乙型肝炎病毒X蛋白及其截短体与损伤DNA结合蛋白1在HBV复制中的作用

Roles of full-length and truncated hepatitis B virus X protein and of interactions with the host-encoded damaged DNA binding protein 1 in HBV repfication

目的探讨乙型肝炎病毒x蛋白（HBx）及其两个截短体（HBx、HBx43～154）与损伤DNA结合蛋白（DDB）1在HBV复制中的作用。方法用质粒瞬时转染HepG2细胞，72h后分别提取细胞总蛋白质和总RNA，通过Westernblot分析验证DDB1蛋白表达，逆转录实时定量PCR在基因的mRNA水平观察HBx蛋白及其截短体对DDB1蛋白表达的影响。提取转染72h后细胞胞质DNA，收集细胞的培养上清液，通过实时定量PCR及酶联免疫吸附试验检测各组细胞胞质HBVDNA复制及培养上清液中病毒标志物HBsAg、HBeAg表达水平，从而观察HBx蛋白及其两个截短体与DDB1对HBV复制的影响。对数据进行单因素方差分析及检验。结果在有HBV复制的HepG2细胞中，缺失HBx转染组细胞中DDB1蛋白在mRNA水平明显下降（相对表达水平为野生型转染组的52．74％±8．95％，f=9．143，P〈0．05），胞质DNA的复制及培养上清液中病毒标志物HBsAg、HBeAg表达水平也明显下降（相对表达水平分别为野生型转染组的55．49％±1．19％、48．05％±2．90％、46．22％±2．20％，P值均〈0．05）。共转染HBxN-端截短体HBx4。后细胞中DDB1蛋白mRNA水平恢复到野生型水平，且能使缺失HBx蛋白的HepG2细胞中胞质HBVDNA的复制及培养上清液中病毒标志物HBsAg、HBeAg的表达恢复到野生型水平。但共转染HBxC-端截短体HBx的细胞中DDB1蛋白在mRNA水平较野生组明显下降（相对表达水平为野生型转染组的59．95％±9．09％，f=7．629，P〈0．05），且不能使转染缺失HBx蛋白的HepG2细胞中胞质HBVDNA的复制及培养上清液中病毒标志物HBsAg、HBeAg的表达恢复到野生型水平（相对表达水平分别为野生型转染组的62．53％±0．67％、63．13％±2．14／0、55．40％±0．99％，P值均〈0．05）。并且各转染组DDB1蛋白mRNA水平变化与胞质HBVDNA复制和培养上清液中病毒标志物HBsAg、HBeAg表达水平是一致的。结论C-端53个氨基酸及DDB1蛋白对于HBV野生型水平的复制是需要的，而N-端42个氨基酸对于HBV复制水平无明显影响。C-端53个氨基酸对HBV复制的影响作用可能是通过影响DDB1蛋白水平实现的。

Objective To investigate the roles of the hepatitis B virus （HBV）-encoded X protein （HBx）, including the full-length and truncated isoforms, and in conjunction with the host-encoded damaged DNA binding protein 1 （DDB1） in HBV replication. Methods Recominant expressionplasmids carrying the wild-type HBV genome （pGEM-HBV1.2） or with deletion of the full-length HBx protein （pHBV-AX）, or carrying the full-length HBx protein （pSI-X） or the HBx1-1 （pSI-XH） or HBx43-154 （pSI-X43-154） isoforms were constructed for transfection into HepG2 cells. The pcDNA6.2-GW/ EmGFP-miR （DDB 1-miRNA） vector was constructed for silencing of the DDB 1 gene in co-transfected HepG2 cells. At 72 h after transfections, DDB1 silencing was confirmed by western blot analysis and realtime quantitive reverse transcription PCR, HBV DNA copies number was assessed by real time PCR, and levels of hepatitis B surface antigen （HbsAg） and hepatitis B e antigen （HbeAg） were determined by ELISA. Differences between groups was statistically analyzed by single-factor analysis of variance and the t-test. Results Transfection with pHBV-AX led to reductions in DDB1 mRNA （to 52.74% of that in the wild-type pGEM-HBV1.2 transfected cells）, HBV replication （to 55.49%）, HBsAg level （48.05%）, and HBeAg level （46.22%）. Co-transfection with pSI-X or pSI-X43-154, but not with pSI-X1-1-1, restored the pHBV-AX-induced reductions in DDB 1 mRNA, HBV replication, HBsAg and HBeAg to wild-type levels. The quantity of DDB 1 mRNA was approximately parallel with the quantity of HBV DNA copies in all the HepG2 transfection groups. Conclusion The COOH-terminal amino acids of HBx are required for HBV replication in hepatocytes, possibly involving the host-encoded DDB 1 protein.