Swine Host Protein Coiled-Coil Domain-Containing 115 (CCDC115) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2 during Virus Replication

Abstract

Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2–CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2–CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.

A β-Hairpin Motif in the Envelope Protein E2 Mediates Receptor Binding of Bovine Viral Diarrhea Virus

“…Porcine cell lines that do not express CD46 were shown to be non-permissive to APPV infection, while porcine pestiviruses such as CSFV and BuPV efficiently infected these cells, indicating a CD46-independent mechanism of entry [ 15 ]. Additional binding partners of CSFV E2 have been identified and mapping of amino acids involved in these interactions has been achieved using yeast-two hybrid system [ 32 , 33 , 34 ]. These factors do not appear to play a role in virus entry, and the regions involved in these interactions fall outside the β-hairpin motif in domain II.…”

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle

“…The interaction between host ACADM and CSFV E2 was also assessed using the Proximity Ligation Assay (PLA) [ 24 ]. The PLA was performed in triplicate using the Duolink-PLA Kit (Sigma-Aldrich, St. Louis, MO, USA) as recommended by the manufactures.…”

“…The random E2 mutant library was then co-transformed into yeast strain AH109 along with ACADM with a transformation rate of at least 1 × 10 6 . Individual colonies were screened as previously reported [ 24 ], where plasmids were recovered and sequenced as previously described [ 25 ]. In recovered plasmids where only individual amino acids were mutated, the E2 mutant plasmids were re-tested individually via co-transformation with ACADM, HPRT1-AD (hypoxanthine phosphoribosyl transferase 1), as previously identified and described [ 26 ].…”

“…In addition, CSFV p7 has been shown to specifically interact with the calcium-signal-modulating cyclosphilin ligand (CALMG), the microtubule-associated protein RP/EB family member 1 (MAPRE1) and the voltage-dependent anion channel 1 (VDAC1) [ 16 , 17 ]. Additionally, E2 has also been demonstrated to interact with several host proteins, such as cellular actin [ 18 ], Anx2 (Annexin 2) [ 19 ], Trx2 (Thioredoxin) [ 20 ], MEK2 (mitogen-activated protein kinase 2) [ 21 ], PPP1CB (protein phosphatase 1 catalytic subunit beta) [ 22 ], SERTAD1 (SERTA-domain-containing protein 1) [ 23 ], CCDC115 (coiled-coil domain-containing 115) [ 24 ] and DCTN6 (dynactin subunit 6) [ 25 ]. Importantly, many of these interactions have been shown to have a critical role in the virus replication cycle or in virus virulence in swine.…”

Anti-Classical Swine Fever Virus Strategies

“…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]. (4) Some host proteins affect both CSFV replication and virulence through interacting with viral proteins, such as CCDC115 [72], SERTAD1 [73], DCTN6 [74], and IQGAP1 [75], which have been proved by both in vivo and in vitro studies. (5) Some host proteins or enzymes are beneficial for viral RNA replication, translation, and assembly, such as RHA [76] and eIF3E [77].…”