To Study of RNA-Protein Interaction in the Human Norovirus 5’- and 3’-Extremities Genome Using Riboproteomics Method

Human Noroviruse (NoV) belong to the members of the family Caliciviridae with single positive stranded RNA genome about 7.5 Kbp. hNoV are the dominant cause of outbreaks of gastroenteritis either in adult or infant acute gastroenteritis (AGE) worldwide since 2006, posing severe threat to public health. However the progress in understanding the molecular characteristics of hNoV and its replication strategies has been hampered due to lack of an in-vitro cell culture system. Based on literatures, the absence of unknown host cellular factors for establishment of reproducible in vitro cell culture system of hNoV might be immune-regulatory proteins; nevertheless, limited knowledge has known on viral-associated host factor. Since hNoV is a positive-stranded RNA virus, its genomes (especially on 5’- or 3’- extremities) usually contain cis-acting RNA sequences and structures that play critical roles in the virus life cycle. To address above issue, we will intend to globally survey the RNA-protein interactions in the human norovirus 5’- and 3’-extremities genome in this grant. Therefore, the objective of this two years’ grant is to reveal the RNA-protein interactions in the human norovirus 5’- and 3’-extremities genome using RNA co-immunoprecipitation following mass spectrometry analysis. We will achieve following specific aims within two years. (1). To construct hNoV 5’- and 3’- extremities genome into T7/or Sp6 based vector; (2). In vitro biotin-labelled transcription of the hNoV 5’- and 3’- extremities following RNA co-immunoprecipitation in Huh7 cells; (3) To identify host factors that interact with the 5’- and 3’-extremities of hNoV genome by mass spectrometry; (4). To functionally analyze interaction between host factors and hNoV 5’- and 3’-extreme genome via luciferase assay. The knowledge obtained from this grant will help us for further insight life cycle of hNoV, which might be helpful for developing anti-viral strategies in the future.

Project ID:PC10308-1334
External Project ID:MOST103-2320-B182-014