Ribavirin-ResistantMutantsof创新.PDFVIP

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture Sara Sadeghipour, Emily J. Bek, Peter C. McMinn Infectious Diseases and Immunology, Sydney Medical School, University of Sydney, New South Wales, Australia It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high repli- cation fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid posi- tion 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 M ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 M ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of D o random mutagenesis after 13 passages in the presence of 400 M ribavirin was nearly 10 times higher in the 26M genome than in w n the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred l o resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G6