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

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro infection of dermal endothelial cells begins with its binding to host cell surface receptor molecules such as heparan sulfate (HS), integrins (α3β1, αVβ3, and αVβ5), xCT, and EphA2 receptor tyrosine kinase (EphA2R). These initial events initiate dynamic host protein-protein interactions involving a multimolecular complex of receptors, signal molecules (focal adhesion kinase [FAK], Src, phosphatidylinositol 3-kinase [PI3-K], and RhoA-GTPase), adaptors (c-Cbl, CIB1, Crk, p130Cas, and GEF-C3G), actin, and myosin II light chain that lead to virus entry via macropinocytosis. Here we discuss how KSHV hijacks c-Cbl, an E3 ubiquitin ligase, to monoubiquitinate the receptors and actin, which acts like a marker for trafficking (similar to zip codes), resulting in the recruitment of the members of the host endosomal sorting complexes required for transport (ESCRT) Hrs, Tsg101, EAP45, and the CHMP5 and -6 proteins (zip code readers) recognizing the ubiquitinated protein and adaptor machinery to traffic through the different endosomal compartments in the cytoplasm to initiate the macropinocytic process and infection.

INTRODUCTION

Members of herpesviruses, infecting a wide range of hosts in the animal kingdom, are characterized by their large double-stranded DNA genome encoding >100 proteins, microRNAs, and long noncoding RNAs, establishment of latent infection in specific cells, and periodic reactivation and reinfection while also maintaining latency (1). Human herpesviruses cause many major ailments, such as encephalitis, keratitis, corneal blindness, sexually transmitted lesions, chickenpox, shingles, birth defects, infectious mononucleosis, B-cell lymphomas, nasopharyngeal carcinoma, Kaposi's sarcoma (KS), primary effusion lymphoma, etc. (1). Mechanistic studies revealing how herpesviruses infect their target cells are under intense scrutiny as blocking the virus entry and infection of target cells is one of the most promising ways to control these diseases. Such studies include analysis of viral and host proteins involved in entry and infection and the mechanism of virus/viral capsid transport in the cytoplasm and delivery of viral nucleic acid to the nucleus.

KSHV UTILIZES MACROPINOCYTOSIS AS ONE OF THE MAJOR ROUTES OF ENTRY IN HUMAN DERMAL ENDOTHELIAL CELLS

Kaposi's sarcoma lesions are characterized by proliferating spindle-shaped vascular endothelial cells in which Kaposi's sarcoma-associated herpesvirus (KSHV) viral DNA is present in the latent form (2). Primary cultures of human dermal microvascular endothelial cells (HMVEC-d) closely resembling the in vivo targets are used as a model system to study the various steps of in vitro KSHV infection, such as binding, entry, and transport in the cytoplasm, nuclear delivery of viral genomes, viral gene expression, and the establishment of in vitro latency (3, 4). KSHV utilizes endocytosis for its entry in HMVEC-d and other target cells, probably because endosomes provide a convenient way for the rapid transport of the virus in the cytoplasm as well as to evade the cytoplasmic innate response effector molecules. Within 5 min of infection of HMVEC-d cells, large endocytic vesicles containing enveloped KSHV particles have been observed by electron microscopy (3), and viral DNA is delivered into the nuclei within 15 to 30 min of infection (3, 4). Studies utilizing light and electron microscopy, inhibitors of endocytosis, and markers of endocytosis such as dextran (macropinocytosis) and transferrin (clathrin-mediated endocytosis) have shown that KSHV utilizes macropinocytosis for its entry into the HMVEC-d cells, which involves the formation of actin-driven spherical membrane protrusion known as blebs. The actin dynamics is also known to play a crucial role during clathrin-mediated endocytosis of KSHV in primary human fibroblast cells (3–5) and endosomal trafficking of KSHV in primary human endothelial and fibroblast cells (3–5).

