Chaperones, Membrane Trafficking and Signal Transduction Proteins Regulate Zaire Ebola Virus trVLPs and Interact With trVLP Elements.

Dong-Shan Yu,Lin-Fang Cheng,Chen-Yu Hu,Yan-Hua Li,Tian-Hao Weng,Hang-Ping Yao,Zhi-Gang Wu,Nan-Ping Wu,Lan-Juan Li

doi:10.3389/fmicb.2018.02724

Abstract

Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle (including viral entry, uncoating, fusion, replication, assembly, and budding). We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins (FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) potentially interacting with trVLP GP, and four (ANXA5, GRP78, HSPA1A, and HSP90AB1) potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, MAPK11, MEK2, and NTRK1) interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle.

Highlights

MATERIALS AND METHODSEbolavirus (EBOV) is a single-stranded, negative-sense RNA virus with a heterogeneous filamentous structure (Jun et al, 2015) that was first reported in 1976 as the cause of Ebola viral disease (EVD) in humans and other primates (Johnson et al, 1977)
The results showed that siRNA knockdown of ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ efficiently inhibited transcription- and replication-competent virus-like particles (trVLPs) replication (p < 0.05; Figure 2A)
Western blotting with anti-matrix protein 40 (VP40) antibody indicated that ANXA5, GRP78, HSPA1A, and HSP90AB1 interacted with VP40, whereas ARFGAP1, HSPA8, MAPK11, MEK2, FLT4, NTRK1, and YWHAZ did not interact with VP40

Summary

Introduction

MATERIALS AND METHODSEbolavirus (EBOV) is a single-stranded, negative-sense RNA virus with a heterogeneous filamentous structure (Jun et al, 2015) that was first reported in 1976 as the cause of Ebola viral disease (EVD) in humans and other primates (Johnson et al, 1977). Based on the EBOV life cycle in host cells, and on previous RNAi screening that identified filovirion-associated and secretory pathway-related host factors (Spurgers et al, 2010; Simpson et al, 2012; Sakurai et al, 2015), we synthesized siRNAs targeting host genes connected to membrane traffic machinery, endoplasmic reticulum-Golgi recycling, inhibition of secretion, and lipid droplet formation, and assessed their role in the EBOV life cycle. These genes are mostly connected with virus life cycle, viral entry, uncoating, fusion, replication, assembly and budding

Results

Discussion

Conclusion