Inhibition Of Self-Cleavage Acitivity Of Hepatitis Delta Virus Genomic Ribozyme By Amimoglycoside

• Main Author: 賈儒馨
• Other Authors: 陳培哲
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/60857002834825094566

碩士 === 國立臺灣大學 === 微生物研究所 === 83 === 　　Hepatitis delta virus(HDV) genome is a single-stranded circular RNA consisting of about 1700 nucleotides. Both genomic and antigenomic HDV RNAs have self-cleavage activity(ribozyme activity), which is believed to play an important role in RNA processing when the RNAs are synthesized through a rolling-circle mechanism. 　　Recently, aminoglycoside antibiotics have been found to act on functional RNA elements both in 16S rRNA and in the group l intron core. There is no nucleotide sequences similarities can be detected by comparing group l introns and rRNA, implying that a three-dimensional structure is recognized by the antibiotics. In this study, we try to examine whether antibiotics could interact with HDV ribozyme, and then assay their effect on the activity of ribozyme. 　　First, a variety of antibiotics have been selected to examine their effects on the self-cleavage activity on HDV subfragment (HDV nucleotides 654 to 770). We found that a subset of aminoglycoside antibiotics, including neomycin, tobramycin, kanamycin, gentamycin and netromycin, can inhibit the ribozyme activity in vitro. The effect of Mg2+ions on such inhibition was also studied. It has been shown that the concentration of antibiotics required for inhibiting 50% of ribozyme activity, namely ID50, is reduced when the reaction are carried out with lower concentration of Mg2+ions. To further narrow down the HDV ribozyme region involved in antibiotic inhibition, a smaller HDV ribozyme(HDV nt 683 to 770) was tested. The results showed that the ID50 of antibiotics was reduced for the smaller HDV ribozyme. 　　Subsequently, we tried to identify the sites of HDV RNA which interact with antibiotics. By using base-specific chemcial probing methods, we found that the modification pattern at position A704 was changed in the presence of tobramycin. A704 is located at the bottom of stem II which may play a role in promoting correct folding and stabilizing the structure. We therefore suggest that antibiotics could result in conformational change of RNA. Finally, we tested the effect of such inhibitory antibiotics on HDV replication in vivo. In tissue oulture system, no obvious effect of antibiotics on HDV replication was observed.