ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase

作者：Karr Jonathan P; Peersen Olve B^*

来源：Biochemistry, 2016, 55(28): 3995-4002.

DOI：10.1021/acs.biochem.6b00467

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 K-m of 24 mu M and the three noncognate nucleotides competitively inhibit the reaction with K-ic values of 500 mu M for GTP, 1300 mu M for ATP, and 3000 mu M for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a K-iu of 1800 mu M, resulting in allosteric modulation of 3D(pol) that slowed the,polymerase elongation rate approximate to 4-fold. ATP uncompetitive inhibition required the beta- and gamma-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.

• 出版日期2016-7-19