Interaction of foot-and-mouth disease virus nonstructural protein 3A with host protein DCTN3 is important for viral virulence in cattle

Abstract

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids in most FMDVs examined to date. The role of 3A in virus growth and virulence within the natural host is not well understood. Using a yeast two-hybrid approach, we identified cellular protein DCTN3 as a specific host binding partner for 3A. DCTN3 is a subunit of the dynactin complex, a cofactor for dynein, a motor protein. The dynactin-dynein duplex has been implicated in several subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction identified by the yeast two-hybrid approach was further confirmed in mammalian cells. Overexpression of DCTN3 or proteins known to disrupt dynein, p150/Glued and 50/dynamitin, resulted in decreased FMDV replication in infected cells. We mapped the critical amino acid residues in the 3A protein that mediate the protein interaction with DCTN3 by mutational analysis and, based on that information, we developed a mutant harboring the same mutations in O1 Campos FMDV (O1C3A-PLDGv). Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in LFBK-αvβ6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a delayed disease in cattle compared to the virulent parental O1Campus (O1Cv). Virus isolated from lesions of animals inoculated with O1C3A-PLDGv virus contained amino acid substitutions in the area of 3A mediating binding to DCTN3. Importantly, 3A protein harboring similar amino acid substitutions regained interaction with DCTN3, supporting the hypothesis that DCTN3 interaction likely contributes to virulence in cattle.

Importance: The objective of this study was to understand the possible role of a FMD virus protein 3A, in causing disease in cattle. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. It was shown that manipulation of DCTN3 has a profound effect in virus replication. We developed a FMDV mutant virus that could not bind DCTN3. This mutant virus exhibited a delayed disease in cattle compared to the parental strain highlighting the role of the 3A-DCTN3 interaction in virulence in cattle. Interestingly, virus isolated from lesions of animals inoculated with mutant virus contained mutations in the area of 3A that allowed binding to DCTN3. This highlights the importance of the 3A-DCTN3 interaction in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

FIG 1

(A and B) Protein-protein interaction of FMDV 3A with bovine DCTN3 in the yeast two-hybrid system (A) and immunofluorescence staining (B). (A) Yeast strain AH109 was transformed with GAL4-binding domain (BD) fused to FMDV 3A (BD-3A) or a negative control, human lamin C (BD-LAM). These strains were then transformed with GAL4 activation domain (AD) fused to DCTN3 (AD-DCTN3) or T antigen (AD-Tag) as indicated above each lane. Spots of strains expressing the indicated constructs containing 2 × 10⁶ yeast cells were placed on selective media to screen for protein-protein interaction in the yeast two-hybrid system: either SD−Ade/His/Leu/Trp plates (−ALTH) or nonselective SD−Leu/Trp (−TL) for plasmid maintenance only. (B) Analysis of the distribution of DCTN3 and FMDV 3A proteins in MCF-10A cells. Cells were infected or mock infected with FMDV O1Cv and processed by immunofluorescence staining at 1.5 hpi as described in Materials and Methods. FMDV 3A was detected with MAb 2C2 and visualized with Alexa Fluor 594 (red). DCTN3 was detected with MAb GTX115607 and visualized with Alexa Fluor 488 (green). Yellow indicates colocalization of Alexa Fluor 594 and 488 in the merged image. (C) Protein-protein interactions by the mammalian two-hybrid system using the indicated plasmids were measured by absorbance at 405 nm as determined by CAT ELISA, as described in Materials and Methods.

FIG 2

Viral yields from FMDV infections in MCF-10A cells overexpressing DCTN3. (A) MCF-10A cells were transfected either with a plasmid encoding DCTN3 (pDCTN3) or GFP (hrGFP) as a control as described in Materials and Methods. (B) After transfection, triplicate plates were infected with FMDV O1Cv (MOI = 0.1). Titers were determined in BHK-21 cells and expressed as log₁₀ TCID₅₀/ml. The Western blot shows the endogenous intracellular levels of DCTN3in in MCF-10A cells transfected with either pDCTN3 or hrGFP.

FIG 3

Effect of disrupting the intracellular dynactin arrangement on FMDV yield. MCF-10A cells were transfected with either plasmid pmEGFP-C1-CC1, pmEGF-N1-p50, or hrGFP, as a control as described in Materials and Methods. (A) After transfection, triplicate plates were infected with FMDV O1Cv (MOI = 0.1). Titers were determined in BHK-21 cells and expressed as log₁₀ TCID₅₀/ml. (B) EGFP expression for the indicated plasmid was monitored by fluorescent microcopy.

FIG 4

Mapping DCTN3 binding site in FMDV 3A using alanine scan mutagenesis. (A) Each alanine 3A mutant name is followed, in parentheses, by the amino acid residues mutated for that mutant. All of the indicated native residues were mutated to an alanine. (B) FMDV 3A alanine mutants were assessed in their binding activity to DCTN3 using the yeast two-hybrid system. Yeast strain AH109 was transformed with either GAL4-binding domain (BD) fused to FMDV 2C (3A-BD), the indicated FMDV 3A mutation, or as a negative-control human lamin C (LAM BD). These strains were then transformed with GAL4 activation domain (AD) fused to DCTN3 (DCTN3-AD). Strains expressing the indicated constructs containing 2 × 10⁶ yeast cells were spotted onto selective media to evaluate protein-protein interaction in the yeast two-hybrid system, using either SD−Ade/His/Leu/Trp plates (−ALTH) or nonselective SD−Leu/Trp plates (−TL) for plasmid maintenance only.

FIG 5

Mapping DCTN3 binding site in FMDV 3A using comparative genomics. (A) Alignment of amino acid sequence among FMDV from different serotypes. The area between amino acid residues 89 and 92 is shaded. (B) FMDV O1C 3A (BD-3A), as well as mutated version of 3A representing polyalanine mutant 3A.13 (BD-3A.13), and mutant 3A.PLDG (BD-3A.PLDG) were tested in their ability to bind bovine DCTN3 in the yeast two-hybrid system. The methodological details are as described in Fig. 2.

FIG 6

Growth characteristics of FMDV O1Cv and O1C3A-PLDGv. (A) In vitro growth curves of O1Cv and O1C3A-PLDGv (MOI = 0.01) in LF-BK-αVβ6 and FBK cells. Samples obtained at the indicated time points were titrated in LF-BK-αVβ6 cells, and virus titers are expressed as TCID₅₀/ml. (B) Plaques corresponding to O1Cv and O1C3A-PLDGv viruses in LF-BK-αVβ6 and FBK cells. Infected cells were incubated at 37°C under a tragacanth overlay and stained with crystal violet at 48 hpi. Plaques from appropriate dilutions are shown.

FIG 7

Assessment of O1Cv and O1C3A-PLDGv virus virulence in cattle. Steers were inoculated with 10⁷ PFU of either O1Cv (animals 72, 73, and 74) or O1C3A-PLDGv (animals 69, 70, and 71) according to the aerosol inoculation method. The presence of clinical signs (clinical scores), and the virus yields (quantified as the log₁₀ of viral RNA copies/ml of sample) in serum and oral and nasal swabs were determined daily during the observational period.

FIG 8

Analysis of DCTN3 binding activity with mutated 3A proteins FMDV O1C 3A (BD-3A), mutant 3A.PLDG (BD-3A.PLDG), and mutants BD-3A.P and BD-3A.L were tested in their ability to bind bovine DCTN3 in the yeast two-hybrid system. The methodological details are as described in Fig. 2.