Domains involved in calcineurin phosphatase inhibition and nuclear localisation in the African swine fever virus A238L protein.
The African swine fever virus A238L protein inhibits calcineurin phosphatase activity and activation of NF-kappaB and p300 co-activator. An 82 amino acid domain containing residues 157 to 238 at the C-terminus of A238L was expressed in E. coli and purified. This purified A238L fragment acted as a potent inhibitor of calcineurin phosphatase in vitro with an IC50 of approximately 70 nM. Two putative nuclear localisation signals were identified between residues 80 to 86 (NLS-1) and between residues 203 to 207 overlapping with the N-terminus of the calcineurin docking motif (NLS-2). Mutation of these motifs independently did not reduce nuclear localisation compared to the wild type A238L protein, whereas mutation of both motifs significantly reduced nuclear localisation of A238L. Mutation of the calcineurin docking motif resulted in a dramatic increase in the nuclear localisation of A238L provided an intact NLS was present. We propose that binding of calcineurin to A238L masks NLS-2 contributing to the cytoplasmic retention of A238L.
Abrams
Charles C
CC
Institute for Animal Health Pirbright Laboratory, Pirbright, UK.
Chapman
Dave A G
DA
Silk
Rhiannon
R
Liverani
Elisabetta
E
Dixon
Linda K
LK
D000358:African Swine Fever Virus / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D013552:Swine; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
C113585:A238L protein, African swine fever virus; D016328:NF-kappa B; D019913:Nuclear Localization Signals; D014764:Viral Proteins; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases