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

Authors: GLADUE, DOUGLAS - University Of Connecticut; O'DONNELL, VIVIAN - University Of Connecticut; BAKER-BRANSTETTER, RYAN - Oak Ridge Institute For Science And Education (ORISE); Pacheco Tobin, Juan; Holinka-Patterson, Lauren; Arzt, Jonathan; Pauszek, Steven; FERNANDEZ-SAINZ, IGNACIO - University Of Connecticut; FLETCHER, PAIGE - Oak Ridge Institute For Science And Education (ORISE); BROCCHI, E - Istituto Zooprofilattico; LU, ZHIQIANG - Us Deparment Of Homeland Security; Rodriguez, Luis; Borca, Manuel

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2013
Publication Date: 3/1/2014
Citation: Gladue, D.P., O'Donnell, V., Baker-Branstetter, R., Pacheco Tobin, J., Holinka-Patterson, L.G., Arzt, J., Pauszek, S.J., Fernandez-Sainz, I., Fletcher, P., Brocchi, E., Lu, Z., Rodriguez, L.L., Borca, M.V. 2014. Interaction of foot-and-mouth disease virus non-structural protein 3A with host protein DCTN3 is important for viral virulence in cattle. Journal of Virology. 88(5):2737-2747.

Interpretive Summary: Foot-and-mouth disease is a devastating disease of livestock. As part of our research program for countermeasure development we are studying the role of different viral proteins and their interactions with cattle proteins, in causing infection and disease. The objective of this work was to understand the possible role of a FMD virus non structural protein called 3A, in causing disease in cattle. Several cattle proteins that interact with FMDV 3A were identified, and the possible role of this interaction in the development of the disease analyzed. We have found that the cellular protein, DCTN3, is a specific binding partner for 3A. DCTN3 provides structure and mobility to the cell. It was shown that manipulation of DCTN3 has a profound effect in virus replication. The area of the non structural protein 3A that actually interacts with DCTN3 was identified. We mutated the 3A protein based on that information, and developed an FMDV mutant that could not bind DCTN3. This mutant virus exhibited significantly 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 in FMD virus virulence and provides possible mechanisms of virus attenuation for the development of improved FMD vaccines.

Technical Abstract: Non-structural 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 co-factor for dynein, a microtubule-based motor protein. The dynactin-dynein duplex has been implicated in several important subcellular functions involving intracellular organelle transport. The 3A-DCTN3 interaction initially identified by the yeast two-hybrid approach was further confirmed using deconvolution microscopy. Over-expression 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 3A mediating the interaction with DCTN3, and we developed a mutant O1 Campos FMDV (O1C3A31 PLDGv) containing these amino acid modifications. Although O1C3A-PLDGv FMDV and its parental virus (O1Cv) grew equally well in the bovine cell line LFBK-alphavBeta6, O1C3A-PLDGv virus exhibited a decreased ability to replicate in primary bovine cell cultures. Importantly, O1C3A-PLDGv virus exhibited a significantly 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.