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

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.

ABSTRACT

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.

INTRODUCTION

Classical swine fever (CSF) virus (CSFV) is the causative agent of a highly contagious disease of swine with important economic consequences. CSFV is a small enveloped virus possessing a positive-stranded RNA genome that, along with bovine viral diarrhea virus (BVDV) and border disease virus (BDV), is a member of the Pestivirus genus within the family Flaviviridae (1). The CSFV genome is 12.5 kb and contains a single open reading frame, which encodes a 3,898-amino-acid polyprotein that yields 11 to 12 final cleavage products (NH₂-Npro-C-E^rns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through processing of the polyprotein by viral and cellular proteases (2). The CSFV virion contains four structural proteins, namely, the core protein and three glycoproteins, E^rns, E1, and E2, which are structurally associated with the virus envelope. The role of these proteins, particularly in the processes of virus replication and virulence, have been studied in past years (3–10).

The identification of host proteins interacting with CSFV proteins during infection is a relatively new field of research. Several host proteins have been shown to interact specifically with structural CSFV proteins. CSFV core protein has been demonstrated to interact with small ubiquitin-related modifier 1 (SUMO1), IQ motif-containing GTPase-activating protein 1 (IQGAP1), ubiquitin-conjugating enzyme 9 (UBC9), and hemoglobin subunit β (HB) proteins (11–14), while E^rns has been shown to interact with the laminin receptor (15). In addition, E2 has been identified as an partner interacting with several different host proteins, including cellular actin (16), annexin 2 (Anx2) (17), thioredoxin 2 (Trx2) (18), mitogen-activated protein kinase kinase 2 (MEK2) (19), and protein phosphatase 1 catalytic subunit β (PPP1CB) (20). In most of these cases, these host-virus protein-protein interactions play a role in regulating the virus replication cycle; in a few cases, these interactions are involved in viral virulence (11–13).

We previously identified several swine host proteins that interact with CSFV E2 by using a yeast two-hybrid approach (21). One of the proteins reported as an E2 partner was dynactin subunit 6 (DCTN6), which forms part of the dynactin complex, an essential component of the microtubule-based cytoplasmic dynein motor activity that is involved in intracellular transport of a variety of cargoes and organelles. Here, we expand our preliminary discovery by characterizing the E2-DCTN6 interaction. The interaction was shown to occur in CSFV-infected swine cells, as independently confirmed by coimmunoprecipitation and proximity ligation assays. E2 residues critical for the interaction with DCTN6 were mapped using a reverse yeast two-hybrid approach, and reverse genetics using an infectious clone of CSFV was then used to create a recombinant CSFV mutant (E2) harboring specific substitutions disrupting the E2-DCTN6 interaction, as assessed with the yeast two-hybrid approach. Although CSFV E2ΔDCTN6v replicates in primary swine macrophages and swine SK6 cells, animals infected with CSFV E2ΔDCTN6v survived infection, indicating that the ability of CSFV E2 to effectively bind host DCTN6 protein during infection may play a critical role in viral virulence.

RESULTS

Interaction between virus structural glycoprotein E2 and swine host protein DCTN6 in CSFV-infected cells.

We previously identified a relatively large set of swine host proteins that specifically interact with the CSFV major structural glycoprotein E2 (21). This result was obtained by using a yeast two-hybrid approach employing a custom-made library based on mRNA from swine macrophages, the main cell target for CSFV during animal infection. DCTN6 was one of those swine host proteins interacting with CSFV E2. DCTN6 forms part of the dynactin complex and is an essential component of the microtubule-based cytoplasmic dynein motor activity in intracellular transport of a variety of cargoes and organelles.

To confirm that the E2-DCTN6 protein interaction discovered using the two-hybrid system in yeast actually occurs during CSFV infection of host cells, coimmunoprecipitation experiments were performed using monoclonal antibodies specifically recognizing both interacting proteins. SK6 cells were infected (multiplicity of infection [MOI] of 10) with CSFV Brescia (derived from an infectious clone of the Brescia strain, a highly pathogenic strain of CSFV). Samples were harvested at 24 h postinfection (hpi) and processed as described in Materials and Methods. SK6 cell lysates were collected from infected or mock-infected cells and immunoprecipitated with an anti-E2 monoclonal antibody (WH303), followed by Western blotting with a DCTN6-specific rabbit antiserum. A single band at the expected molecular mass of DCTN6 (21 kDa) was clearly observed (Fig. 1A). A reverse coimmunoprecipitation experiment was also performed, to confirm the specificity of the one already described. Immunoprecipitation was performed with anti-DCTN6 reagent, and a Western blot was developed with the anti-E2 reagent. Again, a single band of the expected molecular mass of E2 (55 kDa) was observed (Fig. 1B). These results indicated that, in CSFV-infected cells, E2 could be coimmunoprecipitated with DCTN6, confirming the previous yeast two-hybrid finding that suggested that an E2-DCTN6 interaction occurs during viral infection of cell cultures.

