title: Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome.
abstract: Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (ε) located in the preCore region, essential for viral replication. ε stability is enhanced by the presence of preCore variants and ε is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the ε signal was found to be the main constraint for selecting main preCore mutations. Analysis of ε-signal variability suggested the essential nature of the ε structural motif and that certain nucleotides may be involved in ε signal functions.
authors: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Instituto Carlos III Corsega 180, 08036, Barcelona, Spain.
authors: Maria
authors: M
authors: Homs
authors: Maria
authors: M
authors: Buti
authors: Josep
authors: J
authors: Quer
authors: Rosendo
authors: R
authors: Jardí
authors: Melanie
authors: M
authors: Schaper
authors: David
authors: D
authors: Tabernero
authors: Israel
authors: I
authors: Ortega
authors: Alex
authors: A
authors: Sanchez
authors: Rafael
authors: R
authors: Esteban
authors: Francisco
authors: F
authors: Rodriguez-Frias
mesh_terms: D000293:Adolescent; D000328:Adult; D020029:Base Pairing; D001483:Base Sequence; D020134:Catalytic Domain; D003062:Codon; D004252:DNA Mutational Analysis; D015247:Deoxyribonuclease HindIII; D015684:Gene Products, pol / Q000737:chemistry / Q000235:genetics*; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*; D015534:Trans-Activators / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins; D055815:Young Adult
chemical_list: D003062:Codon; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D012367:RNA, Viral; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D015247:Deoxyribonuclease HindIII