Abstract
Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process.
Highlights
Ubiquitination has been linked to diverse cellular functions including directing protein recycling [1,2]
We show that the proteasome activity is required for Kaposi’s sarcoma-associated herpesvirus (KSHV) entry into endothelial cells and intracellular trafficking to nuclei
KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15, as well as activated forms of the E3 ligase c-Cbl
Summary
Ubiquitination has been linked to diverse cellular functions including directing protein recycling [1,2]. Addition of four or more ubiquitin moieties (polyubiquitination) provides the necessary signal for targeting protein for proteasomal degradation. Ubiquitination of target cell surface membrane proteins triggers its internalization and endocytic sorting in addition to proteasomal degradation. Monoubiquitination, the addition of single ubiquitin moieties, of epidermal growth factor receptor (EGFR) cytoplasmic tail induces its internalization and transport to the lysosome for degradation [3,4,5] while ubiquitination of membrane receptors b2 adrenergic receptor and interleukin 2 receptor b chain is required for their correct internalization and processing [6,7,8]. The ubiquitin molecules are transferred to the E2 conjugating enzyme in an ATP-dependent reaction. E2 enzyme interacts with a specific E3 partner and transfers the ubiquitin molecules to the target protein [9]. Mammalian cells express two E1 activating enzymes, up to 40 E2 conjugating enzymes, and hundreds of E3 ligases [10,11]
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