FIG 1.

Coimmunoprecipitation of E2 and DCTN6 in CSFV-infected or mock-infected cells. The input cell lysate is in lanes 1 and 2, and the lysate that was immunoprecipitated (IP) with antibodies to E2 (A) or DCTN6 (B) is in lanes 3 and 4. Protein extracts were blotted for DCTN6 and E2 as indicated in Materials and Methods, with bands observed at the expected molecular sizes of 55 kDa for E2 and 21 kDa for DTN6. Arrows indicate the positions of the DCTN6 and E2 bands.

E2 is abundantly expressed throughout the cell cytoplasm during virus replication. Therefore, it may be difficult to visualize the potential subcellular colocalization of E2 with cellular protein partners. Proximity ligation assays (22) have the advantage of allowing the direct identification of transient protein-protein interactions, without the potential additional background signal occasionally observed in classic colocalization studies. Recognition of proteins E2 and DCTN6 was performed using the same reagents as used for coimmunoprecipitation, i.e., monoclonal antibody WH303 for detection of E2 and a commercial rabbit antiserum for detection of DCTN6 (see Materials and Methods). Results of the proximity ligation assay, analyzed under experimental conditions similar to those used to perform the coimmunoprecipitation studies, confirmed that E2 and DCTN6 interacted in SK6 cell cultures infected with CSFV. This interaction appeared as a distinct punctate pattern throughout the cell cytoplasm (Fig. 2). Therefore, two independent methodologies, namely, coimmunoprecipitation and proximity ligation assays, confirmed the existence of the E2-DCTN6 interaction during CSFV infection of SK6 cell cultures.

FIG 2.

Proximity ligation assay for E2 and DCTN6 in CSFV-infected SK6 cells. The interaction between E2 and DCTN6 was determined by the proximity ligation assay in SK6 cells that were either mock infected or infected for 24 h with CSFV BICv (MOI of 10). Top and bottom panels are two different images of the same treatment. Scale bar, 50 μm.

Identification of CSFV E2 residues critical for interaction with DCTN6.

Identification of the E2 residues mediating interaction with the host protein is a critical step in the development of recombinant CSFVs containing substitution of those residues. These viruses, harboring residue substitutions shown to disrupt the interaction of the virus protein with a host cell partner, can be used as tools to study the role of a particular E2-host protein interaction in several virus functions. Attempts to map the amino acid residues in E2 involved in the interaction with the host proteins described in our previous report were largely unsuccessful (21). An alanine scanning approach, involving a set of 76 CSFV E2 mutant proteins containing sequential stretches of amino acids in which the native amino acid residues were substituted with alanine residues, demonstrated that the E2 binding sites for these cellular proteins are likely to be nonlinear (21). A similar alanine scanning approach has been successfully used in our laboratory to facilitate elucidation of specific areas within foot-and-mouth disease virus (FMDV) and CSFV proteins recognized by host protein partners (13, 23–26).

Therefore, to identify E2 amino acids involved in the interaction with DCTN6, we developed an alternative approach using the yeast two-hybrid system. This methodology permits the detection of E2 residues mediating DCTN6 interaction that are not adjacently located, allowing the random substitution of any amino acid in the sequence of the target protein. This approach was based on an evaluation of the ability of DCTN6 to interact with a library of randomly mutated E2 proteins in which an average of 5 amino acid residues were randomly substituted throughout E2. In this procedure, 1 × 10⁶ different mutations in E2 were tested using the library screening approach described in Materials and Methods. Screening revealed that several mutations in E2 likely disrupted the overall protein structure, as these mutants could not interact with other E2 binding proteins, and thus were not specific mutations that affected DCTN6 (for instance, mutations that caused a frameshift or introduced a premature stop codon in the E2 protein, both of which would explain the loss of protein binding of E2 to DCTN6). E2 mutants lacking reactivity with DCTN6 were also tested for their ability to bind protein HPRT1, another host protein that was found to be a specific host binding partner for E2. This analysis was performed to rule out the possibility that the random mutants had a gross conformational change in E2 that disrupted the overall structure of E2 and could mediate a nonspecific loss of protein binding. Only one E2 mutant was able to fulfill all of the criteria while still decreasing the ability of E2 protein to bind the DCTN6 protein. This E2 mutant, harboring E308G and H335L substitutions, was used to further study the role of DCTN6 binding to E2 in CSFV replication and virulence.

