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-␣v␤6, 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. IMPORTANCEThe 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.

Sequences outside that of residues 93–102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells

“…This is in contrast to those of previous studies showing that chimeric viruses containing 3A of O/TAW/97 grew poorly and cannot form plaques in bovine cell (Beard and Mason, 2000), and that Asia 1 recombinant virus containing deletion at positions 93-102 or 91-104 in 3A failed to replicate and produce plaque in primary bovine kidney cells (Li et al, 2010. However, this conclusion is consistent with a recent report showing that aa substitution at positions 89-92 in 3A affect FMDV replication and plaque formation in primary bovine cells (Gladue et al, 2014). Furthermore, the result of quantification analysis of viral RNA also proves that the deletion at positions 93-102 in 3A, indeed, affect virus replication efficiency in primary cells of bovine origin, but cannot alone GZSB-WT 10 −5 5 5 0 100 10 −6.833 10 −6 5 5 0 100 10 −7 5 2 3 40 account for the inability to replicate in bovine cells.…”

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus

“…Moreover, Pacheco et al reported that an O1Campos variant (O1CaD3A) harboring 20 aa deletion in 3A between residues 87-106 also displayed lower replication rate (approximately 1000-fold) in FBK cells than its parental virus O1Ca (Pacheco et al, 2013). The replication rate of O1CaD3A decreased more obviously than those of the epitopetagged viruses in FBK cells, and one possible reason is that the O1CaD3A lacked the DCTN3 binding site (residues 89-92 in 3A) since the binding between FMDV 3A and host DCTN3 may contribute to the host range specificity of FMDV (Gladue et al, 2014). On the other hand, we also analyzed the growth properties of 3A-tagged viruse (FMDV-FLAG) and its parental virus (r-HN) (Li et al, 2012) in FBK cells, and the results showed that FMDV-FLAG and r-HN replicated poorly in FBK cells with no visible plaque (data not shown).…”

Microtubule Regulation and Function during Virus Infection

“…This interaction with DCTN3 is required for virulence in cattle or cultured primary kidney cells but not in transformed kidney cell lines (30). Intriguingly, during serial passage, NSP3A mutants that reacquired virulence had mutated to reacquire DCTN3 binding (30). This underscores the importance of DCTN3 to FMDV infection in vivo and in normal cells and highlights potential dangers of studying transformed cell lines.…”

“…Foot-and-mouth disease virus (FMDV) nonstructural protein 3A (NSP3A) binds and colocalizes with DCTN3 early in infection. This interaction with DCTN3 is required for virulence in cattle or cultured primary kidney cells but not in transformed kidney cell lines (30). Intriguingly, during serial passage, NSP3A mutants that reacquired virulence had mutated to reacquire DCTN3 binding (30).…”