title: From RNA to quasispecies: a DNA polymerase with proofreading activity is highly recommended for accurate assessment of viral diversity.
abstract: RNA viruses are characterized by their high rates of genetic variation. Their genetic diversity is generally studied by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification and nucleotide (nt) sequence determination. The misinterpretation of viral diversity due to copy errors introduced by the enzymes used in this two-step protocol has not yet been assessed systematically. In order to investigate the impact of such errors, we sought to bypass the intrinsic viral heterogeneity by starting from a homogeneous cDNA template. With this in mind, the hepatitis C virus (HCV) 5' non-coding region (5'NCR) was amplified either by PCR starting from a homopolymeric cDNA template or by RT-PCR starting from the in vitro RNA transcript derived from the same original cDNA template. Amplicons were cloned and the 17-20 individual clones were sequenced in each assay. Different quasispecies patterns were obtained with various commercially available DNA polymerases, resulting in different computed error rates. The non-proofreading Taq DNA polymerase provided the highest error rate which was seven times higher than that obtained with the most reliable of the proofreading polymerases tested. We, therefore, emphasize that the misleading interpretation of the observed heterogeneity for a given viral sample could be due to ignorance of the fidelity of the polymerase used for viral genome amplification, and thus that proofreading DNA polymerases should be preferred for the investigation of natural genetic diversity of RNA viruses.
authors.affiliation: Laboratoire de Virologie, C.E.R.V.I., UPRES EA 2387, Groupe Hôpital Pitié-Salpêtrière, 83 Bd de l'hôpital, 75651 Paris Cedex 13, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Malet
authors.forename: Magali
authors.initials: M
authors.lastname: Belnard
authors.forename: Henri
authors.initials: H
authors.lastname: Agut
authors.forename: Annie
authors.initials: A
authors.lastname: Cahour
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D014644:Genetic Variation; D016174:Hepacivirus / Q000235:genetics; D009154:Mutation; D012328:RNA Viruses / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D004259:DNA-Directed DNA Polymerase