4ZPB

Coxsackievirus B3 Polymerase - F364W mutant

Summary for 4ZPB

• Entry DOI: 10.2210/pdb4zpb/pdb
• Related: 4ZP6 4ZP7 4ZP8 4ZP9 4ZPA 4ZPC 4ZPD 4ZR3 4ZR4
• Descriptor: RNA-directed RNA polymerase (2 entities in total)
• Functional Keywords: rna-dependent rna polymerase, transferase
• Biological source: Coxsackievirus B3
• Cellular location: Host cytoplasm . Host cytoplasmic vesicle . Host cytoplasmic vesicle membrane . Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Virion : Q5UEA2
• Total number of polymer chains: 1
• Total formula weight: 52516.09

Authors

Peersen, O.B.,McDonald, S.M. (deposition date: 2015-05-07, release date: 2016-05-11, Last modification date: 2024-03-06)

Primary citation

McDonald, S.,Block, A.,Beaucourt, S.,Moratorio, G.,Vignuzzi, M.,Peersen, O.B.
Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo.
J.Biol.Chem., 291:13999-14011, 2016

PubMed Abstract: Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.

PubMed: 27137934
DOI: 10.1074/jbc.M116.726596

Experimental method

X-RAY DIFFRACTION (1.797 Å)