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Review
. 2017 Apr 15;234:135-152.
doi: 10.1016/j.virusres.2017.01.006. Epub 2017 Jan 16.

Cystoviral RNA-directed RNA Polymerases: Regulation of RNA Synthesis on Multiple Time and Length Scales

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Free PMC article
Review

Cystoviral RNA-directed RNA Polymerases: Regulation of RNA Synthesis on Multiple Time and Length Scales

Sébastien Alphonse et al. Virus Res. .
Free PMC article

Abstract

P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family. These range from atomic resolution snapshots of P2 trapped in functionally significant states, in complex with catalytic/structural metal ions, polynucleotide templates and substrate nucleoside triphosphates, to P2 in the context of viral capsids providing structural insight into the assembly of supramolecular complexes and regulatory interactions therein. They include in vitro biochemical studies using P2 purified to homogeneity and in vivo studies utilizing infectious core particles. Recent advances in experimental techniques have also allowed access to the temporal dimension and enabled the characterization of dynamics of P2 on the sub-nanosecond to millisecond timescale through measurements of nuclear spin relaxation in solution and single molecule studies of transcription from seconds to minutes. Below we summarize the most significant results that provide critical insight into the role of P2 in regulating RNA synthesis in cystoviruses.

Keywords: Bacteriophage; Cystovirus; RNA-directed RNA polymerase; Replication; Transcription.

