Abstract
Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.
Highlights
The eukaryotic proteasome is a ~2MDa cylindrical shaped protease complex located in the cytoplasm and nucleus and comprises a 20S proteolytic core and one or two 19S regulatory subunits [1,2]
A virulent strain of eastern equine encephalitis virus (EEEV) GA97 was obtained from Dr Jonathan Jacobs (MRIGlobal) and virulent western equine encephalitis virus (WEEV) California 1930 strain was obtained from ATCC
Bortezomib was found to not be toxic to U87MG cells at the concentrations tested. These results indicated that Bortezomib inhibits TC-83 multiplication in a dose-dependent manner in U87MG cells at nontoxic concentrations
Summary
The eukaryotic proteasome is a ~2MDa cylindrical shaped protease complex located in the cytoplasm and nucleus and comprises a 20S proteolytic core and one or two 19S regulatory subunits [1,2]. Proteins destined for degradation are tagged with multiple copies of a small 76 amino acid protein, ubiquitin, making the targeted protein identifiable for degradation by the 19S regulatory component [1,2,3]. Unfolded polyubiquitin-tagged proteins are fed into the 20S core of the proteasome to be cleaved into smaller peptides [1,2]. The proteasome has three types of catalytic activity: chymotryptic, tryptic, and peptidylglutamyl. The mechanism of inhibition was ascribed to Bortezomib being able to form a tetrahedral adduct with an active threonine site within the proteolytic core; making this drug an attractive candidate for drug development [1]. More recently the ubiquitin proteasome system (UPS) has been described to play a role in non-degradative processes such as cell survival, MHC class I antigen presentation, apoptosis, cell division, NF-κB activation, DNA repair, transcriptional regulation, signal transduction, endocytosis and intracellular trafficking, and chemoresistance [1,2,3,7]
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