| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 98 === Hepatitis delta virus (HDV) is a spherical enveloped virus which contains a single-stranded, negative sense circular RNA genome of 1.7 kb in length. Specifically, HDV was considered as a satellite virus with the requirement of its helper virus, hepatitis B virus (HBV), to provide the envelope proteins in forming infectious virus particles. HDV encodes two forms of delta antigen (HDAg). Firstly, the small delta antigen (HDAg-S, 195 amino acids, 24 kDa) is needed for HDV RNA replication. Secondly, the large delta antigen (HDAg-L, 214 amino acids, 27 kDa) is required for viral assembly. According to accumulated evidences, two independent host transcriptional machineries may be necessary for the replication of the viral genomic and antigenomic RNAs. RNA polymerase II is essential for HDV genomic RNA synthesis. The C-terminus of HDAg-S can interact with pol II clamp domain and may affect transcriptional fidelity. However, the synthesis of antigenomic RNA was resistant to α-amanitin. Our laboratory members have previously identified an interaction between HDAg-S and nucleolin, as well as demonstrated that the interaction is critical for the nucleolar targeting of HDAg-S and HDV replication. In addition, the central domain (a.a. 87-165) of HDAg-S interacted with the largest subunit of RNA polymerase I, RPA194, and the RPA194 could be co-precipitated with HDV genomic DNA. Thus, the results indicated that RNA polymerase I may participate in the synthesis of HDV antigenomic RNA. Furthermore, overexpression of HDAg-S inhibited the de novo synthesis of rRNA. Other studies also demonstrated an interaction of RPA194 with the middle domain of human nucleolar phosphoprotein 140 (hNopp140) from amino acid residues 204 to 382. Besides, hNopp140 was regarded as the basic unit of nucleolar structure and was demonstrated to be involved in the synthesis of rRNA. Interestingly, the RPA194-interacting domains of HDAg-S and hNopp140 share certain similarities in sequences, suggesting that HDAg-S may compete with the association among hNopp140, RPA194 and rDNA promoter.
In this study, the interaction between HDAg-S and RPA194 clamp domain (a.a. 1-409, pol IA, predicted by homology modeling) was first demonstrated by pull-down assay. Evolutionarily conserved residues of the similar sequences between HDAg-S and hNopp140 were analyzed. The conserved residues Lys106 and Ala107 of HDAg-S were mutated to Ala and Asp respectively (HDAg-S-106AD), and 125EEE127 were all mutated to Ala (HDAg-S-125AAA). Results from pull-down assay showed that the interaction between HDAg-S-106AD and pol IA was reduced, while HDAg-S-125AAA had a stronger interaction with pol IA. In addition, HDAg-S-106AD had an inhibitory effect on the de novo synthesis of rRNA similar to that of the wild type HDAg-S. Unexpectedly, HDAg-S-125AAA no longer inhibited the de novo synthesis of rRNA. Results from RT real-time PCR indicated that both HDAg-S-106AD and HDAg-S-125AAA did not surpport the viral and antigenomic RNA synthesis. In summary, the conserved residues Lys106, Ala107 and Glu125-127 play differential roles in HDV RNA replication and the inhibitory effect on host rRNA synthesis. However, molecular mechanisms of pol I involved in HDV antigenomic RNA synthesis and the inhibition of rRNA synthesis need to be further studied.
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