Figures

Fig. 1
Fig. 1
Schematic representation of the structural organization of a mature cystovirus virion. The membrane proteins that include the major envelope protein, P9 (90 residues in Φ6), an envelope protein, P10 (42 residues), and a minor membrane protein, P13 (72 residues) are associated with the outer lipid envelope. Exposed to the extracellular medium, is the receptor-binding spike protein, P3 (648 residues) that forms a complex with the fusogenic membrane protein, P6 (167 residues), is able to interact specifically with the type IV pili of the bacterial host in the case of Φ6 (and Φ2954). In some cystoviruses, e.g. in Φ8, P3 comprises of two separate proteins P3a and P3b. The outer protein capsid is formed by 200 trimers of the major outer capsid protein, P8 (149 residues) assembled on a T = 13 lattice. Not all of all the cystoviruses contain a P8 shell e.g. Φ8. The lytic protein P5 (220 residues) that is involved in the degradation of the peptidoglycan layer during internalization into the host cytoplasm is found in the space between the lipid and outer protein layers. The inner protein capsid is formed from asymmetric dimers of the inner capsid protein, P1 (769 residues) arranged on a T = 1 lattice. Associated with the P1 shell are three other proteins all encoded on the genomic L-segment: an RNA-directed RNA polymerase, P2 (664 residues), a hexameric packaging NTPase, P4 (332 residues) and an accessory protein, P7 (161 residues). The proteins P1, P2, P4 and P7 assemble to form the polymerase complex (PX). The three segments, large (L, 6.4 kbp), medium (M, 4.1 kbp) and small (S, 2.9 kbp) of the double-stranded RNA genome are enclosed within the P1 shell. The L-segment encodes for the P1, P2, P4 and P7 proteins, the M-segment encodes for the P3, P6, P10 and P13 proteins; the S-segment encodes P5, P8 and P9 proteins.
Fig. 2
Fig. 2
Structures of viral RNA-directed RNA polymerases. The structures of several viral RdRps are shown in ribbon representation. The structures have been ordered left-to-right with increasing size: poliovirus 3Dpol (PDB: 1RA6, 461 residues, 52.5 kDa) (Thompson and Peersen, 2004), HCV NS5B (PDB: 5CZB, 563 residues, 62.5 kDa) (Pierra Rouvière et al., 2016), Φ6 P2 (PDB: 1HHS, 664 residues, 74.8 kDa) (Butcher et al., 2001); Φ12 P2 (PDB: 4GZK, 659 residues, 75.3 kDa) (Ren et al., 2013) and rotavirus VP1 (PDB: 2R7Q, 1095 residues, 126.1 kDa) (Lu et al., 2008). The fingers, thumb and palm domains are shown in red, blue, and green, respectively. The N- and C-terminal domains, when present, are shown in magenta and yellow, respectively.
Fig. 3
Fig. 3
The template and substrate entry tunnels. The top panel shows two views of P2 with the near orthogonal substrate and template tunnels. The bottom panel shows a third view with the front surface removed to display the basic surfaces of the two tunnels that intersect at the catalytic site. The electrostatic charge surfaces are shown using a red-to-blue gradient.
Fig. 4
Fig. 4
Conserved RdRp sequence motifs. The RdRp sequence motifs A–E (palm) and F (fingers) are mapped onto the structures shown in Fig. 2. Motifs A–F are shown in red, yellow, green, blue, pink and orange, respectively. A subset of key conserved charged residues is shown in ball-and-stick representation and labeled.
Fig. 5
Fig. 5
Phosphodiester bond formation. The substrate NTPs (D1 and D2) that would form the first two bases of the daughter chain are paired with the two bases (T1 and T2) at the 3′-end of the template. This priming platform is stabilized by Y630 from the C-terminal domain that base stacks with D1. The nucleotidyl-transfer reaction requires two Mg2+ ions − one (MgA) that coordinates the 3′-OH of NTP D1 and the α-phosphate of the NTP D2; a second (MgB) that coordinates the β- and γ-phosphates of NTP D2 in addition to the sidechains of the conserved motif A (D324) and motif C (D453) aspartates. Coordination with MgA lowers the pKa of the 3′-OH of NTP D1 enabling a nucleophilic attack on the α-phosphate of D2 creating a phosphodiester bond between the two NTPs. The pyrophosphate product is stabilized by MgB. The nucleotidyl-transfer reaction is shown schematically in the lower panel.
Fig. 6
Fig. 6
Proposed sequence of events in P2 mediated RNA synthesis. I, C, P and S indicate the 2nd NTP binding site (I-site or interrogation-site, formed by motif F), the catalytic NTP binding site (C-site or catalytic-site, formed by the motif C G/SDD triplet), the priming platform (P-site, formed by Y630 on the CTD) and the specificity pocket (S-site, also part of the CTD), respectively. In State I, free P2 is complexed with the structural Mn2+ ion located in its non-catalytic binding-site with the CTD in a closed conformation. Under these conditions P2 rapidly samples states II, II* and III corresponding to NTP and template-bound conformations (pre-initiation). In state III, the template bound-state of P2, the 3′-end of the template overshoots the active site and is locked into the specificity pocket (S-site). Metal ions at non-canonical sites facilitate the transit of NTPs through the substrate portal. Step IV: The template ratchets back to allow Watson-Crick base pairing of its 3′-end with the complimentary NTPs (D1 and D2). At this state the catalytic Mg2+ ions are drawn into their appropriate position and the non-catalytic position is occupied by Mn2+. Step V: The nucleotidyl-transfer reaction occurs with the formation of a phosphodiester bond between D2 and D1 with the release of pyrophosphate. Step VI: In order to allow the nascent dsRNA to move forward in register to prime the system for the next nucleotidyl-transfer reaction, the destabilizing effect of the Mn2+ facilitates the opening of the CTD. Mn2+ ions lost at this stage need to be replaced by external Mn2+. The initiation complex is still not stable enough to transit into