The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Khairunnisa' Mohamed Hussain,Kim Lian Janet Leong,Mary Mah-Lee Ng,Justin Jang Hann Chu

doi:10.1074/jbc.m110.168468

Khairunnisa' Mohamed Hussain, Kim Lian Janet Leong + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.168468

Copy DOI

Abstract

Little is currently known about the infectious entry process of human enterovirus 71 (HEV71) into host cells, which may represent potential anti-viral targeting sites. In this study a targeted small-interfering RNA (siRNA) screening platform assay was established and validated to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics, and endosomal trafficking essential for HEV71 infection. Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis), secondary siRNA-based dosage dependence studies, and drug inhibition assays. The infectious entry of HEV71 into rhabdomyosarcoma cells was shown to be significantly inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis (CME) that include AP2A1, ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1, and WASF1. The functional role of CME was verified by the observation of strong co-localization between HEV71 particles and clathrin as well as dose-dependent inhibition of HEV71 infection upon siRNA knockdown of CME-associated genes. HEV71 entry by CME was further confirmed via inhibition by dominant-negative EPS15 mutants and treatment of CME drug inhibitors, with more than 80% inhibition observed at 20 μm chlorpromazine. Furthermore, HEV71 infection was shown to be sensitive to the disruption of human genes in regulating early to late endosomal trafficking as well as endosomal acidic pH. The identification of clathrin-mediated endocytosis as the entry pathway for HEV71 infection of susceptible host cells contributes to a better understanding of HEV71 pathogenesis and enables future development of anti-viral strategies against HEV71 infection.

Highlights

Human enterovirus 71 (HEV71)2 is a single-stranded, positive-sense RNA virus belonging to the human Enterovirus A (HEV-A) subspecies of the Enterovirus genus in the Picornaviridae family [6]
First identified and characterized in 1969 in California from a stool specimen isolated from an infant with encephalitis [7], ensuing outbreaks of HEV71 have since been reported in various regions of the world, including Australia, Sweden, and Japan
Previous studies have attempted to decipher the entry processes of related enteroviruses, such as poliovirus [13,14,15,16] and echovirus [17,18,19], little is currently known about the specific cellular genes or host factors involved in mediating the infectious entry of HEV71 into human cells

Summary

Introduction

In this study we assessed an array of siRNA libraries that target human genes important for endocytosis processes, trafficking of membrane vesicles, actin polymerization, and cytoskeleton rearrangement to determine the cellular genes or factors that facilitate the infectious entry pathway of HEV71. SiRNA Profiling of Human Endocytic and Membrane Trafficking Genes—An array of 119 siRNA smart pool (Dharmacon) targeting genes known to be directly or indirectly involved in regulating the different endocytic pathways (clathrin, caveolae, macropinocytosis, etc.), polymerization of actin, and cytoskeleton rearrangement and vesicle/cargo trafficking was used to identify host genes necessary for the infectious entry of HEV71.

Results

Conclusion