Abstract
Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7) sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM) were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target.
Highlights
Foot-and-mouth disease virus (FMDV) is the etiological agent of the livestock disease that causes the most severe economic losses in endemic areas, and whose reintroduction poses a threat for disease-free countries
To ensure efficient intracellular delivery, each interfering sequence was extended at the N-terminus with seven Arg (R7) residues conferring cell penetrating peptide (CPP) properties (R7- fused peptides) followed by a 6-aminohexanoic acid flexible spacer residue
Recent structural and biophysical studies show that protein dimerization or oligomerization is a key factor in the regulation of different protein functions [27], including proteins relevant for virus replication [28]
Summary
Foot-and-mouth disease virus (FMDV) is the etiological agent of the livestock disease that causes the most severe economic losses in endemic areas, and whose reintroduction poses a threat for disease-free countries. The FMDV particle encloses a plus stranded RNA genome of about 8500 nucleotides that contains an open reading frame coding for four structural and nine non-structural mature proteins, flanked by non-coding regions at the 3 ́and 5 ́ ends. Non-structural protein 3A plays important roles in virus replication, virulence and host range [1,2,3]. This 153-amino acid protein has a conserved N-terminal and a variable C-terminal region in which several deletions and substitutions have been described to affect viral pathogenesis and virulence [4,5,6].
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