ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase.
The RNA-dependent RNA polymerases from positive-strand RNA viruses, such as picornaviruses and flaviviruses, close their active sites for catalysis via a unique NTP-induced conformational change in the palm domain. Combined with a fully prepositioned templating nucleotide, this mechanism is error-prone and results in a distribution of random mutations in the viral progeny often described as a quasi-species. Here we examine the extent to which noncognate NTPs competitively inhibit single-cycle elongation by coxsackievirus B3 3D(pol), a polymerase that generates three to four mutations per 10 kb of RNA synthesized during viral infection. Using an RNA with a templating guanosine combined with 2-aminopurine fluorescence as a reporter for elongation, we find that the cognate CTP has a Km of 24 μM and the three noncognate nucleotides competitively inhibit the reaction with Kic values of 500 μM for GTP, 1300 μM for ATP, and 3000 μM for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a Kiu of 1800 μM, resulting in allosteric modulation of 3D(pol) that slowed the polymerase elongation rate ≈4-fold. ATP uncompetitive inhibition required the β- and γ-phosphates, and its extent was significantly diminished in two previously characterized low-fidelity polymerases. This led to further mutational analysis and the identification of a putative allosteric binding site below the NTP entry channel at the interface of conserved motifs A and D, although cocrystallization failed to reveal any density for bound ATP in this pocket. The potential role of an ATP allosteric effect during the virus life cycle is discussed.
Department of Biochemistry and Molecular Biology, Colorado State University , Fort Collins, Colorado 80523, United States.
Jonathan P
JP
Karr
Department of Biochemistry and Molecular Biology, Colorado State University , Fort Collins, Colorado 80523, United States.
Olve B
OB
Peersen
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000494:Allosteric Regulation / Q000187:drug effects; D001483:Base Sequence; D020134:Catalytic Domain; D029822:Enterovirus B, Human / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D055029:Inverted Repeat Sequences; D007700:Kinetics; D008958:Models, Molecular; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012313:RNA; D000255:Adenosine Triphosphate; D012324:RNA-Dependent RNA Polymerase