi}6 regulates the switch between replication and transcription in vitro (Frilander et al., 1995; Van Dijk et al., 1995).

HCV.
Recombinant HCV RdRP does not exhibit strict template specificity in vitro. It catalyses various viral and non-viral RNA templates in vitro if the RNA templates have a stable secondary structure and a single-stranded sequence that contains at least one 3' cytidylate (Behrens et al., 1996; Kao et al., 2000; Luo et al., 2000; Oh et al., 1999, 2000; Zhong et al., 2000b). However, Kim et al. (2002) found that native X RNA, which is part of the HCV 3'-untranslated region and plays a major role in the initiation of RNA replication after virus infection, was also an appropriate RdRP substrate, even though it contains a blunt-ended stem at its 3' terminus. However, in vivo the HCV RdRP must discriminate the HCV genomic RNA from other RNAs and catalyse its substrate for viral amplification.

Reovirus.
The reovirus polymerase favours a template G at position T2. It provides a carbonyl for a hydrogen bond with Arg-518 and an amino group for a hydrogen bond with the side-chain of Ser-682. U at this position can also interact favourably with Arg-518 but A and C cannot. Given the conserved 3'-terminal sequences of reovirus RNAs (plus, UCAUC-3'; minus, UAGC-3'), the preference for G or U at T2 promotes synthesis of RNAs of either sense (Tao et al., 2002).

Bacteriophage {pi}6 and other Cystoviridae.
Even though RdRPs of Cystoviridae have different template preferences, they all prefer RNAs with one or several 3'-terminal cytosines (Makeyev & Bamford, 2000b; Yang et al., 2001). The replication efficiency seems to be controlled at the initiation step and RdRPs prefer pyrimidine-rich 3'-terminal initiation sites, C-3' being better than U-3'. The template secondary structure does affect initiation of RNA synthesis. When a stable hairpin–tetraloop structure was added to a template it was replicated one order of magnitude less efficiently than the same template lacking the hairpin (Laurila et al., 2002). For bacteriophage {pi}6 RNAs, the native secondary structure of the 3' end does not favour the formation of back-primed intermediates. The terminal regions of all three segments have a tRNA-like structure, with the five 3'-proximal nucleotides in a single-stranded form (Mindich et al., 1994). This five-nucleotide terminus can span the template channel of the bacteriophage {pi}6 RdRP (Butcher et al., 2001) but cannot loop back. However, when short 3'-terminal extensions are added to the RNA, back priming occurs (Laurila et al., 2002). This suggests that the bacteriophage {pi}6-specific 3'-end secondary structure might be a result of evolutionary selection to ensure accurate de novo initiation.

It is useful to remember that the template preferences may be obscured when saturating amounts of RdRPs are used in vitro and that bacteriophage {pi}6 RdRP and Q{beta} replicase can synthesize a number of unrelated RNAs in vitro – and to some extent in vivo (Avota et al., 1998, and references therein; Makeyev & Bamford, 2000a, b, 2001).


   ROLE OF DIVALENT CATIONS
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Divalent metal ions are essential for the nucleotide polymerization reaction (Fig. 2). In addition, several specific regulatory effects of metal ions on viral RNA synthesis have been described.

Role of Mg2+
The crucial role of Mg2+ ions in the catalysis of phosphodiester bond formation has long been known (Steitz & Steitz, 1993; Pelletier et al., 1994; Steitz, 1998). Mg2+ has also been implicated in stabilizing the daughter strand in replication complexes of bacteriophage T7 DNA polymerase (Doublie et al., 1998), bacillus DNA polymerase (Kiefer et al., 1998) and {pi}6 RdRP (Salgado et al., 2004). The {pi}6 RdRP Mg2+ ions also transiently stabilized the by-product, PPi, before its release via the substrate pore (Salgado et al., 2004).

Role of Mn2+
Manganese ions are known to stimulate a number of RdRPs including those of Q{beta} (Blumenthal, 1980; Blumenthal & Carmichael, 1979), HCV (Alaoui-Ismaili et al., 2000; Zhong et al., 2000b), BMV (Sun et al., 1996), poliovirus (Arnold et al., 1999) and members of the family Cystoviridae (Makeyev & Bamford, 2000b; Yang et al., 2001, 2003a). Mn2+ also stimulates the bacteriophage {pi}6 polymerase complex-based replication and transcription (Emori et al., 1983; Ojala & Bamford, 1995; Van Dijk et al., 1995) and is known to modulate substrate selectivity of DdRPs (Huang et al., 1997; Tabor & Richardson, 1989). The structures of the RdRPs of HCV, bacteriophage {pi}6 and RHDV have revealed that Mn2+ can bind in either the catalytic pocket (Bressanelli et al., 2002; Ng et al., 2002) and/or a specific allosteric position (Butcher et al., 2001).

Analysis of the role of metal ions in RNA-dependent RNA synthesis by three flavivirus recombinant RdRPs, GBV-C, BVDV and HCV, showed that only reactions with exogenously provided Mg2+ and Mn2+ were capable of RNA synthesis. Mg2+ and Mn2+ affected the mode of RNA synthesis of the three RdRPs. Both metals supported GBV-C RdRP de novo-initiated and primer-dependent RNA synthesis. However, Mn2+ significantly increased de novo initiation by HCV and BVDV RdRPs. In the case of HCV RdRP, Mn 2+ reduced the Km for the initiation GTP from 103 to 3 µM. Mn2+ increased de novo initiation, even at GTP concentrations that are comparable with physiological levels (Ranjith-Kumar et al., 2002a).

However, Mn2+ may not play a physiologically relevant role in RNA-dependent RNA synthesis because of its low intracellular concentration (Quamme et al., 1993; Zhang & Ellis, 1989). Furthermore, detecting de novo initiation in vitro does not by itself prove that this process is biologically relevant. Even poliovirus RdRP, which initiates replication in vivo with a protein primer, VPg (Paul et al., 1998), is capable of de novo initiation in vitro in the presence of Mn2+ (Arnold & Cameron, 1999; Arnold et al., 1999). The observation that the HCV RdRP catalytic-site is more occupied when the divalent ion is Mn2+ is suggestive of a stabilizing effect of this ion, favouring the binding of both initiating nucleotides in the active site region (Bressanelli et al., 2002). This may lead, for HCV RdRP also, to de novo initiation via a non-physiological mechanism in some in vitro conditions.

Crotty et al. (2003) recently generated the first viruses with a requirement for an alternative polymerase cation. They studied the function of Asn-297 in the poliovirus RdRP, one of the six core amino acid residues that are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. Viable mutants were identified with non-canonical amino acids at this position that exhibited Mn2+-dependent RNA replication and virus growth. This finding is important since it suggests that drugs targeting this region of RdRPs may still be subjected to the problem of drug-resistant escape mutants.

Role of Ca2+
Ca2+ inhibits in vitro transcription of reovirus (Sargent & Borsa, 1984), RNA polymerase II (Okai, 1982) and bacteriophage {pi}6 (Van Dijk et al., 1995). The crystal structures of {pi}6 RdRP initiation complexes with either Mg2+ or Ca2+ ions revealed key differences that may explain the inhibitory effect of Ca2+ (Butcher et al., 2001; Salgado et al., 2004). In the inhibition complex, the two Mg2+ ions that are present in the {pi}6 initiation complex are substituted by Ca2+ ions. One of the Ca2+ ions (denoted 2 by Salgado et al., 2004) occupies a position equivalent to the corresponding Mg2+ in the initiation complex (Butcher et al., 2001) and interacts with D2. The other Ca2+ ion (denoted 1) has a different coordination sphere from the equivalent Mg2+: it includes the stabilizing Tyr-630 and the triphosphate and base of D1, instead of the phosphate backbones of the two incoming GTPs. The tyrosine side-chain is rotated, disrupting the stacking between the amino acid and the base of the nucleotide. Furthermore, coordination of the phosphate backbone of D1 displaces it away from D2, preventing the necessary nucleophilic attack. Thus, Ca2+ seems to inhibit RNA synthesis by altering the geometry of interactions in the catalytic position.


   TEMPERATURE DEPENDENCE
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ABSTRACT
INTRODUCTION
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TEMPERATURE DEPENDENCE
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Differentiation between the temperature requirements for initiation and elongation of RNA synthesis has been documented for RdRPs. For the Cystoviridae, de novo initiation by RdRPs of bacteriophage {pi}6, {pi}12 and a temperature-sensitive mutant of bacteriophage {pi}12 was more sensitive to increased temperatures than elongation (Yang et al., 2003b). In in vitro assays, the recombinant dengue virus RdRP can either initiate RNA polymerization de novo or extend the template 3' terminus by back priming. At moderate temperatures, the enzyme predominantly uses de novo initiation, whereas back priming dominates at elevated temperatures (Ackermann & Padmanabhan, 2001). Based on these results and the observed structural differences between the poliovirus and HCV RdRP, a steric model was suggested where dengue RdRP can exist in either a closed or an open conformation, with the latter favoured at higher temperatures. The open conformation binds to a fold-back structure at the 3' terminus of the template with the subsequent elongation producing a dimerized product. Conversely, the closed conformation, favoured at lower temperatures, recognizes the single-stranded 3' terminus of the template and initiates de novo synthesis (Ackermann & Padmanabhan, 2001). The data suggest that de novo RNA-dependent RNA synthesis in many virus systems may include a specialized thermolabile state of the RdRP initiation complex.


   ACKNOWLEDGEMENTS
 
We thank Dr Sarah Butcher, Minni Laurila and Paula Salgado for valuable discussions and critical reading of the manuscript. This work was supported by the Academy of Finland (‘Finnish Centre of Excellence Program 2000–2005’, grants 1202855, 1202108 and 172621).