KSHV ENTRY BY MACROPINOCYTOSIS: A CONSEQUENCE OF MULTISTEP INTERACTIONS BETWEEN VIRAL AND HOST PROTEINS AND ACTIVATION OF CELL SIGNALING PATHWAYS

KSHV utilizes the interactions between its envelope glycoproteins gB, gpK8.1A, gH, and open reading frame 4 (ORF4) with the HMVEC-d cell surface ubiquitous heparan sulfate (HS) proteoglycan to bind the cells (6). HS probably acts as the distal receptor to concentrate virus on the cell surface and to enable KSHV-specific interactions with its proximal receptors, such as the integrins α3β1, αVβ3, and αVβ5, the amino acid transporter xCT protein, and EphA2 receptor tyrosine kinase (EphA2R) protein. These interactions probably increase the avidity of binding and receptor clustering leading to the activation of several key cell signaling molecules to create an environment that is conducive for the early stages of infection (4, 7–10). These include focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase (PI3-K), RhoA-GTPase, and reactive oxygen species (ROS), which are essential for signal amplification, actin rearrangement, endocytosis, and acetylation of microtubules. These events are followed by activation of subsequent downstream molecules, such as protein kinase C-ζ (PKC-ζ), MEK, extracellular signal-regulated kinase 1/2 (ERK1/2), NF-κB, and Nrf2, which are essential for virus trafficking and modulation of viral and host gene expression (11) (Fig. 1).

FIG 1.

Schematic representation of KSHV entry by macropinocytosis and trafficking in the human microvascular dermal endothelial cells. (Step 1) Within minutes of KSHV binding to cell surface receptors in the non-lipid raft (NLR) region, FAK, Src, PI3-K, RhoA-GTPase, and ROS signal molecules are induced, leading to the recruitment of adaptor c-Cbl, CIB1, Crk, and p130Cas proteins, and c-Cbl is essential for actin-driven macropinocytosis. (Step 2) c-Cbl-mediated monoubiquitination (zip codes or traffic markers) of receptors or actin results in the rapid KSHV-receptor translocation to the lipid raft (LR) region, and recruitment of ESCRT complex Hrs, Tsg101, EAP45, and CHMP5 and -6 proteins (zip code readers) recognizing the ubiquitinated proteins. (Step 3) KSHV is trafficked via the early Rab5-positive and late Rab7-positive endosomes regulated by the ESCRT and adaptor proteins. (Step 4) Viral capsid released from the late endosome (LE) is rapidly transported to the nuclear periphery using RhoA-GTPase-induced acetylated microtubules and dynein motor proteins, and viral DNA is delivered into the nucleus. (Step 5) Viral gene expression starts and latency is established. c-Cbl knockdown blocks bleb formation, LR translocation of virus, interaction with EphA2R, and productive viral entry and diverts KSHV to a clathrin-lysosomal noninfectious pathway. CIB1 knockdown blocks virus induced blebs, macropinocytosis, and infection induced EphA2R, Src, and ERK1/2 activation. p130Cas knockdown reduces nuclear trafficking of viral DNA and routes KSHV to lysosomal degradation.

KSHV HIJACKS c-Cbl, WHICH ACTS LIKE A ZIP CODE-ADDING MACHINE TO REGULATE VIRUS ENTRY AND INTERACTION WITH SIGNAL ADAPTOR MOLECULES

The initial KSHV-HS, integrin, and xCT interactions occur in the non-lipid raft (NLR) region of HMVEC-d cells, which activates the host preexisting signal molecules within 1 min of infection. Studies to decipher the molecule(s) that regulates the recruitment of signal molecules and macropinocytosis lead to the discovery that KSHV induces the tyrosine phosphorylation of c-Cbl, an adaptor protein, as early as 1 min postinfection (12) (Fig. 1). c-Cbl, an E3 ligase, is reported to facilitate the KSHV internalization along with its receptor integrin β1 during infection of endothelial cells (13). It selectively monoubiquitinates the α3β1 and αVβ3 integrins that serve like traffic markers (similar to adding zip codes), and induces a rapid translocation of KSHV along with α3β1, αVβ3, and x-CT receptors to the lipid raft (LR) region of the host cells, leading to KSHV macropinocytosis and viral gene expression in the nucleus. In contrast, c-Cbl polyubiquitinates αVβ5, leading to clathrin-mediated entry which is targeted to the lysosomes (12). Activated c-Cbl also localizes in the LR region near the macropinocytic blebs and forms a complex with KSHV infection-induced PI3-K–p85 in a time-dependent manner to facilitate the interaction of c-Cbl with downstream molecules (14). The c-Cbl further interacts and adds zip codes (ubiquitination) to the nonmuscle myosin heavy chain IIA (myosin IIA) and actin in the interior of blebs of infected cells, which accelerate the actomyosin contraction and bleb retraction, leading to the formation of macropinosomes having KSHV particles (14) (Fig. 1). This rapid increase in the association of c-Cbl with myosin IIA greatly enhances the membrane blebbing, macropinocytosis, and KSHV internalization (14).

