REPLICATION OF HEPATITIS-B VIRUS IN DIFFERENTIATED ADULT-RAT HEPATOCYTES TRANSFECTED WITH CLONED VIRAL-DNA
REPLICATION OF HEPATITIS-B VIRUS IN DIFFERENTIATED ADULT-RAT HEPATOCYTES TRANSFECTED WITH CLONED VIRAL-DNA
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DOI：
10.1002/jmv.1890360206
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发表时间：
1992-02-01
影响因子：
12.7
通讯作者：
GUGUENGUILLOUZO, C
中科院分区：
文献类型：
--
作者：
DIOT, C;GRIPON, P;GUGUENGUILLOUZO, C
研究了通过分离HBV全基因组，在异源物种（如正常成年大鼠肝细胞）的正常肝细胞中表达人B肝炎病毒（HBV）基因和生产病毒颗粒的可能性。 肝细胞转染的各种技术进行了测定，包括通常的钙-磷酸盐共沉淀技术，帕斯科和费根修改的钙-磷酸盐程序，和脂质转染技术。 通过测量在各种培养条件下HBV表面抗原的产生来确定转染效率。 当在低密度肝细胞铺板后1或2天测定时，转染是最有效的。 在不同的转染程序之间观察到效率的变化很少。 我们表明，在这些培养条件下，HBV的复制可以在分化的成年大鼠肝细胞中实现。 松弛的环状和单链DNA形式以及包括前基因组RNA的病毒转录物的合成发生在细胞中，而病毒抗原以及成熟和未成熟的病毒颗粒被释放到培养基中。 在与大鼠肝上皮细胞共培养的肝细胞中，病毒蛋白的产生总是较高的，并保持在低密度。 相反，通过分离未分化的大鼠肝上皮细胞不能获得病毒复制。 这些结果表明，HBV的复制可以发生在哺乳动物物种的肝细胞非密切相关的灵长类动物，并强烈支持的想法，附着的病毒和其渗透到细胞中的宿主特异性的感染过程中的关键步骤，肝特异性调节因子可能是必不可少的病毒复制。 该模型提供了一个强大的实验系统，易于用于测试HBV DNA序列对正常肝细胞功能活性的作用，特别是那些预期反式激活细胞基因的作用。
The possibility of obtaining expression of human hepatitis B virus (HBV) genes and production of virus particles in normal liver cells from heterologous species like normal adult rat hepatocytes, by transfecting the complete HBV genome, was investigated. Various techniques for hepatocyte transfection were assayed including the usual calcium-phosphate coprecipitation technique, the Pasco and Fagan modified calcium-phosphate procedure, and the lipofection technique. Transfection efficiency was determined by measuring the production of HBV surface antigen under various culture conditions. Transfection was the most efficient when assayed 1 or 2 days after hepatocyte plating at low density. Few variations in the efficiency were observed between the different transfection procedures. We show that under these culture conditions, replication of HBV can be achieved in differentiated adult rat hepatocytes. Synthesis of relaxed circular and single-stranded DNA forms and of viral transcripts including pregenome RNA occurred in the cells whereas viral antigens and mature and immature viral particles were released into the culture medium. The production of viral proteins was always higher in hepatocytes cocultivated with rat liver epithelial cells and maintained at a low density. In contrast, viral replication was not obtained by transfecting undifferentiated rat liver epithelial cells. These results demonstrate that replication of HBV can occur in hepatocytes from mammalian species non-closely related to primates and strongly support the idea that attachment of the virus and its penetration into the cells are critical steps in the host-specificity of the infection process and that hepatic-specific regulating factors could be essential for viral replication. This model provides a powerful experimental system which is easy to use to test the role of HBV DNA sequences on normal hepatocyte functional activity, particularly those expected to transactivate cellular genes.