Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle.

The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence.IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

Dynactin 6 deficiency enhances aging-associated dystrophic neurite formation in mouse brains.

Formation of Reticulon 3 (RTN3)-immunoreactive dystrophic neurites (RIDNs) occurs early during the growth of amyloid plaques in Alzheimer's disease (AD) brains. We have shown that RIDNs in AD and aging mouse brains are composed of abnormally clustered tubular endoplasmic reticulum (ER) and degenerating mitochondria. To understand RTN3-mediated abnormal tubular ER clustering, we aimed to identify proteins that interact with RTN3 and impact accumulation of tubular ER in RIDNs. We found that the N-terminal domain of RTN3, which is unique among RTN family members, specifically interacted with dynactin 6 (DCTN6), a protein involved in dynein-mediated retrograde transport of cargo vesicles. DCTN6 protein levels decrease with aging in the hippocampal regions of WT mice. We found that DCTN6 deficiency enhanced RTN3 protein levels, high molecular weight RTN3 levels, and hippocampus-specific RIDN formation in aging brains of transgenic mice overexpressing RTN3. Our results suggest that the DCTN6-RTN3 interaction mediates tubular ER trafficking in axons, and a DCTN6 deficiency in the hippocampus impairs axonal ER trafficking, leading to abnormal ER accumulation and RIDN formation in brains of aging mice.