Basal Core Promoter and Precore Mutations in the HBV Enhance Replication Effic

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Journal of Virology, August 2004, p. 8524-8535, Vol. 78, No. 16 0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.16.8524-8535.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved. Basal Core Promoter and Precore Mutations in the Hepatitis B Virus Genome Enhance Replication Efficacy of Lamivudine-Resistant Mutants Frank Tacke,1 Christina Gehrke,1 Tom Luedde,1 Albert Heim,2 Michael P. Manns,1 and Christian Trautwein1*

Departments of Gastroenterology, Hepatology and Endocrinology,1 Virology, Hannover Medical School, 30625 Hannover, Germany2

Received 22 January 2004/ Accepted 29 March 2004

During chronic hepatitis B virus (HBV) infection, mutations in the precore (PC) or basal core promoter (BCP) region affecting HBV e antigen (HBeAg) expression occur commonly and represent the predominant virus species in patients with HBeAg-negative chronic hepatitis B. The PC mutation (G1896A+C1858T) creates a translational stop codon resulting in absent HBeAg expression, whereas BCP mutations (A1762T/G1764A) reduce HBeAg expression by transcriptional mechanisms. Treatment of chronic HBV infection with lamivudine (LMV) often selects drug-resistant strains with single (rtM204I) or double (rtL180M+rtM204V) point mutations in the YMDD motif of HBV reverse transcriptase. We cloned replication-competent HBV vectors (genotype A, adw2) combining mutations in the core (wild type [wt], PC, and BCP) and polymerase gene (wt, rtM204I, and rtL180M/M204V) and analyzed virus replication and drug sensitivity in vitro. Resistance to LMV (rtM204I/rtL180M+rtM204V) was accompanied by a reduced replication efficacy as evidenced by reduced pregenomic RNA, encapsidated progeny DNA, polymerase activity, and virion release. PC mutations alone did not alter virus replication but restored replication efficacy of the LMV-resistant mutants without affecting drug resistance. BCP mutants had higher replication capacities than did the wt, also in combination with LMV resistance mutations. All nine HBV constructs showed similar sensitivities to adefovir. In conclusion, BCP-PC mutations directly impact the replication capacity of LMV-resistant mutants. PC mutations compensated for replication inefficiency of LMV-resistant mutants, whereas BCP mutations increased viral replication levels to above the wt baseline values, even in LMV-resistant mutants, without affecting drug sensitivity in vitro. Adefovir may be an effective treatment when combinations of core and polymerase mutations occur.

* Corresponding author. Mailing address: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. Phone: (49) 511-532 6620. Fax: (49) 511-532 4896. E-mail: [email protected].

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慢肝治疗中，前C区变异或基C区启动子 (BCP)区域变异容易影响HBV 的 e抗原表达，表现为E抗原阴性的绝对病毒特征。前C区变异 (G1896A+C1858T) 生成编译停顿的遗传密码，导致E抗原停止表达。而BCP变异 (A1762T/G1764A) 通过降低转录水平从而减少了 HBeAg表达。拉米治疗 HBV (LMV) 经常导致位于 单股 (rtM204I) 或双股 (rtL180M+rtM204V) 链处发生耐药，发生HBV逆转录酶中YMDD型变异。

我们克隆了HBV携带者的完整片断复制 (基因型 A, adw2) 混合了core (wild type [wt], PC, and BCP) 和聚合酶基因 polymerase gene (wt, rtM204I, and rtL180M/M204V) ,并对病毒复制和药敏感程度进行体外分析。对LMV (rtM204I/rtL180M+rtM204V) 的耐药被证实是通过降低 pregenomic RNA, DNA壳体化后裔, 聚合酶活性，和病毒粒子释放等手段来降低复制有效性来完成的。前C区变异本身不改变病毒复制，但是却恢复了耐药变异株复制效率。 和LMV耐药株在一起，BCP 变异具有比野生株更高的复制能力。 所有9种HBV结构显示同样的阿地福韦敏感性。结论， BCP-PC 变异直接影响了拉米耐药株的复制能力。PC 变异弥补了拉米变异株的复制的低效率,而BCP变异则在不影响药物敏感性的前提下，将病毒复制能力提高到野生株，甚至拉米耐药株以上的水平。

阿地福韦可以有效治疗前C变异和聚合酶变异。