In vitro resistance to interferon of hepatitis B virus with precore mutation.
OBJECTIVE
Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-alpha), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro.


METHODS
HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection.


RESULTS
We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind III and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system.


CONCLUSIONS
These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.
Wang
Yan
Y
Hepatology Institute, Peking University People's Hospital, No.11 Xizhimen South Street, Beijing 100044, China.
Wei
Lai
L
Jiang
Dong
D
Cong
Xu
X
Fei
Ran
R
Xiao
Jiang
J
Wang
Yu
Y
D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003470:Culture Media / Q000378:metabolism; D004274:DNA, Recombinant; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral; D016679:Genome, Viral; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D019694:Hepatitis B, Chronic / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D066298:In Vitro Techniques; D016898:Interferon-alpha / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
D000998:Antiviral Agents; D003470:Culture Media; D004274:DNA, Recombinant; D006512:Hepatitis B Core Antigens; D016898:Interferon-alpha