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ABSTRACT
INTRODUCTION
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Ackerman, S., Bunick, D., Zandomeni, R. & Weinmann, R. (1983). RNA polymerase II ternary transcription complexes generated in vitro. Nucleic Acids Res 11, 6041–6064.[Abstract]

Ackermann, M. & Padmanabhan, R. (2001). De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. J Biol Chem 276, 39926–39937.[Abstract/Free Full Text]

Adkins, S., Siegel, R. W., Sun, J. H. & Kao, C. (1997). Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase. RNA 3, 634–647.[Abstract]

Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K. & Miyano, M. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure Fold Des 7, 1417–1426.[Medline]

Alaoui-Ismaili, M. H., Hamel, M., L'Heureux, L., Nicolas, O., Bilimoria, D., Labonte, P., Mounir, S. & Rando, R. F. (2000). The hepatitis C virus NS5B RNA-dependent RNA polymerase activity and susceptibility to inhibitors is modulated by metal cations. J Hum Virol 3, 306–316.[Medline]

Arnold, J. J. & Cameron, C. E. (1999). Poliovirus RNA-dependent RNA polymerase (3Dpol) is sufficient for template switching in vitro. J Biol Chem 274, 2706–2716.[Abstract/Free Full Text]

Arnold, J. J. & Cameron, C. E. (2000). Poliovirus RNA-dependent RNA polymerase (3Dpol). Assembly of stable, elongation-competent complexes by using a symmetrical primer/template substrate (sym/sub). J Biol Chem 275, 5329–5336.[Abstract/Free Full Text]

Arnold, J. J., Ghosh, S. K. & Cameron, C. E. (1999). Poliovirus RNA-dependent RNA polymerase (3Dpol). Divalent cation modulation of primer, template, and nucleotide selection. J Biol Chem 274, 37060–37069.[Abstract/Free Full Text]

Avota, E., Berzins, V., Grens, E., Vishnevsky, Y., Luce, R. & Biebricher, C. K. (1998). The natural 6S RNA found in Q{beta}-infected cells is derived from host and phage RNA. J Mol Biol 276, 7–17.[CrossRef][Medline]

Bamford, D. H. (2003). Do viruses form lineages across different domains of life? Res Microbiol 154, 231–236.[CrossRef][Medline]

Bamford, D. H., Burnett, R. M. & Stuart, D. I. (2002). Evolution of viral structure. Theor Popul Biol 61, 461–470.[CrossRef][Medline]

Behrens, S. E., Tomei, L. & De Francesco, R. (1996). Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J 15, 12–22.[Abstract]

Blumenthal, T. (1980). Q{beta} replicase template specificity: different templates require different GTP concentrations for initiation. Proc Natl Acad Sci U S A 77, 2601–2605.[Abstract]

Blumenthal, T. & Carmichael, G. G. (1979). RNA replication: function and structure of Qbeta-replicase. Annu Rev Biochem 48, 525–548.[CrossRef][Medline]

Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., De Francesco, R. & Rey, F. A. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci U S A 96, 13034–13039.[Abstract/Free Full Text]

Bressanelli, S., Tomei, L., Rey, F. A. & DeFrancesco, R. (2002). Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol 76, 3482–3492.[Abstract/Free Full Text]

Brown, D. & Gold, L. (1996). RNA replication by Q beta replicase: a working model. Proc Natl Acad Sci U S A 93, 11558–11562.[Abstract/Free Full Text]

Bruenn, J. A. (2003). A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases. Nucleic Acids Res 31, 1821–1829.[Abstract/Free Full Text]

Buck, K. W. (1996). Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv Virus Res 47, 159–251.[Medline]

Butcher, S. J., Grimes, J. M., Makeyev, E. V., Bamford, D. H. & Stuart, D. I. (2001). A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240.[CrossRef][Medline]

Carpousis, A. J. & Gralla, J. D. (1980). Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry 19, 3245–3253.[Medline]

Cheetham, G. M. & Steitz, T. A. (2000). Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases. Curr Opin Struct Biol 10, 117–123.[CrossRef][Medline]

Chen, D. & Patton, J. T. (2000). De novo synthesis of minus-strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation. RNA 6, 1455–1467.[Abstract/Free Full Text]

Chen, M.-H., Roossinck, M. & Kao, C. (2000). Efficient and specific initiation of subgenomic RNA synthesis by the cucumber mosaic virus replicase in vitro requires an upstream RNA stem–loop. J Virol 74, 11201–11209.[Abstract/Free Full Text]

Crotty, S., Gohara, D., Gilligan, D. K., Karelsky, S., Cameron, C. E. & Andino, R. (2003). Manganese-dependent polioviruses caused by mutations within the viral polymerase. J Virol 76, 5378–5388.[CrossRef]