Development of the CSFV E2ΔDCTN6v mutant.

To further evaluate the interaction of host DCTN6 and CSFV E2, a recombinant mutant virus based on the virulent strain Brescia was constructed using reverse genetics. E2 amino acid residues identified in the E2-DCTN6 interaction (E308G and H335L substitutions in glycoprotein E2) using the yeast two-hybrid approach were introduced into the infectious clone containing a full-length cDNA copy of CSFV strain Brescia (pBIC) (3), giving rise to the pE2ΔDCTN6 construct. Infectious RNA was in vitro transcribed from full-length infectious clones of pBIC or pE2ΔDCTN6 and was used in SK6 cell transfections. CSFVs BICv and E2ΔDCTN6v were rescued from transfected cells by 4 days postinfection (dpi). Nucleotide sequences of viable rescued virus genomes were identical to those of parental DNA plasmids, confirming that only mutations at predicted mutated sites were present in the rescued viruses.

Replication of the E2ΔDCTN6v mutant in vitro.

In vitro growth characteristics of the mutant virus E2ΔCTN6v were evaluated relative to those of the parental pBIC-derived virus, BICv, in multiple-step growth curves, in both SK6 cells (Fig. 3A) and primary swine macrophage cultures (Fig. 3B). Cell cultures were infected at an MOI of 0.01 times the 50% tissue culture infective dose (TCID₅₀) per cell. Virus was adsorbed for 1 h (time zero), and samples were collected daily until 72 hpi. BICv exhibited similar growth kinetics in SK6 cells and swine macrophages. Interestingly, E2ΔDCTN6v displayed a reduction in virus replication in SK6 cells, compared with the growth ability of parental BICv, and presented a more significant decrease in virus replication in swine macrophages at the 72-h sampling point, with a virus yield approximately 100-fold less than that from BICv-infected macrophages.

FIG 3.

In vitro growth characteristics of CSFV E2ΔDCTN6v. (A and B) Multistep growth curves for E2ΔDCTN6v and BICv on SK6 cell cultures (A) and swine macrophage cell cultures (B). Cell cultures were infected (MOI of 0.01) with CSFV BICv or E2ΔDCTN6v. At the indicated times after infection, samples were collected and titrated for virus yield. Data are means and SDs of three independent experiments. Significant differences (P ≤ 0.05) between the two viruses were found at time points of 24, 48, and 72 h in both cell types (calculated by the unpaired t test). (C) Plaque formation of CSFV E2ΔDCTN6v and BICv on SK6 cell cultures. Cell cultures were infected with either virus, overlaid with 0.5% agarose, incubated at 37°C for 4 days, fixed with 50% (vol/vol) ethanol/acetone, and stained by immunohistochemistry, as described in Materials and Methods.

Accordingly, the plaque size of E2ΔDCTN6v in SK6 cell cultures was reduced by approximately 50%, relative to the BICv plaque size (Fig. 3C). It is evident from the in vitro experiments that disruption of the E2-DCTN6 interaction appears to affect the ability of CSFV to replicate in cell cultures in vitro, particularly in primary swine macrophages.

Evaluation of E2ΔDCTN6v in CSFV virulence in swine.

To examine the effects of disruption of the E2-DCTN6 protein-protein interaction on CSFV virulence, mutant E2ΔDCTN6v or parental BICv was inoculated intranasally into naive swine (10⁵ TCID₅₀) and monitored for clinical disease for up to 21 days. Animals infected with BICv exhibited a characteristic virulent disease with a significant increase in body temperature by 5 dpi, followed by the appearance of classic clinical signs associated with CSF (Table 1 and Fig. 4). As expected, none of the control pigs survived the infection, being euthanized around 7 dpi. Interestingly, mutant E2ΔDCTN6v was completely attenuated in swine. Animals survived the infection and remained clinically normal, with no increase in body temperature, throughout the 21-day observation period (Table 1 and Fig. 4).

