Inhibition of the Ubiquitin-Proteasome System Prevents Vaccinia Virus DNA Replication and Expression of Intermediate and Late Genes

Abstract

The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions (22). Proteins to be degraded are modified by the addition of multiple copies of the 76-amino-acid ubiquitin through the sequential activities of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) (4, 12). The degradation is mediated by the 26S proteasome, a large multiprotein complex containing trypsin-, chymotrypsin-, and postglutamyl peptidyl hydrolytic-like protease activities. In addition, ubiquitylation has nondegradative roles in DNA repair, transcriptional regulation, signal transduction, endocytosis, and intracellular trafficking (48). Viruses belonging to several families utilize or modulate the ubiquitin-proteasome system (2, 13). The inhibition of proteasomal degradation prevents the entry of influenza virus (23) and mouse hepatitis virus (54); the early postentry steps of minute virus of mice (44) and herpes simplex virus (7); and the genome replication or expression of human coxsackie 3B virus (27), adenovirus (5), cytomegalovirus (20), infectious bursal disease virus (26), and vesicular stomatitis virus (40). In some cases the effects may be secondary to the activation of a cellular stress response and signaling pathway (24, 40, 52). Proteasomal inhibitors have an indirect effect on retroviruses and rhabdoviruses by depleting free ubiquitin needed to modify proteins for budding (16).Vaccinia virus (VACV), the repre...

Furin-Processed Antigens Targeted to the Secretory Route Elicit Functional TAP1−/−CD8+ T Lymphocytes In Vivo

“…A slight effect on cells infected with the rVACV expressing the proteasome-independent preprocessed cytosolic miniprotein (Fig. 4C, right) might be explained by a LC effect on vaccinia late gene expression (35). As expected, BFA abrogated K b /SIINFEKL complex formation from all three constructs.…”

Endoplasmic Reticulum Targeting Alters Regulation of Expression and Antigen Presentation of Proinsulin

“…If the processing pathways acting on cytosolic and ER-targeted PI differ, the peptides produced in them also might be distinct; although peptide insulin B [15][16][17][18][19][20][21][22][23] is the optimal peptide recognized by G9C8 cells (16), N-or C-terminally extended variants could be stimulatory. We reasoned that if different peptides result from degradation of PI versus PPI, the stability of the complexes formed with H-2K d molecules might differ.…”

“…This was achieved by 24-h incubation with Doxy followed by a brief acid treatment to remove cell-surface K d -insulin B 15-23 complexes. The generation of new K d -insulin B [15][16][17][18][19][20][21][22][23] complexes from previously synthesized PPI, or from newly synthesized PI, in the presence of protease inhibitors was then monitored.…”

“…Considering a published report that proteasome inhibitors prevent expression of late vaccinia-encoded proteins (20), we used a strong synthetic early/late promoter contained in the pSC65 plasmid to produce the PPI virus. A total of 5 3 10 5 HeLa tet-on (HT)-K d cells (see later) were seeded in six-well plates on day 21.…”

“…Production of antigenic epitopes was read out using TCRtransgenic G9C8 CD8 + T cells with specificity for the H2-K drestricted T cell epitope insulin B [15][16][17][18][19][20][21][22][23] (27), an epitope with immunodominant status in the NOD mouse that is recognized by diabetogenic T cells found in pancreatic islet infiltrates (28). In vitro activated and restimulated G9C8 T cells secrete IFN-g upon stimulation with 10 26 M cognate peptide and H2-K d APCs (Fig.…”