Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon
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A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.
This work was supported by the Fondo Nacional de Ciencia y Tecnología (FONCyT) and Consejo Nacional de Investigaciones Científicas y Tecnológicas of Argentina (CONICET). We are grateful to Dr. Nora Lopez and Dr. Sabrina Foscaldi for providing the pTM1-Renilla plasmid. CL and AG are graduate fellows from CONICET. MW, PRG and NM are at the Scientific Researcher Career in CONICET.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 3.Rueckert R (1985) Picornaviruses and their replication. In: Fields B (ed) Virology. Raven press, New YorkGoogle Scholar
- 5.Nunez JI, Molina N, Baranowski E, Domingo E, Clark S, Burman A, Berryman S, Jackson T, Sobrino F (2007) Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host. J Virol 81(16):8497–8506. https://doi.org/10.1128/JVI.00340-07 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Nunez JI, Baranowski E, Molina N, Ruiz-Jarabo CM, Sanchez C, Domingo E, Sobrino F (2001) A single amino acid substitution in nonstructural protein 3A can mediate adaptation of foot-and-mouth disease virus to the guinea pig. J Virol 75(8):3977–3983. https://doi.org/10.1128/JVI.75.8.3977-3983.2001 CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Gladue DP, O’Donnell V, Baker-Bransetter R, Pacheco JM, Holinka LG, Arzt J, Pauszek S, Fernandez-Sainz I, Fletcher P, Brocchi E, Lu Z, Rodriguez LL, Borca MV (2014) Interaction of foot-and-mouth disease virus nonstructural protein 3A with host protein DCTN3 is important for viral virulence in cattle. J Virol 88(5):2737–2747. https://doi.org/10.1128/JVI.03059-13 CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Moffat K, Howell G, Knox C, Belsham GJ, Monaghan P, Ryan MD, Wileman T (2005) Effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2BC but not 3A blocks endoplasmic reticulum-to-Golgi transport. J Virol 79(7):4382–4395. https://doi.org/10.1128/JVI.79.7.4382-4395.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Gonzalez-Magaldi M, Postigo R, de la Torre BG, Vieira YA, Rodriguez-Pulido M, Lopez-Vinas E, Gomez-Puertas P, Andreu D, Kremer L, Rosas MF, Sobrino F (2012) Mutations that hamper dimerization of foot-and-mouth disease virus 3A protein are detrimental for infectivity. J Virol 86(20):11013–11023. https://doi.org/10.1128/JVI.00580-12 CrossRefPubMedPubMedCentralGoogle Scholar
- 12.O’Donnell VK, Pacheco JM, Henry TM, Mason PW (2001) Subcellular distribution of the foot-and-mouth disease virus 3A protein in cells infected with viruses encoding wild-type and bovine-attenuated forms of 3A. Virology 287(1):151–162. https://doi.org/10.1006/viro.2001.1035 CrossRefPubMedGoogle Scholar
- 13.Midgley R, Moffat K, Berryman S, Hawes P, Simpson J, Fullen D, Stephens DJ, Burman A, Jackson T (2013) A role for endoplasmic reticulum exit sites in foot-and-mouth disease virus infection. J Gen Virol 94(Pt 12):2636–2646. https://doi.org/10.1099/vir.0.055442-0 CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Giraldez A (2013) Diseño de un sistema de genética reversa del virus de la fiebre aftosa. Universidad de Buenos AiresGoogle Scholar
- 21.Tulloch F, Pathania U, Luke GA, Nicholson J, Stonehouse NJ, Rowlands DJ, Jackson T, Tuthill T, Haas J, Lamond AI, Ryan MD (2014) FMDV replicons encoding green fluorescent protein are replication competent. J Virol Methods 209:35–40. https://doi.org/10.1016/j.jviromet.2014.08.020 CrossRefPubMedPubMedCentralGoogle Scholar
- 28.Guarne A, Tormo J, Kirchweger R, Pfistermueller D, Fita I, Skern T (1998) Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition. EMBO J 17(24):7469–7479. https://doi.org/10.1093/emboj/17.24.7469 CrossRefPubMedPubMedCentralGoogle Scholar
- 36.Behura M, Mohapatra JK, Pandey LK, Das B, Bhatt M, Subramaniam S, Pattnaik B (2016) The carboxy-terminal half of nonstructural protein 3A is not essential for foot-and-mouth disease virus replication in cultured cell lines. Adv Virol 161(5):1295–1305. https://doi.org/10.1007/s00705-016-2805-z Google Scholar