TABLE 1.

Swine survival and fever response in animals infected with mutant E2ΔDCTN6v, compared with those infected with parental BICv

Treatmenta	No. of survivors/total no.	Time to death (mean ± SD) (days)	Time to fever onset (mean ± SD (days)	Duration of fever (mean ± SD) (days)	Maximum daily temperature (mean ± SD) (°F)
BICv	0/5	7 ± 0	5.2 ± 0.84	1.8 ± 0.84	105.82 ± 0.44
CSFV E2ΔDCTN6v	5/5				103.52 ± 0.74

^aAll animals were inoculated intranasally with 10⁵ TCID₅₀ of the indicated virus. Animals were observed for 21 days after inoculation.

FIG 4.

Progress of survival (A) and body temperature (B) in animals infected intranasally with 10⁵ TCID₅₀ of either E2ΔDCTN6v (filled symbols) or parental BICv (open symbols). Animals were monitored for an observation period of 21 dpi.

In addition, circulating counts of white blood cells (WBCs), lymphocytes, and platelets, known indicators of CSF severity, were analyzed (Fig. 5). Circulating WBC counts decreased drastically by 4 dpi in BICv-infected animals and remained low or slightly increased until death (7 dpi). E2ΔDCTN6v-infected animals also showed a drastic drop in WBC counts by 4 dpi, with a clear recovery by 11 dpi, followed by oscillating values until the end of the experimental period. Blood lymphocyte counts in BICv-infected animals also dropped by day 4 and continued to decrease until death (7 dpi). A less dramatic decrease was observed in E2ΔDCTN6v-infected animals, with a transient drop in circulating lymphocytes and a return to nearly normal values by 11 dpi, followed by a slow decrease in numbers by the end of the observation period. As expected, platelet counts dropped by 4 and 7 dpi in BICv-infected animals. E2ΔDCTN6v-infected animals had a gradual decrease in platelet numbers throughout the observation period. In summary, E2ΔDCTN6v infection provoked a gradual decrease in hematological values, which never returned to baseline values during the 21-day observation period. This indicates that E2ΔDCTN6v retains some degree of virulence, even though animals do not present clinical signs of the disease.

FIG 5.

Concentrations of circulating WBCs (A), lymphocytes (B), and platelets (PLT) (C) in animals infected intranasally with 10⁵ TCID₅₀ of either E2ΔDCTN6v (filled symbols) or parental BICv (open symbols). Animals were monitored for an observation period of 21 dpi.

Virus replication, detected as viremia, presented with expected kinetics in BICv-inoculated animals. Titers were clearly detectable (average, 10^4.5 TCID₅₀/ml; standard deviation [SD], 0.92 TCID₅₀/ml) by 4 dpi, markedly increasing after that (average, 10^7.2 TCID₅₀/ml; SD, 0.47 TCID₅₀/ml) and remaining high until the animals were euthanized by 7 to 8 dpi. Viremia levels in animals inoculated with E2ΔDCTN6v were similar to those in BICv-inoculated animals at 4 dpi (average, 10^4.5 TCID₅₀/ml; SD, 0.32 TCID₅₀/ml), but by 7 dpi they were ∼1,000-fold lower (average, 10^4.1 TCID₅₀/ml; SD, 0.32 TCID₅₀/ml) than those in BICv-infected animals. Viremia levels remained stable but detectable in CSFV E2ΔDCTN6v-infected animals until the end of the experimental period (Fig. 6). It should be noted that all E2ΔDCTN6v-infected animals developed an anti-E2 antibody response, as detected with a commercial kit (Idexx Laboratories, Inc., Hoofddorp, Netherlands). Based on the appearance of clinical signs associated with CSF, hematological values, and levels of virus replication in animals infected with CSFV E2ΔDCTN6v, a correlation exists between disruption of the E2-DCTN6 interaction in the yeast two-hybrid system and attenuation of viral virulence during infection in domestic swine.

FIG 6.

Virus titers in blood samples obtained from animals infected intranasally with 10⁵ TCID₅₀ of either E2ΔDCTN6v (filled symbols) or parental BICv (open symbols). The sensitivity of virus detection was ≥10^1.8 TCID₅₀/ml.

