Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity.

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid, Spain.
Journal of Virology (Impact Factor: 4.44). 07/2012; 86(20):11013-23. DOI: 10.1128/JVI.00580-12
Source: PubMed


Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.

Download full-text


Available from: Paulino Gomez-Puertas, Oct 13, 2015
1 Follower
24 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Foot-and-mouth disease virus non-structural protein 3A plays important roles in virus replication, virulence and host-range; nevertheless little is known on the interactions that this protein can establish with different cell components. In this work, we have performed in vivo dynamic studies from cells transiently expressing the green fluorescent protein (GFP) fused to the complete 3A (GFP3A) and versions including different 3A mutations. The results revealed the presence of a mobile fraction of GFP3A, which was found increased in most of the mutants analyzed, and the location of 3A in a continuous compartment in the cytoplasm. A dual behavior was also observed for GFP3A upon cell fractionation, being the protein equally recovered from the cytosolic and membrane fractions, a ratio that was also observed when the insoluble fraction was further fractioned, even in the presence of detergent. Similar results were observed in the fractionation of GFP3ABBB, a 3A protein precursor required for initiating RNA replication. A nonintegral membrane protein topology of FMDV 3A was supported by the lack of glycosylation of versions of 3A in which each of the protein termini was fused to a glycosylation acceptor tag, as well as by their accessibility to degradation by proteases. According to this model 3A would interact with membranes through its central hydrophobic region exposing its N- and C- termini to the cytosol, where interactions between viral and cellular proteins required for virus replication are expected to occur.
    PLoS ONE 10/2014; 9(9):e106685. DOI:10.1371/journal.pone.0106685 · 3.23 Impact Factor