Analyses of Hepatitis D Viral Antigen Interacting Molecules in Caneorhabditis Elegans

Caenorhabditis elegans is a soil free-living round worm containing 959 somatic cells in adult hermaphrodites. It provides a good model for studying many important biological questions; for example, the pathways of apoptosis and RNAi, are first discovered in C. elegans and then extended to other organisms. Each year, an increasing number of life scientists use C. elegans as a model organism because of its short life-cycle, complete genome being sequenced and easy handling. It has a great advantage using C. elegans for forward and reverse genetic study, especially, by RNAi feeding to knock down specific gene. This simple method has not been able to be demonstrated in other organisms. In addition to being a model for studying genetics, cell biology, neurobiology and behavior in the past, recently, it becomes a model for studying bacterial and viral pathogenesis. In the past five years, we have established the C. elegans system to study the effects of hepatitis viral antigens on worm growth and development. We first created dicistronic vectors to co-express fluorescence proteins (GFP or RFP) and various hepatitis viral proteins in entire worms or specific tissues. We have obtained three integration lines, expressing the HDV large antigen (LDAg) and HBV surface antigen (HBsAg), respectively. The phenotypes of slow growth or dumpy appearance in transgenic worms allow us to ask the location of post-translational modification of LDAg, the signal pathway that induces the exportation of LDAg outside of the nucleus by the ER resided HBsAg, and molecules interacting with LDAg to cause the phenotypes. Therefore, in this three-year grant proposal, we aim to determine the LDAg interacting molecules, it could be either proteins or RNAs, to characterize the sites that for LDAg post-translational modification, and to delineate the signal pathway for LDAg exportation out of the nucleus induced by ER-resided HBsAg. To reach the aims we will (1) pull down the tag-LDAg from transgenic worms and to analyze the complex by MALTI-TOF and RT-PCR to see what proteins and RNAs are associated with LDAg to resulting dumpy and slow growing phenotypes, (2) by RNAi feeding to knock down known modifying enzyme gene expression to study where are LDAg accumulated, and (3) generate double expressing LDAg and HBsAg transgenic worms by crossing two different viral gene expressing lines and to delineate the pathway involving the exportation of LDAg. Any positive results obtained will be applied to human corresponding gene in human hepatoma cell lines to verify whether the phenomenon is true in humans. By the end of the project, we hope we will understand more about the behavior of LDAg and to know the HDV pathogenesis better.

Project ID:PC10007-0367
External Project ID:NSC100-2320-B182-021