Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection.

Chun-Chun Liu,Jiang-Fei Zhou,Rui-Cong Sun,Jing Chen,Yun Young Go,Xi Chen,Huan Chen,Jin Zhang,Ya-Yun Liu,Jian-Chao Wei,Bin Zhou,Jia-Huan Hu,Eiji Morita,Yolanda Revilla

doi:10.1371/journal.ppat.1010294

Abstract

As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.

Highlights

A virus composed of genetic material with a protein shell usually exploits specific viral transport protein on the host cell membrane for endocytosis and releases a large amount of viral genetic materials for replication [1]
We uncover the novel roles of ESCRT-III and VPS4A in virus replication complex (VRC) assembly and update the additional subunits involved in the intracellular trafficking of classical swine fever virus (CSFV)
These data indicate that the ESCRT machinery promotes CSFV replication by forming VRC, which making it become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA

Summary

Introduction

A virus composed of genetic material with a protein shell usually exploits specific viral transport protein on the host cell membrane for endocytosis and releases a large amount of viral genetic materials for replication [1]. The virus requires cellular factors to replicate its genome and usurps cell function to serve itself. Both enveloped and non-enveloped viruses depend on the host endocytic pathways for entry and advance this processes through different ways, allowing themselves to move from the cell periphery to the perinuclear space [2,3,4]. Viruses take advantages of host factors for replication and spread [5]. A map of the interaction between host cells and viral proteins has been drawn through proteomics and transcriptomics studies of infected cells, and systematic RNA interference (RNAi) screening has been developed to identify host factors involved in viral replication. The interaction between viral and host factors leads to the formation of replication compartments, such as the virus replication complex (VRC) [8,9]

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