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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.


Related in: MedlinePlus

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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ijms-16-12943-f004: 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.

Mentions: In contrast to HCV NS5B, JEV NS5 and BVDV NS5B each has an N-terminal region that may provide in cis regulation of the core polymerase. This difference is reflected in the crystal structures of these RdRPs to some extent (Figure 4). The structure of HCV NS5B exhibits a canonical RdRP fold (Figure 4a, bottom panel) with all seven catalytic motifs properly arranged around the active site [50]. By contrast, the crystal structures of the Flavivirus NS5 in the absence of the N-terminal MTase consistently exhibit fingers domain disorder or non-canonical folding, particularly for motifs F and G (Figure 4b, JEV-ii, JEV-iii, WNV, and DENV-ii structures) [14,15,60]. It was until the report of the first full-length NS5 crystal structure that the intact and canonically folded Flavivirus polymerase was observed (Figure 4a, top panel). In this full-length JEV NS5 structure, motif F participates in an intra-molecular interface between MTase and polymerase and contributes Phe467 to the formation of a six-residue hydrophobic network [17]. These hydrophobic residues are highly conserved in genus Flavivirus, implying the functional significance of the interface. When polar or charged residue mutations were introduced into these sites, virus replication levels were significantly affected [20]. Furthermore, the introduction of negatively charged Asp mutation into these sites could affect in vitro polymerase activities at both initiation and elongation [19]. Taken together, the MTase-polymerase interface observed in the full-length JEV NS5 structure play auxiliary roles to fingers domain folding and polymerase catalysis. In DENV NS5 studies, the removal of the MTase domain and/or further removal of the linker residues were shown to alter polymerase activities [61,62]. Very recently, a full-length DENV NS5 crystal structure was reported, and a different interface between MTase and polymerase was observed with less hydrophobic features [18]. In such a structure, motif F no longer participates in the interface and both motif F and motif G are largely disordered (Figure 4b, DENV structure) as with those MTase-less NS5 structures. As a natural fusion of MTase and polymerase, the full-length Flavivirus NS5 may have the MTase interact with the polymerase in different ways. However, the conformational state observed in the full-length JEV NS5 crystal structure with canonically folded motifs F and G may be regarded as the ground state for polymerase catalysis, as the highly conserved K/E/R residues in motif F (JEV NS5 residues 459, 461, 474 or equivalent) and the structurally critical residue pair in motif G (JEV NS5 409–410 or equivalent) are in-line for NTP/+1 templating nucleotide interactions and for template RNA binding, respectively (Figure 2b and Figure 4a). Although the binding of NTP or an inhibitor molecule could affect the folding of motif F in JEV RdRP structures [60] or motif G in a DENV RdRP structure [63], respectively, the conformations of the NTP/inhibitor-induced regions apparently deviate from the canonical folds (see Figure 4b and JEV-iii for the conformation of the JEV RdRP-GTP structure).


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

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

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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Related In: Results  -  Collection

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ijms-16-12943-f004: 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.
Mentions: In contrast to HCV NS5B, JEV NS5 and BVDV NS5B each has an N-terminal region that may provide in cis regulation of the core polymerase. This difference is reflected in the crystal structures of these RdRPs to some extent (Figure 4). The structure of HCV NS5B exhibits a canonical RdRP fold (Figure 4a, bottom panel) with all seven catalytic motifs properly arranged around the active site [50]. By contrast, the crystal structures of the Flavivirus NS5 in the absence of the N-terminal MTase consistently exhibit fingers domain disorder or non-canonical folding, particularly for motifs F and G (Figure 4b, JEV-ii, JEV-iii, WNV, and DENV-ii structures) [14,15,60]. It was until the report of the first full-length NS5 crystal structure that the intact and canonically folded Flavivirus polymerase was observed (Figure 4a, top panel). In this full-length JEV NS5 structure, motif F participates in an intra-molecular interface between MTase and polymerase and contributes Phe467 to the formation of a six-residue hydrophobic network [17]. These hydrophobic residues are highly conserved in genus Flavivirus, implying the functional significance of the interface. When polar or charged residue mutations were introduced into these sites, virus replication levels were significantly affected [20]. Furthermore, the introduction of negatively charged Asp mutation into these sites could affect in vitro polymerase activities at both initiation and elongation [19]. Taken together, the MTase-polymerase interface observed in the full-length JEV NS5 structure play auxiliary roles to fingers domain folding and polymerase catalysis. In DENV NS5 studies, the removal of the MTase domain and/or further removal of the linker residues were shown to alter polymerase activities [61,62]. Very recently, a full-length DENV NS5 crystal structure was reported, and a different interface between MTase and polymerase was observed with less hydrophobic features [18]. In such a structure, motif F no longer participates in the interface and both motif F and motif G are largely disordered (Figure 4b, DENV structure) as with those MTase-less NS5 structures. As a natural fusion of MTase and polymerase, the full-length Flavivirus NS5 may have the MTase interact with the polymerase in different ways. However, the conformational state observed in the full-length JEV NS5 crystal structure with canonically folded motifs F and G may be regarded as the ground state for polymerase catalysis, as the highly conserved K/E/R residues in motif F (JEV NS5 residues 459, 461, 474 or equivalent) and the structurally critical residue pair in motif G (JEV NS5 409–410 or equivalent) are in-line for NTP/+1 templating nucleotide interactions and for template RNA binding, respectively (Figure 2b and Figure 4a). Although the binding of NTP or an inhibitor molecule could affect the folding of motif F in JEV RdRP structures [60] or motif G in a DENV RdRP structure [63], respectively, the conformations of the NTP/inhibitor-induced regions apparently deviate from the canonical folds (see Figure 4b and JEV-iii for the conformation of the JEV RdRP-GTP structure).

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.


Related in: MedlinePlus