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

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.

The multifaceted interactions between pathogens and host ESCRT machinery

“…Using an siRNA screen, the authors identified several ESCRT components that contribute to CSFV infection at different stages of infectious cycle, including entry. Comparable to what has been observed for JEV, the ESCRT-III components CHMP2 and CHMP4 localized to CSFV VRCs [ 53 , 60 ]. Although ESCRT components such as HRS, TSG101, VPS28 (ESCRT-I), EAP20 (ESCRT-II), ALIX, CHMP7, and VPS4 also contribute to VRC formation through their interactions with the nonstructural proteins NS3, NS4B, NS5A, and NS5B [ 60 ], the molecular mechanism by which these nonstructural proteins interact with host ESCRT components remains to be elucidated.…”

“…A more comprehensive depiction of the mechanism for ESCRT-dependent VRC formation was provided in a recent study of classical swine fever virus (CSFV) infection [ 60 ]. Using an siRNA screen, the authors identified several ESCRT components that contribute to CSFV infection at different stages of infectious cycle, including entry.…”

ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses

“…Many studies have also reported on the formation of viral replication complexes (VRC) by ESCRT, such as classical swine fever virus (CSFV) [ 22 ] and brome mosaic virus (BMV) [ 23 ]. As alphacoronavirus rearrange the ER of host cells to form DMVs, a platforms for viral replication [ 12 ], we thus speculated that ALIX and TSG101 may be recruited to DMV to promote virus replication.…”

“…In fact, the Crimean-Congo hemorrhagic fever virus relies on ESCRT components to enter cells via MVB [ 28 ]. Meanwhile, TSG101, VPS25, CHMP4B, and CHMP7 play important roles in clathrin-mediated endocytosis of classical swine fever virus [ 22 , 29 ]. Hence, it follows that ESCRT is also involved in viral entry.…”

Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B

“…ESCRT-III contains 12 α-helical charged multivesicular body proteins known as CHMP1A, CHMP1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP5, CHMP6, CHMP7, and IST1, and it plays a key role in mediating cellular membrane constriction ( 17 , 18 ). The ESCRT machinery not only is required in cellular processes like plasma membrane repair but also is hijacked by some viruses in different stages of propagation, especially the budding stage ( 19 – 22 ). In these cases, viruses usually encode some conserved peptides, called the L-domain, as an adaptor to recruit ESCRT-associated proteins and utilize them to catalyze membrane fission events.…”