Emergence of a novel mutation in the FLLA region of hepatitis B virus during lamivudine therapy

Abstract

The emergence of resistance to lamivudine has been one of the major stumbling blocks to successful treatment and control of hepatitis B virus (HBV) infections. The major mechanism of resistance has been attributed to the alteration in the YMDD motif of the HBV polymerase due to an amino acid change of rtM204 to V/I and an accompanying rtL180M conversion. A novel mutation pattern in a patient having clinical breakthrough under lamivudine therapy was discovered. The mutant had a rtL180C/M204I genotype and was detected after 2 years of therapy with lamivudine. To characterize this novel variant, site-directed mutagenesis was performed using a vector construct containing the HBV genome. Transient transfection studies in human hepatoma cells with HBV carrying the new mutant demonstrated that the rtL180C/M204I mutant was resistant to lamivudine up to 10 microM. The resistance profile was comparable to that of the previously reported rtL180 M/M204I-containing virus. These observations were further confirmed by generation of stable cultures transfected with the mutant virus.

FIG. 1.

Evolution of the novel HBV variant from the wild-type virus. Virological assays and determinations were performed as described in Materials and Methods.

FIG. 2.

DNA sequence alignment of pol region of mutant HBV generated by site-directed mutagenesis. Generation of the plasmids and PCR was performed as described in Materials and Methods. The sequence of the pol region flanking the FLLA region and the YMDD motif is depicted.

FIG. 3.

HBV copy numbers from wild-type (rtM204M) and mutant (rtM204I and rtL180C/M204I) viruses from transiently transfected Huh-7 cells. HBV abundance was determined using real-time PCR technology. Cells transfected with each of the vector constructs were treated with 10 μM of lamivudine for 5 days. Untreated cultures served as controls. The DNA copy numbers in each group were normalized to their HBsAg values.

FIG. 4.

HBV copy numbers from wild-type (rtL180L/M204M) and FLLA mutant (rtL180C/M204M and rtL180M/M204M) viruses from transiently transfected Huh-7 cells. HBV DNA quantification was done using real-time PCR technology. Cells transfected with the vector and a plasmid carrying the β-galactosidase gene construct were treated with 10 μM of lamivudine (3TC) for 5 days. Untreated cultures served as controls. The DNA copy numbers in each group were normalized to β-galactosidase activity.

FIG. 5.

A. Replicative intermediates of rtL180C/M204I HBV from transiently transfected Huh-7 cells. Southern analysis of HBV DNA from rtL180C/M204I HBV transfections was performed as described in Materials and Methods. OC: open circular HBV DNA; DS: double-stranded HBV DNA; SS: single-stranded HBV DNA; NT: nontransfected. The positions of the size markers of HindIII-digested lambda markers (4 kb and 2 kb) are indicated. B. β-Galactosidase activity from rtL180C/M204I HBV transient transfection. β-Galactosidase activity from the cells cotransfected with a vector carrying the β-gal gene was performed as described in the Materials and Methods section.

FIG. 6.

A. rtM204M, rtM204I, and rtL180C/M204I HBVs from stably transfected HepG2 cells. The arrow indicates PCR-amplified HBV product from the supernatants of cell cultures. Lanes: 1, product from rtM204M; 2, rtM204I HBV; and 3 and 4, product from rtL180C/M204I. Lambda DNA digested with HindIII was used as the size marker. B. Effect of lamivudine on HBV DNA production from stably transfected cell cultures. Virus quantification was performed by real-time PCR methodology as previously described (7).