ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalInteraction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na ؉ /H ؉ exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs-and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. IMPORTANCEMacropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membraneassociated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV. K aposi's sarcoma-associated herpesvirus (KSHV) entry into its in vitro adherent target cells is a multistage process which involves binding of viral glycoproteins to cell surface heparan sulfate receptors followed by interaction with specific entry receptors, induction of cell signaling pathways, and endocytosis. KSHV exploits multiple host cell surface receptors, including integrins ␣3␤1, ␣V␤3, ␣V␤5, and ␣9␤1 and nonintegrins CD98/xCT and EphA2, to enter the adherent target cells such as human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblast (HFF) cells (1-6). The interaction of KSHV with its specific entry receptors leads to the formation of a multimolecular receptor complex consisting of integrins, xCT, and EphA2 (2, 4, 7). Receptor engagement and multimolecular receptor complex formation result in autophosphorylation of focal adhesion kinase (FAK) and activation of Src, phosphatidylinositol 3-kinase (PI3-K), and Rho GTPases, and all of these molecules are targeted to specific entry sites on the plasma membrane (8-10). Our previous studies have demonstrated that the signal transduction pathways induced by KSHV and the consequent activation of their downstream molecules play a central role in coordinating the actin dynamics and the membrane protein assembly required for the successful entry of the virus into the cytoplasm (8-10). The

Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of Macropinocytosis

“…Considering the implication of late endosomal components in LASV cell entry (31,35,36), it will be of interest to see if and at which point the macropinocytosis-related pathway described here merges into the classical late endosomal route. Interestingly, a very recent study revealed that cell entry of Kaposi's sarcoma-associated herpesvirus via macropinocytosis is promoted by Hrs, a component of the endosomal sorting complex required for transport, linking macropinocytosis to the multivesicular endosome (83) A targeted screen of kinase inhibitors of rLCMV-LASVGP entry, followed by suitable counterscreens, identified a set of specific candidates. The identified target kinases included PI3K and PKC, which are well-known to be involved in the regulation of macropinocytosis of many viruses and cellular cargo (12,64).…”

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

“…Studies of in vitro KSHV infection have provided several pieces of novel information to virology research, such as the utilization of integrins for target cell infection by a herpesvirus (7), utilization of bleb-associated macropinocytosis for herpesvirus entry (4,15), c-Cbl playing an essential role in the bleb formation and macropinocytosis of KSHV (12,14), adaptor proteins CIB1, p130Cas, and Crk playing roles in macropinocytosis and trafficking of KSHV (12,14), and the association of cytosolic ESCRT Hrs protein with the plasma membrane and KSHV-containing macropinosomes (15). 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).…”

“…KSHV infection induces the translocation of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) protein, a prime component of the ESCRT-0 complex, to the plasma membrane near the KSHV-induced membrane blebs (15). This event is further amplified by the triggering of Rho-associated protein kinase 1 (ROCK1), which phosphorylates the sodium/hydrogen exchanger 1 (NHE1) to adjust a local pH change that is a critical step in macropinocytosis.…”

“…This event is further amplified by the triggering of Rho-associated protein kinase 1 (ROCK1), which phosphorylates the sodium/hydrogen exchanger 1 (NHE1) to adjust a local pH change that is a critical step in macropinocytosis. In the absence of Hrs (knockdown), the KSHV entry and gene expression are severely impacted (15) (Fig. 1).…”

“…Our recent studies demonstrate that KSHV indeed utilizes the ESCRT complex for efficient macropinocytic entry and trafficking in the cytoplasm of endothelial cells (15). KSHV infection induces the translocation of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) protein, a prime component of the ESCRT-0 complex, to the plasma membrane near the KSHV-induced membrane blebs (15).…”