The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.     

The “Trojan horse” model-delivery of anti-HBV small interfering RNAs by a recombinant HBV vector

Hepatitis B virus (HBV) is a small virus that infects the liver. The major obstacle in applying the RNA interference method as an anti-HBV weapon is the challenge to deliver the small interfering RNA molecules to the liver efficiently and specifically. Here we show that HBV-specific short hairpin RNAs (shRNAs) are efficiently expressed from a recombinant HBV into which an shRNA-expressing cassette was inserted, resulting in a significant knock-down of HBV gene expression. Notably, this recombinant HBV still expresses the HBV Core protein, which is targeted by the shRNAs produced by the same vector. Our results set the stage for further use of this recombinant HBV virus with the potential to function as a “Trojan horse”; one that specifically targets the liver and uses the resident virus as an helper for its own propagation, and at the same time eliminate itself and the resident HBV by knocking-down their gene expression.     

应用RNAi技术治疗HBV的研究进展

乙型肝炎病毒（hepatitis B virus,HBV）感染是一种全球性的健康问题。全球大约有4亿HBV慢性感染患者,尤其在中国,感染率高达9.8%。RNAi（RNA interference）技术具有特异性地抑制,甚至关闭基因表达的特点,在治疗HBV感染方面有很大的应用前景。近年来,研究者针对HBV基因设计或筛选有效siRNA靶点做了很多研究,最近还出现了针对与HBV代谢相关的宿主基因设计或筛选siRNA靶点治疗HBV的新策略。该文对RNAi技术在HBV治疗方面取得的研究进展作一综述。     

Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.     

乙肝病毒感染导致Mrell下调及基因组断裂

[目的]乙肝病毒的持续感染与肝细胞癌的发生密切相关.本文探讨了乙肝病毒感染后导致宿主蛋白Mrell的变化,并研究这种蛋白的变化可能导致肝癌发生的机制.[方法]本文通过Westernblot检测了乙肝病毒感染HL7702细胞及肝癌组织标本的Mrell蛋白的变化,并且通过RNA干涉的方法干扰了Mrell的表达,用连接介导PCR检测基因组的变化.[结果]本研究发现乙肝病毒感染细胞导致Mrell表达下调,肝癌组织也可以发现Mrell表达下调;下调Mrell表达可以导致细胞基因组的断裂增多.[结论]HBV感染导致Mrell表达下调导致基因组不稳定,这可能与HBV感染导致细胞恶性转化相关.     

Identification of two distinct elements in the long terminal repeat of HTLV-I responsible for maximum gene expression

Human T-cell leukemia virus type I has a unique sequence, pX, between env and the 3' long terminal repeat (LTR). One of its products, p40, activates gene expression directed by the LTR in a trans-acting manner. We have analysed the mechanism of this trans-activation mediated by p40 in human T cells co-transfected with a plasmid expressing p40 using the transient CAT gene expression. We identified two distinct elements in the LTR which are involved in maximum gene expression. The first was present in a 230-bp fragment upstream from TATA box in the U3 region and behaved as a classical enhancer. This region was also shown to be responsible for trans-activation by p40. This element alone together with functional p40 could direct the gene expression at only approximately 10% of the level achieved by the complete LTR and p40. The second element was present within a 300-bp fragment downstream from the RNA start site and profoundly enhanced the gene expression in a way independent from trans-activation mechanism. This enhancement was observed only when the element was located immediately downstream from the RNA start site without orientation preference. These two elements participate independently in the enhancement of gene expression.     

复制型HBV转基因小鼠遗传稳定性研究

目的：提高复制型HBV转基因小鼠的遗传稳定性。方法：应用回交传代及双杂交育种法,经荧光定量PCR、ELISA和化学发光法研究HBV基因在小鼠体内的复制与表达。结果：HBV转基因小鼠已稳定传至第23代,血清HBsAg达4122.31±2044.74IU/ml,93.93%的转基因小鼠血清HBV DNA达104-106copies/ml,表达复制水平较早期有显著提高并稳定传代;雌雄小鼠之间表达水平无显著性差异。结论：该转基因小鼠经过培育传代,已成为一个高表达且遗传稳定的复制型HBV小鼠模型。     

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner.

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.