title: Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication.
abstract: Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
authors.forename: Alastair
authors.initials: A
authors.lastname: Teale
authors.forename: Stephanie
authors.initials: S
authors.lastname: Campbell
authors.forename: Nick
authors.initials: N
authors.lastname: Van Buuren
authors.forename: Wendy C
authors.initials: WC
authors.lastname: Magee
authors.forename: Kelly
authors.initials: K
authors.lastname: Watmough
authors.forename: Brianne
authors.initials: B
authors.lastname: Couturier
authors.forename: Robyn
authors.initials: R
authors.lastname: Shipclark
authors.forename: Michele
authors.initials: M
authors.lastname: Barry
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018155:Orthopoxvirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D004279:DNA, Viral; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D014764:Viral Proteins; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes