The Experimental Study On Effect Of The Deletion Of La Protein Binding Site On Stability Of HBV RNA

The lupus autoantigen protein (La protein) is a nuclear phosphoprotein. It was first identified as an autoantigen in serum of patients suffering from systemic lupus erythematosus and Sjogren's syndrome. La protein interacts with a large variety of cellular and viral RNAs, and involves in their metabolism. In recent years, some evidences for the association between La protein and HBV RNA were found.【Objective】To investigate the effect of the deletion of La protein binding site in HBV RNA on the stability of HBV RNA in host cell for further study on the significance of the site in HBV life cycle, and searching a new anti-HBV target.【Methods】The HBV expression vectors with mutation related with the La protein binding site were constructed by a molecular clone and PCR based site directed mutagenensis in vitro, named pHBV-mLa. The HBV RNA secondary structure of the site was calculated with the program RNAdraw V1.1b2 by computer. The normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamineam2000. In two groups, the HBV RNA level were analyzed by semi-quantitative RT-PCR, and HBV antigens secretion into culture media were tested by ELISA.【Results】The HBV vectors with mutation related with the La protein binding site were successfully constructed, it was confirmed by the identification of restriction analysis and sequencing. In La protein binding site, The HBV RNA secondary structure of the mutant vectors was completely different to the stem-loop structure of the normal HBV vectors. After transfection into HepG2 cells, Semi-quantitative RT-PCR and ELISA showed that, both the level of HBsAg-mRNA and HBeAg-mRNA in the mutant vectors group were significantly lower than that in the normal HBV vectors group(t'=12.703, P<0.05, and t'=11.085, P<0.05), and both the expression efficacy of HBsAg and HBeAg in mutant group were reduced(t=44.648,P<0.01 t=42.048,P<0.01), compared with the nomal group.【Conclusion】The change of La protein binding site in HBV RNA due to the mutation in HBV DNA disorganizes the predicated stem-loop structure in the site shared by all HBV RNAs. As a result of the structural change, La protein cannot bind the site and stabilize HBV RNAs, including in HBsAg-mRNA and HBeAg-mRNA, which are exposed the cleavage site in the upstream of the stem-loop structure to endoribonuclease. It contributes to HBV RNA decay and effects expression of HBV gene. This study shows, it is necessary for the formation of the stem-loop structure in La protein binding site that the sequence of this site in HBV DNA is conserved; the disorganization of the stem-loop structure affects the HBV RNA posttranscriptional stability in host cell. La protein binding site in HBV RNA as well as the special secondary structure in the site is crucial to Hepatitis B Virus life cycle.