Identification of host factors involved in hepatitis E virus RNA replication

• Applicant: Viet Loan Dao Thi, Ph.D.
• Subject Area: Virology
• Term: from 2014 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 253648794

Hepatitis E virus (HEV) is believed to be the most common cause of acute hepatitis in the world. Until recently, it has been considered to affect only developing countries. However, autochthonous hepatitis E due to zoonotic transmission of HEV has been recognized as an emerging challenge also in industrialized countries. Despite a growing interest, current knowledge of the molecular virology of HEV is scarce. Its positive-strand RNA genome harbours three open reading frames (ORF 1-3). While ORF2 and ORF3 encode for virus structural proteins mediating HEV assembly and secretion, ORF1 encodes for nonstructural proteins which are responsible for viral genome replication.The aim of the proposed study is to identify cellular host factors that are involved in HEV RNA replication, which is an essential viral life cycle step. To this end, two successive high-throughput screens will be performed. First, a novel protein-protein interaction assay (BioID) that is based on a promiscuous biotin ligase which will be fused to the HEV RNA-dependent RNA polymerase (RdRp), the viral protein being responsible for HEV RNA synthesis. The fused biotin ligase biotinylates adjacent and/or interacting proteins that will be identified through affinity tag purification and mass spectrometry. Identified cellular candidates from this interaction screen will be then subjected to a small interfering RNA (siRNA) screen on a HEV genome replicating cell line to assess their implication in RNA replication. The relevance of these candidates will be confirmed using an infectious HEV cell culture system. The final step includes the detailed investigation of their interaction with the RdRp and their effect on its enzymatic activity, including the different steps of HEV RNA synthesis. This study should lead to a better understanding of HEV RNA replication and to the identification of involved host factors as potential drug targets against HEV infection.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Charles M. Rice, Ph.D.