Abstract
In recent years it has been well established that two major constituent parts of the ubiquitin proteasome system (UPS)—the proteasome holoenzymes and a number of ubiquitin ligases—play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of human immunodeficiency virus type 1 (HIV-1). However, the role in HIV-1 replication of the third major component, the deubiquitinating enzymes (DUBs), has remained largely unknown. In this study, we show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the therapeutic effect of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and the major histocompatibility complex (MHC)-I pathway. In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells.
Highlights
The homeostasis of eukaryotic cells is maintained by a well-tuned balance between biosynthesis and degradation of proteins
In order to investigate of whether deubiquitinating enzymes (DUBs) activities play any role in late steps of human immunodeficiency virus type 1 (HIV-1) replication, we analyzed virus-like particle (VLP) release and Gag processing after DI treatment
Cells were treated with the following DIs: P22077, which inhibits the DUBs USP7 and USP47 [59], the DI P5091, which only inhibits
Summary
The homeostasis of eukaryotic cells is maintained by a well-tuned balance between biosynthesis and degradation of proteins. Ubiquitin (Ub) is attached to target proteins by the cascade-like catalytic action of. The ubiquitin molecule is activated by an E1 ligase, followed by the transfer to one of numerous conjugating enzymes, and an E3 ligase catalyzes the transfer of ubiquitin onto the ε-amino group of a Lysine residue within the target protein (for review see [1]). Viruses 2017, 9, 222 attachment of single Ub molecules can lead to monoubiquitination or multiple monoubiquitination, whereas binding of at least 4 ubiquitin moieties, conjugated via the internal Lysine 48 or 63 of ubiquitin, is called polyubiquitination. Monoubiquitination as well as Lysine 63-linked polyubiquitination regulates protein functions in processes such as DNA repair, signal transduction, endocytosis and many others. K48-linked polyubiquitination represents the canonical signal for degradation of the target protein by the 26S proteasome (for review see [2]). All ubiquitin modifications can be reversed by the isopeptide-bond specific proteolytic activity of DUBs [3]
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