TY - JOUR
T1 - Identification of a ribavirin-resistant NS5B mutation of hepatitis C virus during ribavirin monotherapy
AU - Young, Kung Chia
AU - Lindsay, Karen L.
AU - Lee, Ki Jeong
AU - Liu, Wen Chun
AU - He, Jian Wen
AU - Milstein, Susan L.
AU - Lai, Michael M.C.
N1 - Funding Information:
Abbreviations: RBV, ribavirin; IFN-α, interferon alfa; HCV, hepatitis C virus; RdRp, RNA-dependent RNA polymerase; NS, nonstructural; ALT, alanine aminotransferase; RT, reverse transcription; PCR, polymerase chain reaction. From the 1Department of Molecular Microbiology and Immunology, 2Howard Hughes Medical Institute, and 4Department of Medicine, University of Southern California, Keck School of Medicine, Los Angeles, CA; and the 3Department of Medical Technology and 5Institute of Basic Medical Sciences, Medical College, National Cheng Kung University, Tainan, Taiwan, Republic of China. Received February 14, 2003; accepted August 4, 2003. Supported by grants AI 47348 and AI 40038 from the National Institutes of Health. Address reprint requests to: Michael M. C. Lai, M.D., Ph.D., Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, 2011 Zonal Avenue, HMR-401, Los Angeles, CA 90089-9094. E-mail: michlai@hsc.usc.edu; fax: 323-442-1721. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3804-0012$30.00/0 doi:10.1053/jhep.2003.50445
PY - 2003/10/1
Y1 - 2003/10/1
N2 - Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or interferon alfa (IFN-α) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN-α or placebo group. RBV caused preferentially A-to-G and U-to-A mutations. Interestingly, an NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose-dependent manner. Thus, NS5B F415Y mutation represents an RBV-resistant variant. The 3-dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template-primer duplex in the putative RNA-binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore, the selection of an RBV-resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity.
AB - Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or interferon alfa (IFN-α) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN-α or placebo group. RBV caused preferentially A-to-G and U-to-A mutations. Interestingly, an NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose-dependent manner. Thus, NS5B F415Y mutation represents an RBV-resistant variant. The 3-dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template-primer duplex in the putative RNA-binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore, the selection of an RBV-resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity.
UR - http://www.scopus.com/inward/record.url?scp=0141755411&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0141755411&partnerID=8YFLogxK
U2 - 10.1053/jhep.2003.50445
DO - 10.1053/jhep.2003.50445
M3 - Article
C2 - 14512874
AN - SCOPUS:0141755411
VL - 38
SP - 869
EP - 878
JO - Hepatology
JF - Hepatology
SN - 0270-9139
IS - 4
ER -