DISCUSSION

The molecular mechanisms used by CSFV to facilitate its own replication and to evade the host immune response are not completely understood. The direct interaction between virus and host proteins may modulate the host cell response, allowing the virus to manipulate host cellular pathways to favor its own replication. Our preceding work was oriented to studying cellular proteins interacting with several CSFV proteins. We demonstrated that structural CSFV core protein interacts with host SUMO1, IQGAP1, and UBC9 (11–13); more recently, we described the interaction of major structural glycoprotein E2 with PPP1CB (20) and viroporin p7 with calcium-modulating ligand (CAMLG) (26). In addition to modulating the virus replication cycle, these host-virus protein interactions are also virulence determinants in swine (11–13). Here, we report the characterization of the interaction between CSFV E2 protein and DCTN6, a subunit of the dynactin complex that acts as a cofactor for the microtubule-based dynein motor.

DCTN6 (subunit p27) is part of the activator complex dynactin, which enhances dynein-dependent motility, possibly through interactions with microtubules and vesicles (27). Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of 11 different polypeptides, of which 8 are unique to this complex, namely, dynactin 1 [p150(Glued)], dynactin 2 (p50 or dynamitin), dynactin 3 (DCTN3 or p24), dynactin 4 (DCTN4 or p62), dynactin 5 (DCTN5 or p25), DCTN6, and the actin-related proteins Arp1 and Arp10 (Arp11) (28). DCTN6 is part of the pointed-end subcomplex in dynactin, which also includes DCTN4, DCTN5, and Arp11. DCTN6 forms a heterodimer with DCTN5, and codepletion studies with DCTN5 and DCTN6 determined that both components were nonessential for dynactin functionality, although there were a reduced ability for dynactin to bind membranes and impaired early and recycling endosome movements, suggesting that DCTN5/6 forms a selective endomembrane cargo-targeting module (29). Interestingly, CSFV has been associated with early endosomes (30) after virus internalization, suggesting the possibility that DCTN6-E2 could be facilitating CSFV through the endocytic pathway.

There are several examples of viruses modulating the dynein pathway, indicating that the dynactin complex may be required for critical virus functions such as replication. Dynein has been shown to be involved in the mechanisms of viral entry and egress of dengue virus; inhibition of expression of dynein light chain or overexpression of dynein p50 decreased viral yield (31, 32). Adenovirus recruits and binds dynein directly (33) and competes for binding of dynein to lysosomes, facilitating intracellular movement of the viral capsid (34), and disruption of dynein resulted in decreased viral yield (35). In African swine fever virus, dynein has been shown to play an important role in virus transport by binding to structural viral proteins (36–38). We recently reported that FMDV nonstructural protein 3A specifically binds DCTN3 (25). Mapping of 3A critical residues interacting with DCTN3 allowed us to develop a mutant virus that disrupted the 3A-DCTN3 interaction. Interestingly, although the mutant virus replicated at a rate similar to that of the parental virus in cell cultures in vitro, it showed significantly decreased virulence in cattle. Interestingly, surviving animals presented persistent viremia that lasted until the end of the observation period. Although this is an unusual event, we previously observed 21-day-long viremia in animals infected with recombinant CSFV derived from the Brescia strain (11).

The results reported here characterize, for the first time, the cellular protein DCTN6 as a protein interaction partner for CSFV protein E2 in infected cells. These results indicate that the E2-DCTN6 protein interaction is important for CSFV replication in cell cultures in vitro. Importantly, the E2-DCTN6 protein interaction may be critical for viral virulence during CSFV infection in the natural host, domestic swine. This presents new possibilities for exploring CSFV pathogenesis and the viral requirements for virus-host interactions necessary to produce disease.

MATERIALS AND METHODS

Viruses and cells.

