The ESCRT-I Subunit Tsg101 Plays Novel Dual Roles in Entry and Replication of Classical Swine Fever Virus

Abstract

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the highly contagious diseases of swine with high morbidity and mortality, that negatively affects the pig industry worldwide, in particular China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. Endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological, metabolic processes including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT regulates the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in Clathrin-mediated endocytosis of CSFV and also involved in CSFV trafficking. Tsg101 assisted the virus in entering the host cell through the late endosome (Rab7 and Rab9), and finally to reach the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and dsRNA or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not Golgi in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genus Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both Clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for Pestivirus inhibitors.

RPLP1, an NS4B-interacting protein, enhances production of CSFV through promoting translation of viral genome

“…Fatty acid synthase (FASN) interacts with CSFV NS4B and facilitates the virus proliferation by regulating the formation of lipid droplets (LDs) [ 54 ]. Recently, tumor susceptibility gene 101 (TSG101) has been reported to promote CSFV replication by interacting with NS4B and NS5B to form replication complexes [ 55 ]. In current study, CSFV production was apparently improved by overexpression of RPLP1 ( Figure 4 ); in contrast, it was drastically diminished by recombinant lentivirus-mediated RPLP1 knockdown ( Figure 3 ), which indicated that RPLP1 could positively modulate CSFV propagation.…”

Transboundary Animal Diseases, an Overview of 17 Diseases with Potential for Global Spread and Serious Consequences

“…Naturally occurring strains are non-cytopathic in cell culture [173,175,177]. Important factors in transmission and virulence include attachment to host cells, viral replication, immunomodulatory effects, and inhibition of cell apoptosis [178][179][180][181][182][183][184]. There are three distinct genotypes with three or four sub-genotypes [83,173,175].…”

Anti-Classical Swine Fever Virus Strategies

“…(3) Some host proteins can help CSFV enter cells. For example, Tsg101 participates in clathrin-mediated endocytosis of CSFV [68]; LamR is a cellular attachment receptor for CSFV [69]; Anx2 is a cellular membrane protein likely associated with CSFV entry [70]; Integrin β3 is membrane-bound signal mediator related to the CSFV infection [71]. Some members of the small Rab GTPase family are also involved in virus entry; Rab5, Rab7, and Rab11 are needed for caveola-dependent endocytosis of CSFV in porcine alveolar macrophages [62].…”