Deiman, B. A. L. M., Koenen, A. K., Verlann, P. W. G. & Pleij, P. W. G. (1998). Minimal template requirement for initiation of minus-strand synthesis in vitro by the RNA-dependent RNA polymerase of turnip yellow mosaic virus. J Virol 72, 3965–3972.[Abstract/Free Full Text]

Deiman, B. A., Verlaan, P. W. & Pleij, C. W. (2000). In vitro transcription by the turnip yellow mosaic virus RNA polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases. J Virol 74, 264–271.[Abstract/Free Full Text]

Doublie, S., Tabor, S., Long, A. M., Richardson, C. C. & Ellenberger, T. (1998). Crystal structure of a bacteriophage T7 DNA replication complex at 2·2 Å resolution. Nature 391, 251–258.[CrossRef][Medline]

Doublie, S., Sawaya, M. R. & Ellenberger, T. (1999). An open and closed case for all polymerases. Structure Fold Des 7, R31–R35.[Medline]

Downing, K. M., Jurmark, B. S. & So, A. G. (1971). Determination of nucleotide sequences at promoter regions by use of dinucleotides. Biochemistry 10, 4970–4975.[Medline]

Emerson, S. U. (1982). Reconstitution studies detect a single polymerase entry site on the vesicular stomatitis virus genome. Cell 31, 635–642.[Medline]

Emori, Y., Iba, H. & Okada, Y. (1983). Transcriptional regulation of three double-stranded RNA segments of bacteriophage {pi}6 in vitro. J Virol 46, 196–203.[Medline]

Fechter, P., Rudinger-Thirion, J., Florentz, C. & Giege, R. (2001). Novel features in the tRNA-like world of plant viral RNAs. Cell Mol Life Sci 58, 1547–1561.[Medline]

Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T. P. & Roizman, B. (1996). Multiplication of viruses. In Fields Virology, 3rd edn, pp. 87–94. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.

Frilander, M., Poranen, M. & Bamford, D. H. (1995). The large genome segment of dsRNA bacteriophage {pi}6 is the key regulator in the in vitro minus and plus strand synthesis. RNA 1, 510–518.[Abstract]

Gaal, T., Bartlett, M. S., Ross, W., Turnbough, C. L. & Gourse, R. L. (1997). Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria. Science 278, 2092–2097.[Abstract/Free Full Text]

Garcin, D. & Kolakofsky, D. (1990). A novel mechanism for the initiation of Tacaribe arenavirus genome replication. J Virol 64, 6196–6203.[Medline]

Garcin, D. & Kolakofsky, D. (1992). Tacaribe arenavirus RNA synthesis in vitro is primer-dependent and suggests a novel mechanism for the initiation of genome replication. J Virol 66, 1370–1376.[Abstract]

Garcin, D., Lezzi, M., Dobbs, M., Elliot, R. M., Schmaljohn, C., Kang, C. Y. & Kolakofsky, D. (1995). The 5' end of Hantaan virus (Bunyaviridae) RNAs suggests a prime-and-realign mechanism for the initiation of RNA synthesis. J Virol 69, 5754–5762.[Abstract]

Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A. & Kornberg, R. D. (2001). Structural basis of transcription: an RNA polymerase II elongation complex at 3·3 Å resolution. Science 292, 1876–1882.[Abstract/Free Full Text]

Goldbach, R., LeGall, O. & Wellink, J. (1991). Alpha-like viruses in plants. Semin Virol 2, 19–25.

Gong, Y., Shannon, A., Westaway, E. G. & Gowans, E. J. (1998a). The replicative intermediate molecule of bovine viral diarrhoea virus contains multiple nascent strands. Arch Virol 143, 399–404.[CrossRef][Medline]

Gong, Y., Trowbridge, R., Macnaughton, T. B., Westaway, E. G., Shannon, A. D. & Gowans, E. J. (1998b). Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhoea virus. J Gen Virol 77, 2729–2736.

Guyatt, K. J., Westaway, E. G. & Khromykh, A. A. (2001). Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin. J Virol Methods 92, 37–44.[CrossRef][Medline]

Hagen, M., Tiley, L., Chung, T. D. Y. & Krystal, M. (1995). The role of template–primer interactions in cleavage and initiation by the influenza virus polymerase. J Gen Virol 76, 603–611.[Abstract]

Hansen, J. L., Long, A. M. & Schultz, S. C. (1997). Structure of the RNA-dependent RNA polymerase of poliovirus. Structure 5, 1109–1122.[Medline]

Hobson, S. D., Rosenblum, E. S., Richards, O. C., Richmond, K., Kirkegaard, K. & Schultz, S. C. (2001). Oligomeric structures of poliovirus polymerase are important for function. EMBO J 20, 1153–1163.[Abstract/Free Full Text]

Hofmann, M. A. & Brian, D. A. (1991). The 5' end of coronavirus minus-strand RNAs contains a short poly(U) tract. J Virol 65, 6331–6333.[Medline]