After translocating to LR (integrin-virus complex) via its gH-gL proteins, KSHV interacts and activates the LR-located receptor tyrosine kinase EphrinA2 (EphA2R), which associates with KSHV and integrins (α3β1 and αVβ3) in LRs early during infection and augments the virus-induced signaling (10). Calcium and integrin-binding protein-1 (CIB1), a key adaptor effector molecule, is also induced during KSHV infection, which promotes the KSHV-EphA2R-associated Src and ERK1/2 signal amplification. CIB1 also assists in the bleb formation by facilitating the association of EphA2R with myosin IIA and alpha-actinin 4 and is also associated with KSHV-containing Rab5-positive early endosomes (EEs) (3). Interactions of KSHV with the integrin molecules induce the CIB1, p130Cas, and Crk adaptor molecules to the virus-containing LR bleb regions, phosphorylation of p130Cas, assembly of the c-Cbl-EphA2R-CIB1-Crk signalosome, and activation of GEF-C3G molecule to aid in macropinocytosis and trafficking (4). Knockdown of c-Cbl blocks KSHV LR translocation, EphA2R interaction, signal amplification, bleb formation, and macropinocytosis (12, 14). Live cell and confocal studies demonstrating the targeting of macropinosomes with the virus to the lysosomes in the absence of p130Cas indicate a role for the adaptor proteins for avoiding the lysosomes during macropinocytic entry in the endothelial cells (4) (Fig. 1).

KSHV UTILIZES THE HOST ESCRT PROTEIN COMPLEX FOR ENTERING AND POSTMACROPINOCYTIC TRAFFICKING IN INFECTED CELLS

The key questions are (i) how the zip codes (ubiquitination-traffic markers) added by c-Cbl are recognized, and (ii) how virus-containing macropinosome is properly trafficked in the cytoplasm. ESCRT proteins play crucial role by recognizing the ubiquitinated cargos destined for lysosomal degradation. The complex is comprised of ESCRT-0, -I, -II, and -III and the VPS4 group of proteins primarily involved in recognizing and sorting the ubiquitinated protein via the classical endocytic pathway comprised of early endosomes, maturing endosomes, late endosomes (LEs), and the endolysosome. The sequential assembly of these complexes on endosomal membrane is primed by ESCRT-0 recognizing the ubiquitinated proteins.

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). 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).

KSHV infection-induced Hrs localization to the plasma membrane initiates the association of the tumor susceptibility gene 101 (Tsg101), a component of ESCRT-I complex, with the ubiquitinated integrin–virus–EphA2R–c-Cbl-adaptor molecules (4). 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. 1). 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. 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).

The role of ESCRT complex proteins in the entry of other herpesviruses is not known at this time. However, studies demonstrate the utilization of ESCRT proteins in the viral egress stage of infection. For example, the Epstein-Barr Virus (EBV) recruits ESCRT components for efficient nuclear egress (17), while herpes simplex virus 1 (HSV-1) has been shown to utilize them for the cytoplasmic budding and not for nuclear egress (18). The human cytomegalovirus (HCMV) utilizes ESCRT proteins during its final stages of replication (19). The ESCRT machinery also plays a pivotal role in the egress of many enveloped retroviruses, such as HIV, equine infectious anemia virus, and murine leukemia virus (20).

OUTLOOK

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). 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. (i) How does the assembly of ESCRT proteins on the virus-containing endosomes prevent their interaction with lysosome? (ii) Do the Hrs-induced ROCK1-NHE1-mediated pH change and signal activated by KSHV binding and entry stages play roles in the transition of early to late endosome and the fusion of viral envelope with the endosomal membrane?

Overall, these studies revealed for the first time that c-Cbl and ESCRT proteins play a role in the macropinocytic and postmacropinocytic step of KSHV infection and suggest that KSHV infection-induced signal and adaptor molecules can serve as important targets for therapeutic interventions against KSHV infection.