Characterization of a ubiquitinated protein which is externally located in African swine fever virions.
An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located. Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections. This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated. This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions. The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid. The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded. The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector. Recombinant PIG1 was ubiquitinated in the presence of E. coli expressed UBCv1 in vitro. These results suggest that PIG1 may be a substrate for UBCv1. The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions. Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein.
Pirbright Laboratory, Institute for Animal Health, Woking, Surrey, United Kingdom.
P M
PM
Hingamp
M L
ML
Leyland
J
J
Webb
S
S
Twigger
R J
RJ
Mayer
L K
LK
Dixon
D000358:African Swine Fever Virus / Q000648:ultrastructure*; D000595:Amino Acid Sequence; D001483:Base Sequence; D005814:Genes, Viral; D007150:Immunohistochemistry; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics; D014771:Virion / Q000648:ultrastructure*
C091943:PIG1 protein, African swine fever virus; D014452:Ubiquitins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D044763:Ubiquitin-Conjugating Enzymes; C091944:ubiquitin-conjugating enzyme UBCv1, African swine fever virus; D008025:Ligases