its processive, elongation stage. Step VII: A new substrate NTP (D3) complementary to the third template base from the 3′-end (T3) now forms a new initiation complex culminating in a repeat of IV–VI and leading to a phosphodiester bond formation between D3 and D2. At this stage (or in cases after a few more cycles) the complex is stable, the CTD is fully open and the Mn2+ is stably bound at the non-catalytic binding site. At this point P2 enters the elongation stage where the entire template can be read through. The alphanumeric codes in the bottom left-hand corner of each box, where present, represent the PDB accession codes of the structures, where available), used to characterize the corresponding step. Modified from Wright et al. (Wright et al., 2012) with permission. Step VII: after the entire daughter strand complimentary to the template has been synthesized, the TNTase activity of P2 attaches a nucleotide to its 5′-end. Step VII: The dsRNA leaves P2. Steps VII and VIII have been proposed to comprise the termination step in RNA synthesis (Poranen et al., 2008a).
Fig. 7
Fig. 7
Fast timescale dynamics. Flexibility measured from spin-relaxation utilizing the methyl positions of leucine, valine, methionine and isoleucine (δ1 only) residues in Φ12 P2 and isoleucine (δ1) for Φ6 P2. Σ values represent flexibility on the fast, nanoseconds timescale with residues with Σ = 0.5 having average flexibility for a given residue type and Σ > 0.5 and Σ < 0.5 being less and more flexible than average, respectively (Alphonse et al., 2015). The inset on the top panel shows the distribution of the least (largely distributed around the template and substrate tunnels) and most flexible positions. The bottom panel shows the flexibility of measured positions in the various conserved RdRp sequence motifs for Φ12 and Φ6 P2. Positions for motifs D and F are overall the most dynamic. The Σ values are represented using a red-to-blue gradient ranging from the most-to-least dynamic elements.
Fig. 8
Fig. 8
Slow timescale dynamics in P2. Dynamics measurements on two separate stalled initiation complexes one 5′-UUUCC-3′ with GMPCPP (ICA) and a second with a 5′-UUUUU-3′ template with AMPCPP (ICB). The 5′-UUUUU-3′ and 5′-UUUCC-3′ templates have affinities that are ∼50-fold different (fluorescence anisotropy traces with increasing concentration shown on the bottom left panel) but only the latter for which the last two nucleotides correspond to the S and M 3′-ends, shows dynamics on a catalytically relevant timescale in its catalytic elements. Slow timescale dynamics for ICA (black trace) using the δ1 position of the Motif C residue I449 (also shown is the SDD triad) as probe is reflected in a change in the relaxation rate with field. No such dynamics is seen for ICB as reflected by the flat trace (in red) (Ren and Ghose, 2011).
Fig. 9
Fig. 9
Mechanism for the nucleotide addition cycle. A kinetic scheme that is applicable to nucleic acids polymerases is shown. The first step involves binding of the substrate NTP to the enzyme in complex with RNA at the nth stage of elongation i.e. the daughter chain is n nucleotides long. The second step involves a conformation change (shown by the red arrows) of the ERn•NTP complex into a “closed” state that is capable of catalysis. The third step is the chemistry step (shown by the blue arrows) extending the daughter chain by one nucleotide (n + 1). The fourth step (shown by the red arrows) is a second conformational change that enables the release of the pyrophosphate product. The fifth and the final step involves release of pyrophosphate that is likely coupled to movement of the template forward in register to prime it for the next nucleotidyl-transfer reaction.
Fig. 10
Fig. 10
Measurement of elongation at the single molecule level. Schematic depiction of the magnetic tweezers setup to P2 mediated transcription (top left panel). Array of multiple tweezers setup in a homogeneous magnetic field to allow data collection in a highly multiplexed fashion (top middle panel). The setup comprises of an RNA strand that this largely in duplex form tethered to a magnetic bead at one extremity and to a surface on the other. The RNA contains a short 15-nt overhang at the 3′-end to allow transcriptional initiation in the presence of NTPs. The inset on the top right depicts three typical traces (displaying three distinct types of dynamic behaviors − no pauses, a few short pauses, fast transcription interrupted by a very long pause) that represent the increase in product length versus time due to transcription under a constant force (F) in the presence of an optimal NTP concentration. The lower panel depicts a dwell time distribution (DTD) plot displaying four regions: rapid addition without pause (a Γ-distribution), two sets of short pauses (Pause 1 and Pause 2 described by exponential distributions) and a power-law distribution representing long pauses due to back-tracking. Modified from (Dulin et al., 2015b) with permission.
Fig. 11
Fig. 11
Kinetic view of the elongation cycle. The P2 elongation complex samples two specific conformations, one with an optimal conformation of the catalytic elements (A, a High-Fidelity Conformation or HFC) that allows rapid NTP incorporation (with the correct NTP) leading to B; and an unfavorable conformation of the catalytic pocket (D, a Low Fidelity Conformation or LFC) which has a lower rate of NTP incorporation leading to pauses (Pause 1). In the presence of an incorrect nucleotide, the system enters a Terminal Mismatch Conformation (TMC) that leads to the longest pauses (Pause 2) with the slowest rate of incorporation (misincorporation). The arrows symbolize the transition between the different states with the thick arrows representing the preferred path. Adapted from Dulin et al. (2015b).

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