Honda, A., Mizumoto, K. & Ishihama, A. (1986). RNA polymerase of influenza virus: dinucleotide-primed initiation of transcription at specific positions on viral RNA. J Biol Chem 261, 5987–5991.[Abstract/Free Full Text]

Hong, Z., Cameron, C. E., Walker, M. P., Castro, C., Yao, N., Lau, J. Y. N. & Zhong, W. (2001). A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase. Virology 285, 6–11.[CrossRef][Medline]

Huang, Y., Beaudry, A., McSwiggen, J. & Sousa, R. (1997). Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts. Biochemistry 36, 13718–13728.[CrossRef][Medline]

Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998). Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669–1675.[Abstract/Free Full Text]

Iyer, L. M., Koonin, E. V. & Aravind, L. (2003). Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases. BMC Struct Biol 3, 1–23.[CrossRef][Medline]

Jin, H. & Elliott, R. M. (1993). Nonviral sequences at the 5' end of Dugbe nairovirus S mRNAs. J Gen Virol 74, 2293–2297.[Abstract]

Jorgensen, S. E., Buch, L. B. & Nierlich, D. P. (1969). Nucleoside triphosphate termini from RNA synthesized in vivo by Escherichia coli. Science 164, 1067–1070.[Medline]

Joyce, C. M. (1997). Choosing the right sugar: how polymerases select a nucleotide substrate. Proc Natl Acad Sci U S A 94, 1619–1622.[Free Full Text]

Joyce, C. M. & Steitz, T. A. (1995). Polymerase structures and function: variations on a theme? J Bacteriol 177, 6321–6329.[Free Full Text]

Kao, C. & Sun, J. H. (1996). Initiation of minus-strand RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase: use of oligoribonucleotide primers. J Virol 70, 6826–6830.[Abstract]

Kao, C. C., Del Vecchio, A. M. & Zhong, W. (1999). De novo initiation of RNA synthesis by a recombinant flaviviridae RNA-dependent RNA polymerase. Virology 253, 1–7.[CrossRef][Medline]

Kao, C. C., Yang, X., Kline, A., May Wang, Q., Barket, D. & Heinz, B. A. (2000). Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase. J Virol 74, 11121–11128.[Abstract/Free Full Text]

Kao, C. C., Singh, P. & Ecker, D. J. (2001). De novo initiation of viral RNA-dependent RNA synthesis. Virology 287, 251–260.[CrossRef][Medline]

Kiefer, J. R., Mao, C., Braman, J. C. & Beese, L. S. (1998). Visualizing DNA replication in a catalytically active bacillus DNA polymerase crystal. Nature 391, 304–307.[CrossRef][Medline]

Kim, M. J., Zhong, W., Hong, Z. & Kao, C. C. (2000). Template nucleotide moieties required for de novo initiation of RNA synthesis by a recombinant viral RNA-dependent RNA polymerase. J Virol 74, 10312–10322.[Abstract/Free Full Text]

Kim, M., Kim, H., Cho, S.-P. & Min, M.-K. (2002). Template requirements for de novo RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase on the viral X RNA. J Virol 76, 6944–6956.[Abstract/Free Full Text]

Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol 72, 2197–2206.[Abstract]

Labonte, P., Axelrod, V., Agarwal, A., Aulabaugh, A., Amin, A. & Mak, P. (2002). Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis. J Biol Chem 277, 38838–38846.[Abstract/Free Full Text]

Lahser, F. C., Marsh, L. E. & Hall, T. C. (1993). Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation. J Virol 67, 3295–3303.[Abstract]

Lai, M. M. C. (1998). Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244, 1–12.[CrossRef][Medline]

Lai, V. C. H., Kao, C. C., Ferrari, E., Park, J., Uss, A. S., Wright-Minogue, J., Hong, Z. & Lau, J. Y. N. (1999). Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. J Virol 73, 10129–10136.[Abstract/Free Full Text]

Laurila, M. R., Makeyev, E. V. & Bamford, D. H. (2002). Bacteriophage {pi}6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer. J Biol Chem 277, 17117–17124.[Abstract/Free Full Text]

Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F. & Weber, P. C. (1999). Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 6, 937–943.[CrossRef][Medline]

Leveque, V. J., Johnson, R. B., Parsons, S., Ren, J., Xie, C., Zhang, F. & Wang, Q. M. (2003). Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis. J Virol 77, 9020–9028.[Abstract/Free Full Text]

López Vázquez, A., Martín Alonso, J. M. & Parra, F. (2001). Characterization of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli. Arch Virol 146, 59–69.[CrossRef][Medline]

Losick, R. & Chamberlin, M. (1976). RNA Polymerase. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Luo, G., Hamatake, R. K., Mathis, D. M., Racela, J., Rigat, K. L., Lemm, J. & Colonno, R. J. (2000). De novo initiation of RNA synthesis by the RNA dependent RNA polymerase (NS5B) of hepatitis C virus. J Virol 74, 851–863.[Abstract/Free Full Text]

Makeyev, E. V. & Bamford, D. H. (2000a). Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage {pi}6. EMBO J 19, 124–133.[Abstract/Free Full Text]

Makeyev, E. V. & Bamford, D. H. (2000b). The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J 19, 6275–6284.[Abstract/Free Full Text]

Makeyev, E. V. & Bamford, D. H. (2001). Primer-independent RNA sequencing with bacteriophage {pi}6 RNA polymerase and chain terminators. RNA 7, 774–781.[Abstract/Free Full Text]

Makeyev, E. V. & Grimes, J. M. (2004). RNA-dependent RNA polymerases of dsRNA bacteriophages. Virus Res (in press).

