Attachment, Entry, and Intracellular Trafficking of Classical Swine Fever Virus

Abstract

Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the Pestivirus genus in the Flaviviridae family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.

Keywords: attachment; classical swine fever virus; entry; intracellular trafficking.

Figure 1

Basic features of CSFV and the organization of CSFV genome. (A) Schematic outline of a CSFV particle. CSFV is an enveloped, single-stranded, and positive-sense RNA virus. Three envelope proteins of CSFV-E^rns, E1, and E2 are located on the external part of viral particles. As transmembrane proteins, E2 is present as homodimers or forms E1–E2 heterodimers on the virion. Unlike E1 buried underneath, E^rns is exposed on the outside surface of the envelope with E2 and exists as a dimer. C proteins condense viral RNA genome with an unspecific affinity for nucleic acids. (B) Schematic organization of CSFV genome. The length of the genome is approximately 12.3 kb. The viral genome is composed of a large open reading frame (ORF), a 5’-noncoding region (5′-UTR), and a 3’-noncoding region (3’-UTR). The ORF encodes a precursor polyprotein, which is cleaved into four structural proteins (C, E^rns, E1, and E2) and eight nonstructural proteins (N^pro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B).

Figure 2

Model of CSFV entry and intracellular transport. The attachment to receptors (HS, LamR, CD46, MERTK, ADAM17, etc.) mediated by CSFV E^rns or E2 facilitates penetration and entry ① The CSFV particles are endocytosed within clathrin-coated endosomes (in PK-15 cells) or internalized via a caveolae-dependent pathway (in 3D4/21 cells), followed by dynamin-mediated scission from the plasma membrane, during which actin cytoskeleton is required and ESCRT proteins (Tsg101, VPS25, CHMP4B, CHMP7, etc.) are involved. Clathrin interacts with Tsg101, CHMP4B, VPS25, and CHMP7 during CSFV entry, and is then transported to the EEs ②. Subsequently, CSFV transports from EEs to LEs along microfilaments with the assistance of Rabs, ESCRT proteins (Tsg101, CHMP4B, CHMP7), and other factors (GBF1, ARF1, COP I, VCP, etc.). Among them, Rab5 and Rab7 function in coordinating the transition from early to late endosomes. Rab9 participates in the migration of cargo between late endosomes and the trans-Golgi network, and Rab11 guides the transit to REs. In the EEs, VCP/CHMP4B/CHMP7 interact with Rab5 and assist in CSFV transportation to the LEs ③. In the LEs, Tsg101/CHMP7/CHMP4B interact with Rab9 and cooperate in CSFV transportation to the lysosomes. COP I, together with GBF1 and ARF1, facilitates CSFV entry and regulates virion transport from early to late endosomes ④. In the lysosomes, the CSFV envelope fuses with the endosomal membrane for uncoating and nucleic acid releasement, with the assistance of Tsg101 and CHMP4B ⑤. In the ER lumens, ESCRT proteins interact with CSFV nonstructural proteins (NS4B/5B) forming the virus replication complex (VRC) for viral genome replication ⑥. Finally, transfer to the Golgi proceeds for processing and assembly before virion budding from the host cells ⑦ and ⑧.