A Template RNA Entry Channel in the Fingers Domain of the Poliovirus Polymerase

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• 作者：Matthew G. Kortus¹ ; Brian J. Kempf² ; Kevin G. Haworth¹ ; David J. Barton² ; Olve B. Peersen¹ ; ^{Olve.Peersen@ColoState.edu}
• 关键词：RdRP ; RNA-dependent RNA polymerase ; FP ; fluorescence polarization ; EC ; elongation complex ; PFU ; plaque-forming units ; PETE ; polymerase elongation template element ; TF ; total fluorescence ; hpt ; hours post-transfection
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：6 April, 2012
• 卷：417
• 期：4
• 页码：263-278
• 文件大小：1428 K

摘要

Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase, 3D^pol, that is required for viral RNA replication. Structures of 3D^pol from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a series of alanine substitution mutations at these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex (EC) stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding, while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for EC stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3D^pol pinky finger drastically decreased the rate of initiation as well as EC stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or EC stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA ECs.