Swine kidney (SK6) cells (3), free of BVDV, were cultured in Dulbecco’s minimal essential medium (DMEM) (Gibco, Grand Island, NY, USA) with 10% fetal calf serum (FCS) (Atlas Biologicals, Fort Collins, CO, USA). CSFV Brescia strain was propagated in SK6 cells and used for the construction of an infectious cDNA clone (3). Growth kinetics were assessed on both SK6 cells and primary swine macrophage cell cultures, prepared as described previously (3). Titration of CSFV from clinical samples was performed using SK6 cells in 96-well plates (Costar, Cambridge, MA, USA). Viral infectivity was detected after 4 days in culture, by an immunoperoxidase assay using the CSFV monoclonal antibody WH303 (39) and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Titers were calculated and expressed as TCID₅₀ per milliliter, as described previously (40). As performed, titer sensitivity was ≥1.8 TCID₅₀/ml. Plaque assays were performed using SK6 cells in 6-well plates (Costar). SK6 cell monolayers were infected, overlaid with 0.5% agarose, and incubated at 37°C for 3 days. Plates were fixed with 50% (vol/vol) ethanol/acetone and stained by immunohistochemistry with monoclonal antibody WH303 (39).

Immunoblotting and antibodies.

For immunoblotting, infected and mock-infected (control) cell monolayers were washed in ice-cold phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer (Teknova, Hollister, CA, USA) in the presence of a protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were resolved on NuPAGE Bis-Tris gels (4 to 12% [wt/vol]; Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes, following the manufacturer’s instructions. Immunodetection was performed using the following antibodies: anti-DCTN6 monoclonal antibody (product no. 197913; Abcam, Cambridge, UK), anti-CSFV E2 protein monoclonal antibody WH303 (39), and Pierce goat anti-mouse IgG and anti-rabbit IgG peroxidase-conjugated secondary antibody reagents (product no. 31430 and 31460, respectively; Thermo Fisher Scientific, Waltham, MA, USA). Western blots were imaged using an Azure C300 imaging system, and results were analyzed with cSeries capture software (Azure Biosystems, Dublin, CA, USA).

Immunoprecipitation.

Immunoprecipitation procedures were performed in triplicate using the Pierce coimmunoprecipitation kit, following the manufacturer’s instructions (Thermo Fisher Scientific). In brief, cell suspensions were washed in ice-cold PBS and lysed with Pierce lysis buffer plus protease inhibitor cocktail (Roche, Basel, Switzerland). Anti-E2 WH303 was conjugated to the Pierce beads and incubated with precleared lysate overnight. The beads were then washed with Pierce lysis buffer with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and protease inhibitor cocktail (Roche) and were eluted with Pierce elution buffer.

Proximity ligation assay.

The proximity ligation assay was performed in triplicate using guidelines from the Duolink proximity ligation assay kit (Sigma-Aldrich). Specific details describing this technology can be found at the manufacturer’s website (https://www.sigmaaldrich.com/technical-documents/protocols/biology/how-pla-works.html). Specifically, SK6 cells were plated onto 12-mm round coverslips (Thomas Scientific, Swedesboro, NJ, USA), in a 24-well plate (Corning, Corning, NY, USA), at a density of 25,000 cells/well and were incubated at 37°C in 5% CO₂. Twenty-four hours later, cells were infected (MOI of 10) with CSFV. At 24 hpi, cells were washed with PBS and fixed for 20 min at room temperature with 500 μl of 4% (wt/vol) formaldehyde diluted in PBS. Cells were then permeabilized for 10 min at room temperature using permeabilization buffer (0.3% Triton X-100 in PBS). The permeabilized cells were blocked for 30 min at 37°C with Duolink blocking buffer, followed by incubation for 1 h at 4°C with primary antibodies anti-E2 WH303 (39) and anti-DCTN6 (product no. ab197913; Abcam) or anti-pan pS/T (product no. ab117253; Abcam). Cells were then washed twice with Duolink wash buffer A (Sigma-Aldrich) and incubated for 1 h at 37°C with the plus and minus assay probes, followed by two washes with Duolink wash buffer A and incubation for 30 min at 37°C with Duolink ligase in ligation buffer. The fixed cells were washed twice with Duolink wash buffer, followed by incubation for 100 min at 37°C with Duolink polymerase in amplification buffer. The fixed cells were then washed twice with Duolink wash buffer B, mounted with Duolink proximity ligation assay mounting medium with 4′,6-diamidino-2-phenylindole (DAPI), and observed using a Zeiss Axio Observer A1 microscope for the presence of fluorescence.

Yeast two-hybrid screening for disruption of DCTN6 binding to E2.

