Use of the hepatitis B virus recombinant baculovirus-HepG2 system to studythe effects of (-)-beta-2 ',3 '-dideoxy-3 '-thiacytidine on replication ofhepatitis B virus and accumulation of covalently closed circular DNA

(-)-beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside a nalog which effectively interferes with the replication of hepatitis B viru s (HBV) DNA in vitro and in vivo. We have investigated the antiviral proper ties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovir us. Different types of information can be obtained with the HBV baculovirus -HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that ca n be obtained from conventional HBV-expressing cell lines, (ii) cultures ca n be manipulated and/or treated prior to or during the initiation of HBV ex pression, and (iii) high levels of HBV replication allow the rapid detectio n of HBV products including covalently closed circular (CCC) HBV DNA from l ow numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by r eductions in the levels of both extracellular HBV DNA and intracellular rep licative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we obs erved agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC, Importantly, the HBV baculo virus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. W e also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with t he highest concentration of 3TC tested in this study (2 mu M) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DN A, whereas treatment with the same concentration of 3TC at a time when CCC HBV DNA pools were established within the cells was considerably less effec tive. In addition, our results suggest that in HepG2 cells, non-protein-ass ociated relaxed circular HBV DNA and particularly CCC HBV DNA are considera bly more resistant to 3TC treatment than other forms of HBV DNA, including replicative intermediates and extracellular DNA, We conclude from these stu dies that the HBV baculovirus-HepG2 system has specific advantages for drug studies and can be used to complement other in vitro model systems current ly used for testing antiviral compounds.