Inhibition of hepatitis C virus (HCV) RNA polymerase by DNA aptamers: mechanism of inhibition of in vitro RNA synthesis and effect on HCV-infected cells.
We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.
Bellecave
Pantxika
P
CNRS UMR5234, Bordeaux 33076 Cedex, France.
Cazenave
Christian
C
Rumi
Julie
J
Staedel
Cathy
C
Cosnefroy
Ophélie
O
Andreola
Marie-Line
ML
Ventura
Michel
M
Tarrago-Litvak
Laura
L
Astier-Gin
Thérèse
T
D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis* / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D052156:SELEX Aptamer Technique; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014771:Virion / Q000378:metabolism; D014779:Virus Replication
D052157:Aptamers, Nucleotide; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase