Host protein CD63 enhances viral RNA replication by interacting with human astrovirus nonstructural protein nsP1a/4

Abstract

ABSTRACTHuman astrovirus nonstructural protein nsP1a/4, located at the C-terminal end of nsP1a, is thought to be involved in the regulation of RNA replication and capsid maturation;however, its rolesviral growth and virulence are not well understood. We investigated the intracellular host proteins that interact with nsP1a and explored the potential roles of the interaction in the pathogenesis of human astrovirus infection. We screened 14 independent proteins with a cDNA library derived from Caco-2 cells using a yeast two-hybrid technique. Deletion analysis revealed that interaction between the nsP1a/4 domain and the large extracellular loop (LEL) domain of the human protein CD63 is necessary for astrovirus replication. The interaction was confirmed by glutathione-S-transferase (GST) pull-down assays and co-immunoprecipitation assays. Confocal microscopy showed that nsP1a/4 and CD63 co-localized in the cytoplasm of infected cells. Over expression of CD63 promoted viral RNA synthesis, whereas knockdown of CD63 markedly decreased viral RNA levels. Those results suggest that CD63 plays a critical role in human astrovirus RNA replication. The interaction between CD63 and nsP1a/4 provides a channel to further understand the roles of interactions between host and virus proteins in astrovirus infection and release.IMPORTANCEHuman astroviruses cause gastroenteritis in young children and immunocompromised patients. In this study, we provide evidence that nsP1a/4, a nonstructural protein located at the C-terminal end of the human astrovirus nsP1a polyprotein, interacts with the host protein CD63. Over expression of CD63 promoted viral RNA replication, whereas knockdown of CD63 decreased virus RNA replication, indicating that CD63 plays a critical role in the human astrovirus life cycle.