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Review
. 2015 Jun 8;16(6):12943-57.
doi: 10.3390/ijms160612943.

A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

Jiqin Wu  1   2   3 , Weichi Liu  4   5   6 , Peng Gong  7   8
Affiliations
Review

A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

Jiqin Wu et al. Int J Mol Sci. .

Abstract

RNA-dependent RNA polymerases (RdRPs) from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A-E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

Keywords: Flaviviridae; RNA-dependent RNA polymerase; catalytic motif; de novo initiation; elongation; in cis regulation.

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Figures

Figure 1
Figure 1
Structural comparison of representative Flaviviridae RdRPs. (a) A schematic of Flaviviridae RdRPs defining functional regions; (b) Stereo-pair images of Flaviviridae RdRP structures (pdb entries: 4K6M, 1S4F and 1NB4) viewing down into the polymerase active site. The RdRP signature sequence XGDD is shown in magenta (also indicated by a black triangle). Red dots in the JEV structure indicates disordered residues 271–273 in the MTase-RdRP linker and the S-adenosyl-l-homocysteine (SAH) bound in the MTase domain is shown as sticks. In the BVDV structure, residues 92–127 from a second molecule are connected to the C-terminal part of the protein by the green dots to present the likely canonical fold. Structures were superimposed using program THESEUS [22].
Figure 2
Figure 2
Catalytic motifs of Flaviviridae RdRPs. (a) Stereo-pair images of spatial organization of the JEV RdRP catalytic motifs A–G (pdb entry: 4K6M). Seven RdRP motifs are shown as thick noodles. The color-coding is as in Figure 1b; (b) A structure-based sequence alignment depicting the conservation of RdRP motifs (pdb entries: 4K6M, 1S4F, 1NB4, 3OL6, 3BSO, 3AVT [36], 1HI0, 2PGG [37], 4WRT [38], 1RTD [39], and 3DU6 [40]). Three RdRPs from other positive-strand RNA viruses (PICO/PV: Picornaviridae/poliovirus; CALI/NV: Caliciviridae/norovirus; LEVI/QB: Leviviridae/bacteriophage Qβ), two from double-stranded RNA viruses (CYST/PHI6: Cystoviridae/bacteriophage ϕ6; BIRN/BIRV: Birnaviridae/birnavirus), one from negative-stranded RNA viruses (ORTH/IBV: Orthomyxoviridae/influenza virus B), and two reverse transcriptases (RTs) (RETR/HIV1: Retroviridae/human immunodeficiency virus 1; TERT: Telomerase RT) were chosen as representatives for alignment and/or comparison. Only the structurally conserved segment of motif D is included in this alignment. Some important RdRP consensus residues are highlighted in red. Two motif G residues interacting with the +1/+2 junction of the RNA template are indicated by a red box. The two universal aspartic acid residues are indicated by asterisks. Numbers in parenthesis indicate the number of residues not shown. The alignment of IBV polymerase motif G is of lower confidence due to a lower level of structural similarity to motif G in other structures.
Figure 3
Figure 3
The crystal structures of de novo RdRP initiation complex (IC). (a) Bacteriophage ϕ6 polymerase IC structure (pdb entry: 1HI0). Template RNA is in cyan. Initiation NTPs and residues Q629 and Y630 are shown as sticks, and magnesium ions are shown as cyan spheres; (b) HCV NS5B IC structure (pdb entry: 4WTJ). Template RNA is in cyan and dinucleotide primer is in green. ADP and residues Y448 and G449 are shown as sticks, and manganese ions are shown as cyan spheres. Structure superimpositions were carried out using motif C residues and the least-square method. For both panels, the +1 templating nucleotide is shown in orange. Capital letters with dark grey background indicate RdRP catalytic motifs.
Figure 4
Figure 4
Conformational heterogeneity of Flaviviridae polymerase structure may be related to in cis regulations. (a) Representative Flaviviridae polymerase structure adopting the canonical conformations (pdb entries: JEV/4K6M, BVDV/1S4F, HCV/1NB4); (b) Disorder and/or alternative folding of motifs F and G observed in Flaviviridae polymerases (pdb entries: JEV-ii/4MTP [60], JEV-iii/4HDG, WNV/2HFZ, DENV/4V0Q, DENV-ii/2J7U, BVDV-ii/2CJQ). Motifs F and G are shown as thick noodles and the pinky finger residues flanking motif G are shown as thin noodles. Note that in the apo JEV RdRP structure (JEV-ii) and GTP-bound JEV RdRP structure, the NTP entry channel is blocked by the non-canonically folded motif F. Color coding is as in Figure 1b and Figure 3b. A 4 nt RNA template, a dinucleotide primer, and an ADP molecule taken from an HCV IC structure (pdb entry: 4WTJ) were modeled into all structures for comparison. The α-carbons of JEV NS5 residues 409–410 in motif G and 459, 461, 474 in motif F and their equivalents in other polymerases are shown as spheres to help distinguish canonical and alternative folding of these two motifs. Double arrows are used to connect polymerases from the same viral species or from the same genus. The MTase in the full-length Flavivirus NS5 structures are shown as surface representations. Structure superimpositions were carried out as in Figure 1b.
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