[The deletion of La protein binding site in HBV RNA leads to instability of S gene mRNA].
OBJECTIVE
To investigate the effect of deletion of the La protein binding site of HBV RNA, caused by its mutation, on the HBV S-mRNA stability of S gene, to study the role of the site in hepatitis B virus life cycle, and to try to find a new anti-HBV target in the future.


METHODS
A HBV vector with mutation related to the La protein binding site was constructed using molecular cloning and PCR based site directed mutagenesis, and the vector was named pHBV-mLa. The HBV RNA secondary structure of the site was calculated using a computer. Normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamine 2000. HBV S-mRNA levels in the two groups were analyzed using semi-quantitative RT-PCR, and HBsAg secretion into the culture media was tested using ELISA.


RESULTS
A HBV vector with mutation related to the La protein binding site was successfully constructed, and it was identified and confirmed using restriction analysis and sequencing. The HBV RNA secondary structure of the mutant vector was completely different to the stem-loop structure of the normal HBV vector. Semi-quantitative RT-PCR and ELISA analyses showed that the level of HBV S-mRNA in the mutant vector group was significantly lower than that in the normal HBV vector group (t'=12.703, P<0.05), and the expression efficacy of HBsAg was reduced in the mutant vector group (t= 44.648, P<0.01).


CONCLUSIONS
The change of La protein binding site in the HBV RNA caused by the mutation in HBV DNA disorganizes the stem-loop structure in the HBV RNA site. With the structural change, the La protein cannot bind the site and stabilize the HBV RNA (HBV S-mRNA), as the cleavage site in the upstream of the stem-loop structure is exposed to endoribonuclease. This results in HBV S-mRNA decay and affects the expression of the S gene. This study shows that only the sequence of this site in the HBV DNA is reserved, then the stem-loop structure in the La protein binding site will remain intact, and the disorganization of the stem-loop structure affects the stability of the transcripted HBV RNA. The La protein binding site in HBV RNA and the special secondary structure of the site are crucial to the life cycle of the hepatitis B virus.
Lin
Yuan-yuan
YY
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Lin
Ju-sheng
JS
Xie
Na
N
Zhou
Xiu-min
XM
Song
Yu-hu
YH
D001665:Binding Sites; D045744:Cell Line, Tumor; D017353:Gene Deletion; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics*
C096362:L protein, hepatitis B virus; D012333:RNA, Messenger; D012367:RNA, Viral; C118669:S envelope protein, hepatitis B virus; D014759:Viral Envelope Proteins