| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 101 === Hepatitis delta virus (HDV) is a defective human pathogen. It obtains envelope proteins from helper hepatitis B virus and relies on host RNA polymerase for genome replication. Furthermore, HDV genome is a 1.7-kb negative-sense circular RNA, forms an un-branched rod-like structure, and has ribozyme activity. Due to these unique features of HDV, it is reasonable to propose that HDV is extremely reliant upon host cellular proteins to facilitate its life cycle. Several host proteins have been published to be able to interact with the potential HDV RNA promoter located at one end of HDV rod RNA. In this study, a surprising data were obtained when nt 303-537 in a genomic orientation, a region with unknown biological function and has no rod-like RNA structure, was used as a bait to study the HDV RNA-host factors interactions. Many cellular proteins, including nucleo-cytoplasmic shuttling proteins heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A2/B1, were identified as HDV RNA partners. The interactions were validated and the specificity of the binding was confirmed by competition assays. Importantly, hnRNP A1 was captured in the nucleus in HDV replicating cells under actinomycin D treatment. The binding of hnRNP A1 and A2/B1 across the whole genome was then examined. HDV RNAs transcribed from constructs containing part of genome with or without rod structure were used to perform pull down-Western analyses. Interestingly, sequence and binding analyses indicated that hnRNP A1 and A2/B1 predominantly bind to genomic HDV RNAs covering nt 1638-173 and 303-424. Furthermore, HDV RNA replication increased when the expression of hnRNP A2/B1 was knockdown. In contrast, the expression of viral protein delta antigen was not affected by hnRNP A2/B1 knockdown. These data suggested that hnRNP A1 and A2/B1 play important role in HDV RNA life cycle. The significance of HDV RNA-hnRNP A1 and A2/B1 interactions in HDV biology is thereby a new research field remains to be investigated.
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