Plasmids E2-BD and DCTN6-AD were previously identified or constructed (21). E2-BD was randomly mutated using a mutagenic PCR approach to give an average of 5 nucleotide substitutions across the E2 open reading frame (this random mutant library was constructed by Epoch Bioscience [Bothell, WA]). The random E2 mutant library was then cotransformed into yeast strain AH109 with DCTN6-AD with a transformation rate of at least 1 × 10⁶ individual colonies, representing full coverage of the E2 mutagenic library. Transformed yeast colonies were initially selected on SD agar lacking leucine and tryptophan (SD-TL), the selection markers for DCTN6-AD and E2-BD, respectively. The yeast was then replica plated onto agar plates also lacking histidine and adenine (SD-ALTH) and incubated at 30°C. Colonies that grew on SD-ALTH plates contained a mutant E2-BD plasmid that maintained the ability of E2 to interact with DCTN6. However, individual yeast colonies that were not able to grow on SD-ALTH plates and grew only on SD-TL agar plates were tested further. Individual colonies that met these criteria were further tested by growth in SD-TL liquid medium overnight at 30°C, were spot plated, as described previously (11), on both SD-TL and SD-ALTH agar plates at 30°C, and were observed daily for growth, in comparison with colonies with native E2-BD and DCTN6. Individual colonies that were negative for growth on SD-ALTH plates were then subjected to previously described plasmid recovery procedures (11), through which the plasmid could be isolated using a Qiagen miniprep kit (Qiagen, Hilden, Germany) and Sanger sequenced. Sanger sequencing often revealed that the mutated E2 protein contained stop codons or out-of-frame mutations, thus explaining why the loss of E2-DCTN6 interaction occurred. Plasmids that contained stop codons or out-of-frame mutations were discarded and not studied further. In cases in which only individual amino acids were mutated, the E2 mutant plasmids were tested individually by cotransformation with DCTN6-AD, HPRT1-AD (encoding hypoxanthine phosphoribosyl transferase 1, a previously identified [21] positive E2 protein interaction partner), or PGADT7 (negative control) and selected on SD-TL plates. Individual colonies were grown overnight in SD-TL liquid medium at 30°C and spot plated on both SD-TL and SD-ALTH plates, to assess the ability of individual E2 mutants to bind both positive and negative controls (HPRT1-AD and PGADT7) and to confirm the reduction of binding to DCTN6. This second selection was performed to discard any mutant E2 proteins that lost the ability to bind DCTN6 because of a gross structural change in E2.

Construction of the CSFV E2ΔDCTN6v mutant.

A full-length infectious clone of the virulent CSFV Brescia strain (pBIC) (3) was used as a template in which E2 amino acid substitutions disrupting the E2-DCTN6 interaction, as mapped by reverse yeast two-hybrid methods, were included. Therefore, residue substitutions E308G and H335L were introduced into the native E2 sequence in the designed pBICΔDCTN6 construct. The pBICΔDCTN6 plasmid was obtained by DNA synthesis (Epoch Life Sciences, Sugar Land, TX, USA).

The CSFV pBICΔDCTN6 full-length genomic clone was linearized with SrfI and in vitro transcribed using the T7 MEGAscript system (Ambion, Austin, TX). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation (at 500 V, 720 Ω, and 100 W) with a BTX 630 electroporator (BTX, San Diego, CA). Cells were seeded in 12-well plates and incubated for 4 days at 37°C in 5% CO₂. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at –70°C or below. The full-length genome of in vitro rescued CSFV E2ΔDCTN6 virus (E2ΔDCTN6v) was completely sequenced by next-generation sequencing.

Animal infections.

The E2ΔDCTN6v mutant was evaluated for its virulence phenotype in swine relative to the virulent Brescia strain. Animal experiments were performed under biosafety level 3Ag conditions in the animal facilities at Plum Island Animal Disease Center, following a protocol approved by the Plum Island Animal Disease Center Institutional Animal Care and Use Committee of the U.S. Department of Agriculture and the U.S. Department of Homeland Security (protocol 171.04-15-R; approved 19 November 2015). Swine used in all animal studies were 10- to 12-week-old, 40-pound, commercial breed pigs. Five animals were inoculated intranasally with 10⁵ TCID₅₀ of either E2ΔDCTN6v or wild-type parental virus (BICv). Clinical signs (anorexia, depression, purple skin discoloration, staggering gait, diarrhea, and cough) and changes in body temperature were recorded daily throughout the 21-day experiment. Total and differential WBC, lymphocyte, and platelet counts were obtained using a Hemavet HV950FS system (Drew Scientific, Miami Lakes, FL).