Abstract
Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host’s ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.
Highlights
The ubiquitin-proteasome (UP) system is an important non-lysosomal protein degradation system in eukaryotic cells [1]
Vero (ATCC CCL-81, Richmond, VA, USA) and Cos-7 (ATCC CRL-1651, Richmond, VA, USA) cell lines were maintained in Dulbeccos modified Eagle medium (DMEM) containing 100 IU/ml penicillin, 100 g/ml streptomycin, 2 mM GlutaMAX and supplemented with 5% and 10% of heat-inactivated fetal bovine serum (FBS), respectively
After 16 hpi, African swine fever virus (ASFV) infectivity was analysed by flow cytometry by early p30 and late p72 protein expression using monoclonal antibodies against these viral proteins
Summary
The ubiquitin-proteasome (UP) system is an important non-lysosomal protein degradation system in eukaryotic cells [1]. Ubiquitin is a small (76 amino acids; aa) and highly conserved protein present in almost all eukaryotic cells. The addition of these few aa, so-called ubiquitination (UB), is the signal to direct proteins to the proteasome [2]. Ubiquitination of particular targets is involved in a diversity of cellular processes such as transcription, transduction, regulation of the immune response, control of cell division [3], development, endocytosis [4], cellular trafficking [5], and cell survival control [6]. The length (poly- versus monoubiquitination) and the site of ubiquitination define whether the protein would be sent to the proteasome, or if ubiquitination regulates any target protein activity.
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