Variable influence of mutational patterns in reverse-transcriptase domain on replication capacity of hepatitis B virus isolates from antiviral-experienced patients.
BACKGROUND
Various mutations in reverse-transcriptase domain (RT) of hepatitis B virus (HBV) polymerase may develop during antiviral therapy. The influence of these mutational patterns on HBV replication capacity remains to be fully clarified.


METHODS
Nine clones containing complete HBV genomes were isolated from 5 patients with chronic hepatitis B who had received antiviral treatment. Viral replication capacity was measured by quantitation of HBV replicative intermediates using vector-free transfer of paired mutant and wild-type HBV genomes into human hepatoma cell lines HepG2 and Huh7. HBV pgRNA was quantitated by real-time PCR and Southern blot analysis.


RESULTS
A real-time PCR assay with high sensitivity and small variation was developed for quantitation of HBV replicative intermediates. Compared to wild-type counterpart, mutant rtL217P produced 1.98-fold higher replicative intermediate level, and mutant rtM204I+rtL217P increased the replicative intermediate level to 1.20 fold. Other mutational patterns (rtV173M, rtA181S/V, rtM204I, rtQ215H, rtL229M, rtN238H, rtV84M+rtA181S+rtM204I, rtV84M+rtM204I, rtA181S+rtM204I, rtA181V+rtL229M, rtQ215H+rtN238H) reduced viral replication capacity to different extents.


CONCLUSIONS
The study offers a practical measurement assay and novel information for replication features of mutant strains; especially, rtL217P substitution likely represents an energetic replication-compensatory mutation.
Viral Hepatitis Research Laboratory, Institute of Infectious Diseases, Beijing 302 Hospital, Beijing, China.
Dong
D
Ji
Yan
Y
Liu
Lan-Lan
LL
Si
Le
L
Li
Guo-Feng
GF
Chen
Shao-Jie
SJ
Xin
Jin-Min
JM
Zhao
Dongping
D
Xu
D000998:Antiviral Agents / Q000627:therapeutic use*; D002138:Calibration; D016679:Genome, Viral / Q000235:genetics; D005838:Genotype; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D014779:Virus Replication
D000998:Antiviral Agents; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase