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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.


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].
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ijms-16-12943-f001: 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].

Mentions: The viral proteins that carry out RdRP function in family Flaviviridae vary in size, with about 600–900 residues encoded. Among these, the nonstructural protein 5 (NS5) of Flavivirus is the largest, having a ≈260-residue S-adenosyl-l-methionine (SAM)-dependent methyltransferase (MTase) fused to the N-terminus of the polymerase module through a short flexible linker (Figure 1a). Based on consensus folding of the MTase and RdRP in numerous crystal structures [13,14,15,16] and the first full-length NS5 crystal structure in JEV [17], the linker spans about ten residues following a highly consensus GTR sequence in the C-terminus of the MTase. It exhibits tendency to be disordered or to adopt an extended conformation in the majority of structures. As an exception, a helical fold was observed in this region in a recent report of the full-length DENV NS5 structure [18], suggesting its potentials to coordinate domain motions of the regions it is tethering. The interactions and regulations between the MTase and RdRP had remained enigmatic until the first full-length NS5 crystal structure was reported in 2013, starting to unravel the rather complicated nature of the MTase-RdRP interplay [17]. This JEV NS5 structure defines a medium size MTase-RdRP interface with key residues highly conserved in Flavivirus and recently proven to be functionally important [19,20]. In contrast to Flavivirus NS5, the nonstructural protein 5B (NS5B) of HCV is essentially the polymerase module with a C-terminal 21-residue “membrane anchor” (Figure 1a). The Pestivirus NS5B appears to be a “hybrid” with its C-terminal hydrophobic region resembling that of HCV NS5B and its N-terminal 90 residues reminiscent of the Flavivirus NS5 MTase (Figure 1a). The Flavivirus NS5 and Pestivirus NS5B also share a 20–28-residue region first defined in the full-length JEV NS5 structure as “the N-terminal extension” to the core polymerase and recently shown to be non-dispensable to regular polymerase catalysis in JEV NS5 (Figure 1a) [19]. Although sequence similarity of this N-terminal extension is quite low between the two genera, they adopt a similar fold, and if properly presented, were structurally integrated into the core polymerase to form one entirety (Figure 1b) [14,15,17,21].


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

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

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].
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Related In: Results  -  Collection

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

ijms-16-12943-f001: 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].
Mentions: The viral proteins that carry out RdRP function in family Flaviviridae vary in size, with about 600–900 residues encoded. Among these, the nonstructural protein 5 (NS5) of Flavivirus is the largest, having a ≈260-residue S-adenosyl-l-methionine (SAM)-dependent methyltransferase (MTase) fused to the N-terminus of the polymerase module through a short flexible linker (Figure 1a). Based on consensus folding of the MTase and RdRP in numerous crystal structures [13,14,15,16] and the first full-length NS5 crystal structure in JEV [17], the linker spans about ten residues following a highly consensus GTR sequence in the C-terminus of the MTase. It exhibits tendency to be disordered or to adopt an extended conformation in the majority of structures. As an exception, a helical fold was observed in this region in a recent report of the full-length DENV NS5 structure [18], suggesting its potentials to coordinate domain motions of the regions it is tethering. The interactions and regulations between the MTase and RdRP had remained enigmatic until the first full-length NS5 crystal structure was reported in 2013, starting to unravel the rather complicated nature of the MTase-RdRP interplay [17]. This JEV NS5 structure defines a medium size MTase-RdRP interface with key residues highly conserved in Flavivirus and recently proven to be functionally important [19,20]. In contrast to Flavivirus NS5, the nonstructural protein 5B (NS5B) of HCV is essentially the polymerase module with a C-terminal 21-residue “membrane anchor” (Figure 1a). The Pestivirus NS5B appears to be a “hybrid” with its C-terminal hydrophobic region resembling that of HCV NS5B and its N-terminal 90 residues reminiscent of the Flavivirus NS5 MTase (Figure 1a). The Flavivirus NS5 and Pestivirus NS5B also share a 20–28-residue region first defined in the full-length JEV NS5 structure as “the N-terminal extension” to the core polymerase and recently shown to be non-dispensable to regular polymerase catalysis in JEV NS5 (Figure 1a) [19]. Although sequence similarity of this N-terminal extension is quite low between the two genera, they adopt a similar fold, and if properly presented, were structurally integrated into the core polymerase to form one entirety (Figure 1b) [14,15,17,21].

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.