ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.

Insight into the Roles of E3 Ubiquitin Ligase c-Cbl, ESCRT Machinery, and Host Cell Signaling in Kaposi's Sarcoma-Associated Herpesvirus Entry and Trafficking

“…Current studies add to this list and demonstrate that ubiquitination of the KSHV receptors, integrin and EphA2R, by c-Cbl is recognized by the ESCRT-0 Hrs protein, which interacts with the virus-induced bleb region and virus-containing macropinosome (16). Hrs then associates with ESCRT-I Tsg101, which in turn associates with its downstream ESCRT-II and -III proteins in the macropinosome to direct the proper trafficking and transition of early to late endosome (16). These studies opened up the following questions which need to be investigated further.…”

“…Tsg101 has been shown to assist in the macropinocytic entry of the Crimean-Congo hemorrhagic fever virus, human papillomavirus, and echovirus-1 (4). The Tsg101 knockdown, although it shows an insignificant difference in KSHV entry, severely impacts the nuclear delivery of viral genome and subsequent gene expression in various endothelial cells (4,16). A proximity ligation assay and immunofluorescence and immunoprecipitation assays demonstrating the close association of KSHV glycoprotein gB with Tsg101 suggest the greater chance of Tsg101 actually recognizing the ubiquitinated EphA2R, which in turn interacts with the viral glycoproteins (16) (Fig.…”

“…Studies demonstrate that the Tsg101 interacts with EphA2R, c-Cbl, p130Cas, and Crk molecules that are involved in macropinocytosis and this interaction increases significantly with time. KSHV is associated with Tsg101 while trafficking through Rab5 containing early endosomes to Rab7 containing late endosomal compartments (16). During viral infection, Tsg101 interacts with its upstream protein Hrs (ESCRT-0) and downstream proteins EAP45 (ESCRT-II), CHMP5, and CHMP6 (ESCRT-III), and this interaction increases markedly upon KSHV infection.…”

“…During viral infection, Tsg101 interacts with its upstream protein Hrs (ESCRT-0) and downstream proteins EAP45 (ESCRT-II), CHMP5, and CHMP6 (ESCRT-III), and this interaction increases markedly upon KSHV infection. In the absence of Tsg101, although KSHV traffics normally through the early endosomal stages, it fails to transit to the late endosomal compartments (16), thus demonstrating that Tsg101 facilitates the transition of virus-containing early endosome to late endosome in HMVEC-d as well as in the human umbilical vein endothelial cells (HUVECs) (16) (Fig. 1).…”

Metastatic Kaposi’s Sarcoma Causing Gastrointestinal Bleeding: Report of an Unusual Case

“…The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins are recruited to assist in viral entry and trafficking. In addition, KSHV interacting with the cell surface receptors also induces the host transcription factors NF-κB, Nrf2, and ERK1/2 during infection to initiate and modulate viral and host gene expression [14][15].…”