Martin, C. T., Muller, D. K. & Coleman, J. E. (1988). Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry 27, 3966–3974.[Medline]

McClure, W. R. (1985). Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem 54, 171–204.[CrossRef][Medline]

McKnight, K. & Lemon, S. M. (1998). The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication. RNA 4, 1569–1584.[Abstract/Free Full Text]

Miller, W. A. & Koev, G. (2000). Synthesis of subgenomic RNAs by positive-strand RNA viruses. Virology 273, 1–8.[CrossRef][Medline]

Mindich, L. (1999). Precise packaging of the three genomic segments of the double-stranded-RNA bacteriophage {pi}6. Microbiol Mol Biol Rev 63, 149–160.[Abstract/Free Full Text]

Mindich, L., Qiao, X., Onodera, S., Gottlieb, P. & Frilander, M. (1994). RNA structural requirements for stability and minus-strand synthesis in the dsRNA bacteriophage {pi}6. Virology 202, 258–263.[CrossRef][Medline]

Murakami, K. S. & Darst, S. A. (2003). Bacterial RNA polymerases: the whole story. Curr Opin Struct Biol 13, 31–39.[CrossRef][Medline]

Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O. & Darst, S. A. (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme–DNA complex. Science 296, 1285–1290.[Abstract/Free Full Text]

Nagy, P. D., Carpenter, C. D. & Simon, A. E. (1997). A novel 3'-end repair mechanism in an RNA virus. Proc Natl Acad Sci U S A 94, 1113–1118.[Abstract/Free Full Text]

Netolitzky, D. J., Schmaltz, F. L., Parker, M. D., Rayner, G. A., Fisher, G. R., Trent, D. W., Bader, D. E. & Nagata, L. P. (2000). Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes. J Gen Virol 81, 151–159.[Abstract/Free Full Text]

Ng, K. K. S., Cherney, M. M., Vázquez, A. L., Machín, A., Alonso, J. M. M., Parra, F. & James, M. N. G. (2002). Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase. J Biol Chem 277, 1381–1387.[Abstract/Free Full Text]

O'Farrell, D., Trowbridge, R., Rowlands, D. & Jäger, J. (2003). Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): structural evidence for nucleotide import and de-novo initiation. J Mol Biol 326, 1025–1035.[CrossRef][Medline]

Oh, J. W., Ito, T. & Lai, M. M. (1999). A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J Virol 73, 7694–7702.[Abstract/Free Full Text]

Oh, J. W., Sheu, G. T. & Lai, M. M. (2000). Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template. J Biol Chem 275, 17710–17717.[Abstract/Free Full Text]

Ojala, P. M. & Bamford, D. H. (1995). In vitro transcription of the double-stranded RNA bacteriophage {pi}6 is influenced by purine NTPs and calcium. Virology 207, 400–408.[CrossRef][Medline]

Okai, Y. (1982). Calcium effects on free and chromatin-bound RNA polymerase II reactions. FEBS Lett 140, 139–141.[CrossRef][Medline]

Ollis, D. L., Kline, C. & Steitz, T. A. (1985). Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase. Nature 313, 818–819.[Medline]

Osman, T. A. M., Hemenway, C. L. & Buck, K. W. (2000). Role of the 3' tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase in vitro. J Virol 74, 11671–11680.[Abstract/Free Full Text]

Panavas, T., Pogany, J. & Nagy, P. D. (2002). Internal initiation by the cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences. Virology 296, 275–287.[CrossRef][Medline]

Patton, J. T., Jones, M. T., Kalbach, A. N., He, Y.-W. & Xiaobo, J. (1997). Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome. J Virol 71, 9618–9626.[Abstract]

Paul, A., van Boom, J. H., Fillippov, D. & Wimmer, E. (1998). Protein primed RNA synthesis by purified RNA polymerase. Nature 393, 280–284.[CrossRef][Medline]

Pellerin, C., Lefebvre, S., Little, M. J., McKercher, G., Lamarre, D. & Kukolj, G. (2002). Internal initiation sites of de novo RNA synthesis within the hepatitis C virus polypyrimidine tract. Biochem Biophys Res Commun 295, 682–688.[CrossRef][Medline]

Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. & Kraut, J. (1994). Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264, 1891–1903.[Medline]

Pirttimaa, M. J., Paatero, A. O., Frilander, M. J. & Bamford, D. H. (2002). Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage {pi}6 is required for single-stranded RNA packaging and transcription. J Virol 76, 10122–10127.[Abstract/Free Full Text]

Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J 12, 3867–3874.

