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

Wu J, Liu W, Gong P - Int J Mol Sci (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, No. 44 Xiao Hong Shan, Wuchang District, Wuhan 430071, China. wujiqin2010@163.com.

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.

No MeSH data available.


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.
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ijms-16-12943-f003: 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.

Mentions: Analogous to DNA-dependent RNA polymerases [47,48,49], an initiation complex (IC) of Flaviviridae RdRP is unstable, and tends to release short RNA products and re-initiate [27]. However, precise and relatively efficient initiation can be achieved with the help of the priming element. Note that the priming element is structurally quite diverse across different viruses (Figure 1b). It could comprise solely an extended loop structure, as in Flavivirus NS5 [14,15,17], or two peptide segments that contain a β-hairpin structure, as in HCV NS5B [50], or even a helical module, as in bacteriophage ϕ6 polymerase [28]. Crystal structures of the de novo RdRP IC from ϕ6 and HCV have been reported, providing the structural basis for how the early stages of RNA synthesis is generally achieved with the assistance of the priming element (Figure 3). In the ϕ6 polymerase IC structure, residues Q629 and Y630 of the priming element were placed at the immediate upstream position of the initiation site, stacking onto the −1 templating nucleotide and the priming NTP, respectively (Figure 3a) [28]. Such an arrangement likely ensures terminal initiation for faithful replication, and may also stabilize the priming NTP for catalysis. In the recently reported set of HCV NS5B IC structures, different dinucleotides were used as short primers to mimic the situation in de novo initiation [42]. Similar to the ϕ6 polymerase IC, priming element residues Y448 and G449 interact with the upstream end (now position −2 vs. −1 in ϕ6 structure) of the template and product strands, respectively (Figure 3b). Note that the priming element could contribute to efficient initiation, or precise start site selection, or both. In an earlier report, when the β-hairpin of the HCV NS5B priming element was truncated for eight residues (residues 444–447 and 450–453 were removed, and residues 448–449 were replace by two glycines to accommodate the truncation), the de novo synthesis activity was not much affected, but this mutant NS5B lost the specificity of terminal initiation and could initiate from internal sites of a template [51]. In a very recent report, mutation of hydrophobic and charged residues within the priming element of HCV NS5B resulted in a substantial decrease of denovo initiation activities [52]. These data indicate that the priming element does play essential roles in de novo initiation, although the mechanistic details remain to be further clarified. Similar initiation mechanisms may be taken by RdRPs from Flavivirus and Pestivirus genera, and several residues within the priming element of DENV NS5 has been proposed to contribute to the initiation-related processes [53]. However, how the initiation platform is set up in these RdRPs still awaits clarification from relevant IC structures. In contrast to de novo viral RdRPs that take advantage of the priming element at initiation, primer-dependent viral RdRPs represented by PV 3Dpol utilize a protein primer (termed VPg for “viral protein genome-linked”) or its uridylylated forms to initiate RNA synthesis [54,55]. To date, RdRP-VPg complex crystal structures have been reported in foot-and-mouth disease virus (FMDV), coxsackievirus B3 (CV B3), and enterovirus 71 (EV71), each displaying a distinct mode of interactions between the polymerase and VPg [56,57,58]. However, how VPg or uridylylated VPg is utilized in the template-directed polymerase initiation remains elusive, as none of the three polymerase-VPg structures includes an RNA template or provides clear clues for how such an IC is spatially arranged.


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

Wu J, Liu W, Gong P - Int J Mol Sci (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490480&req=5

ijms-16-12943-f003: 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.
Mentions: Analogous to DNA-dependent RNA polymerases [47,48,49], an initiation complex (IC) of Flaviviridae RdRP is unstable, and tends to release short RNA products and re-initiate [27]. However, precise and relatively efficient initiation can be achieved with the help of the priming element. Note that the priming element is structurally quite diverse across different viruses (Figure 1b). It could comprise solely an extended loop structure, as in Flavivirus NS5 [14,15,17], or two peptide segments that contain a β-hairpin structure, as in HCV NS5B [50], or even a helical module, as in bacteriophage ϕ6 polymerase [28]. Crystal structures of the de novo RdRP IC from ϕ6 and HCV have been reported, providing the structural basis for how the early stages of RNA synthesis is generally achieved with the assistance of the priming element (Figure 3). In the ϕ6 polymerase IC structure, residues Q629 and Y630 of the priming element were placed at the immediate upstream position of the initiation site, stacking onto the −1 templating nucleotide and the priming NTP, respectively (Figure 3a) [28]. Such an arrangement likely ensures terminal initiation for faithful replication, and may also stabilize the priming NTP for catalysis. In the recently reported set of HCV NS5B IC structures, different dinucleotides were used as short primers to mimic the situation in de novo initiation [42]. Similar to the ϕ6 polymerase IC, priming element residues Y448 and G449 interact with the upstream end (now position −2 vs. −1 in ϕ6 structure) of the template and product strands, respectively (Figure 3b). Note that the priming element could contribute to efficient initiation, or precise start site selection, or both. In an earlier report, when the β-hairpin of the HCV NS5B priming element was truncated for eight residues (residues 444–447 and 450–453 were removed, and residues 448–449 were replace by two glycines to accommodate the truncation), the de novo synthesis activity was not much affected, but this mutant NS5B lost the specificity of terminal initiation and could initiate from internal sites of a template [51]. In a very recent report, mutation of hydrophobic and charged residues within the priming element of HCV NS5B resulted in a substantial decrease of denovo initiation activities [52]. These data indicate that the priming element does play essential roles in de novo initiation, although the mechanistic details remain to be further clarified. Similar initiation mechanisms may be taken by RdRPs from Flavivirus and Pestivirus genera, and several residues within the priming element of DENV NS5 has been proposed to contribute to the initiation-related processes [53]. However, how the initiation platform is set up in these RdRPs still awaits clarification from relevant IC structures. In contrast to de novo viral RdRPs that take advantage of the priming element at initiation, primer-dependent viral RdRPs represented by PV 3Dpol utilize a protein primer (termed VPg for “viral protein genome-linked”) or its uridylylated forms to initiate RNA synthesis [54,55]. To date, RdRP-VPg complex crystal structures have been reported in foot-and-mouth disease virus (FMDV), coxsackievirus B3 (CV B3), and enterovirus 71 (EV71), each displaying a distinct mode of interactions between the polymerase and VPg [56,57,58]. However, how VPg or uridylylated VPg is utilized in the template-directed polymerase initiation remains elusive, as none of the three polymerase-VPg structures includes an RNA template or provides clear clues for how such an IC is spatially arranged.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, No. 44 Xiao Hong Shan, Wuchang District, Wuhan 430071, China. wujiqin2010@163.com.

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.

No MeSH data available.