Studies of the ubiquitin conjugating (UBCv) enzyme encoded by African swine fever virus

Abstract

Ubiquitin conjugating (UBC) enzymes play a key role in eukaryotes during the posttranslational modification of proteins by covalent attachment of ubiquitin. A gene was identified in the double stranded DNA genome of African swine fever virus (ASFV) which was predicted to encode a protein with high homology to eukaryotic UBC enzymes. This ASFV encoded enzyme (UBCv) was expressed in E. coli and was shown to have ubiquitin conjugating activity in vitro. Antisera against recombinant UBCv were used to detect UBCv in ASFV infected cells. UBCv was shown to be a cytosolic protein present throughout the early and late stages of ASFV replication and was packaged in ASPV virions. Attempts to inhibit UBCv activity during ASFV infection using antisense oligonucleotides were unsuccessful, and a recombinant ASPV mutant with the UBCv gene disrupted by the luciferase reporter could not be isolated. However, ASFV replication was impaired late in infection in TS20 cells at a temperature which inhibits the ubiquitin conjugating pathway. No novel ubiquitinated proteins could be detected in ASFV infected cells by immunoblotting, although an unspecific increase of cellular ubiquitin conjugation was observed in early infection. However, virus factories were intensely stained late in ASFV infection by immunofluorescence using anti-ubiquitin antisera. In addition, several ubiquitinated structural proteins were detected in purified ASFV extracellular particles by both immunoblotting and immunogold electron microscopy. An 18 kDa ubiquitinated structural protein, probably localized in the virion periphery, was purified to homogeneity and the sequence of its N-terminal 10 amino acids was determined. The N-terminal sequence of this protein matched exactly the product of a gene of unknown function encoded by the ASPV genome