Qin, W., Luo, H., Nomura, T., Hayashi, N., Yamashita, T. & Murakami, S. (2002). Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase. J Biol Chem 277, 2132–2137.[Abstract/Free Full Text]

Quamme, G. A., Dai, L.-J. & Rabkin, S. (1993). Dynamics of intracellular free Mg2+ changes in a vascular smooth muscle cell line. Am J Physiol 265, H281–H288.[Medline]

Ranjith-Kumar, C. T., Kim, Y.-C., Gutshall, L., Silverman, C., Khandekar, S., Sarisky, R. T. & Kao, C. C. (2002a). Mechanism of de novo initiation by the hepatitis C virus RNA-dependent RNA polymerase: role of divalent metals. J Virol 76, 12513–12525.[Abstract/Free Full Text]

Ranjith-Kumar, C. T., Gutshall, L., Kim, M.-J., Sarisky, R. T. & Kao, C. C. (2002b). Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases. J Virol 76, 12526–12536.[Abstract/Free Full Text]

Ranjith-Kumar, C. T., Gajewski, J., Gutshall, L., Maley, D., Sarisky, R. T. & Kao, C. C. (2002c). Terminal nucleotidyl transferase activity of recombinant flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis. J Virol 75, 8615–8623.[CrossRef]

Ranjith-Kumar, C. T., Zhang, X. & Kao, C. C. (2003). Enhancer-like activity of a brome mosaic virus RNA promoter. J Virol 77, 1830–1839.[Abstract/Free Full Text]

Reddy, P. S. & Chatterji, D. (1994). Evidence for a pyrimidine-nucleotide specific initiation site (the i site) on Escherichia coli RNA polymerase: proximity relationship with the inhibitor binding domain. Eur J Biochem 225, 737–745.[Abstract]

Salas, M. (1991). Protein-priming of DNA replication. Annu Rev Biochem 60, 39–71.[CrossRef][Medline]

Salgado, P. S., Makeyev, E. V., Butcher, S., Bamford, D., Stuart, D. I. & Grimes, J. M. (2004). The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase. Structure (in press).

Sargent, M. D. & Borsa, J. (1984). Effects of Ca2+ and Mg2+ on the switch-on of transcriptase function in reovirus in vitro. Can J Biochem Cell Biol 62, 162–169.[Medline]

Sawicki, D. & Gomatos, P. J. (1976). Replication of Semliki Forest virus: polyadenylates in plus-strand RNA and polyuridylate in minus-strand RNA. J Virol 20, 446–464.[Medline]

Schibler, U. & Perry, R. P. (1977). The 5'-termini of heterogeneous nuclear RNA: a comparison among molecules of different sizes and ages. Nucleic Acids Res 4, 133–149.

Seal, B. S., Neill, J. D. & Ridpath, J. F. (1994). Predicted stem–loop structures and variation in nucleotide sequence of 3' noncoding regions among animal calicivirus genomes. Virus Genes 8, 243–247.[Medline]

Siegel, R. W., Bellon, L., Beigelman, L. & Kao, C. (1998). Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A 95, 11613–11618.[Abstract/Free Full Text]

Singh, R. N. & Dreher, T. W. (1997). Turnip yellow mosaic virus RNA-dependent RNA polymerase: initiation of minus-strand synthesis in vitro. Virology 233, 430–439.[CrossRef][Medline]

Singh, R. N. & Dreher, T. W. (1998). Specific site selection in RNA resulting from a combination of non-specific secondary structure and -CCR- boxes: initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase. RNA 4, 1083–1095.[Abstract/Free Full Text]

Sit, T. L., Vaewhong, S. & Lommel, S. (1998). RNA-mediated transactivation of transcription from a viral RNA. Science 281, 829–832.[Abstract/Free Full Text]

Sivakumaran, K. & Kao, C. C. (1999). Initiation of genomic positive strand synthesis from DNA and RNA templates by a viral RNA-dependent RNA polymerase. J Virol 73, 6415–6423.[Abstract/Free Full Text]

Steitz, T. A. (1998). A mechanism for all polymerases. Nature 391, 231–232.[CrossRef][Medline]

Steitz, T. A. & Steitz, J. A. (1993). A general two-metal-ion mechanism for catalytic RNA. Proc Natl Acad Sci U S A 90, 6498–6502.[Abstract]

Strauss, J. H. & Strauss, E. G. (1994). The alphaviruses: gene expression, replication and evolution. Microbiol Rev 58, 491–562.[Medline]

Sun, J. H. & Kao, C. C. (1997a). RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase: transition from initiation to elongation. Virology 233, 63–73.[CrossRef][Medline]

Sun, J. & Kao, C. C. (1997b). Characterization of RNA products associated with or aborted by a viral RNA-dependent RNA polymerase. Virology 236, 348–353.[CrossRef][Medline]

Sun, J., Adkins, S., Faurote, G. & Kao, C. C. (1996). Initiation of (–)-strand RNA synthesis catalyzed by the brome mosaic virus RNA-dependent RNA polymerase: synthesis of oligonucleotides. Virology 226, 1–12.[CrossRef][Medline]

Sun, X. L., Johnson, R. B., Hockman, M. A. & Wang, Q. M. (2000). De novo initiation catalyzed by HCV RNA-dependent RNA polymerase. Biochem Biophys Res Commun 268, 798–803.[CrossRef][Medline]

Tabor, S. & Richardson, C. (1989). Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A 86, 4076–4080.[Abstract]

Tao, Y., Farsetta, D. L., Nibert, M. L. & Harrison, S. C. (2002). RNA synthesis in a cage – structural studies of reovirus polymerase 83. Cell 111, 733–745.[Medline]

Testa, D. & Banerjee, A. K. (1979). Initiation of RNA synthesis in vitro by vesicular stomatitis virus. Role of ATP. J Biol Chem 254, 2053–2058.[Abstract]

Tomei, L., Vitale, R. L., Incitti, I., Serafini, S., Altamura, S., Vitelli, A. & De Francesco, R. (2000). Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence. J Gen Virol 81, 759–767.[Abstract/Free Full Text]

Van Belkum, A., Abrahams, J. P., Pleij, C. W. A. & Bosch, L. (1985). Five pseudoknots at the 204 nucleotides long 3' noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res 13, 7673–7686.[Abstract]

Van Dijk, A. A., Frilander, M. & Bamford, D. H. (1995). Differentiation between minus- and plus-strand synthesis: polymerase activity of dsRNA bacteriophage {pi}6 in an in vitro packaging and replication system. Virology 211, 320–323.[CrossRef][Medline]

Whelan, S. P. J. & Wertz, G. W. (2002). Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome. Proc Natl Acad Sci U S A 99, 9178–9183.[Abstract/Free Full Text]

Yamakawa, M., Furuichi, Y., Nakashima, K., La Fiandra, A. J. & Shatkin, A. J. (1981). Excess synthesis of viral mRNA 5-terminal oligonucleotides by reovirus transcriptase. J Biol Chem 256, 6507–6514.[Abstract/Free Full Text]

Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K. & Murakami, S. (1998). RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C terminal region. J Biol Chem 273, 15479–15486.[Abstract/Free Full Text]

Yang, H., Makeyev, E. V. & Bamford, D. H. (2001). Comparison of polymerase subunits from double-stranded RNA bacteriophages. J Virol 75, 11088–11095.[Abstract/Free Full Text]

Yang, H., Makeyev, E. V., Butcher, S. J., Gaidelyte, A. & Bamford, D. H. (2003a). Two distinct mechanisms ensure transcriptional polarity in double-stranded RNA bacteriophages. J Virol 77, 1195–1203.[CrossRef][Medline]

Yang, H., Gottlieb, P., Wei, H., Bamford, D. H. & Makeyev, E. V. (2003b). Temperature requirements for initiation of RNA-dependent RNA polymerization. Virology 314, 706–715.[CrossRef][Medline]

Yoshinari, S. & Dreher, T. W. (2000). Internal and 3' RNA initiation by Q{beta} replicase directed by CCA boxes. Virology 271, 363–370.[CrossRef][Medline]

Yoshinari, S., Nagy, P., Simon, A. E. & Dreher, T. W. (2000). CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases. RNA 6, 698–707.[Abstract/Free Full Text]

You, S. & Padmanabhan, R. (1999). A novel in vitro replication system for Dengue virus. J Biol Chem 274, 33714–33722.[Abstract/Free Full Text]

Zhang, R. & Ellis, K. (1989). In vivo measurement of total body magnesium and manganese in rats. Am J Physiol 257, R1136–R1140.[Medline]

Zhong, W., Gutshall, L. L. & Del Vecchio, A. M. (1998). Identification and characterization of an RNA-dependent RNA polymerase activity within the nonstructural protein 5B region of bovine viral diarrhea virus. J Virol 72, 9365–9369.[Abstract/Free Full Text]

Zhong, W., Uss, A. S., Ferrari, E., Lau, J. Y. & Hong, Z. (2000a). De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J Virol 74, 2017–2022.[Abstract/Free Full Text]

Zhong, W., Ferrari, E., Lesburg, C. A., Maag, D., Gosh, A. K. B., Cameron, C. E., Lau, J. Y. N. & Hong, Z. (2000b). Template–primer requirements and single-nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase. J Virol 74, 9134–9143.[Abstract/Free Full Text]