Sayan Chakraborty

Kaposi's Sarcoma-Associated Herpesvirus Latency in Endothelial and B Cells Activates Gamma Interferon-Inducible Protein 16-Mediated Inflammasomes

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) infections of endothelial and B cells are etiologically linked with Kaposis sarcoma (KS) and primary effusion B-cell lymphoma (PEL), respectively. KS endothelial and PEL B cells carry multiple copies of the nuclear episomal latent KSHV genome and secrete a variety of inflammatory cytokines, including interleukin-1β (IL-1β) and IL-18. The maturation of IL-1β and IL-18 depends upon active caspase-1, which is regulated by a multiprotein inflammasome complex induced by sensing of danger signals. During primary KSHV infection of endothelial cells, acting as a nuclear pattern recognition receptor, gamma interferon-inducible protein 16 (IFI16) colocalized with the KSHV genome in the nuclei and interacted with ASC and procaspase-1 to form a functional inflammasome (Kerur N et al., Cell Host Microbe 9:363-375, 2011). Here, we demonstrate that endothelial telomerase-immortalized human umbilical cells (TIVE) supporting KSHV stable latency (TIVE-LTC cells) and PEL (cavity-based B-cell lymphoma 1 [BCBL-1]) cells show evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β and pro-IL-18. Interaction of ASC with IFI16 but not with AIM2 or NOD-like receptor P3 (NLRP3) was detected. The KSHV latency-associated viral FLIP (vFLIP) gene induced the expression of IL-1β, IL-18, and caspase-1 mRNAs in an NF-κB-dependent manner. IFI16 and cleaved IL-1β were detected in the exosomes released from BCBL-1 cells. Exosomal release could be a KSHV-mediated strategy to subvert IL-1β functions. In fluorescent in situ hybridization analyses, IFI16 colocalized with multiple copies of the KSHV genome in BCBL-1 cells. IFI16 colocalization with ASC was also detected in lung PEL sections from patients. Taken together, these findings demonstrated the constant sensing of the latent KSHV genome by IFI16-mediated innate defense and unraveled a potential mechanism of inflammation induction associated with KS and PEL lesions.

Kaposi’s sarcoma-associated herpesvirus interacts with EphrinA2 receptor to amplify signaling essential for productive infection

Kaposi’s sarcoma-associated herpesvirus (KSHV), etiologically associated with Kaposi’s sarcoma, uses integrins (α3β1, αVβ3, and αVβ5) and associated signaling to enter human dermal microvascular endothelial cells (HMVEC-d), an in vivo target of infection. KSHV infection activated c-Cbl, which induced the selective translocation of KSHV into lipid rafts (LRs) along with the α3β1, αVβ3, and xCT receptors, but not αVβ5. LR-translocated receptors were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR–associated αVβ5 was polyubiquitinated, leading to clathrin-mediated entry that was targeted to lysosomes. Because the molecule(s) that integrate signal pathways and productive KSHV macropinocytosis were unknown, we immunoprecipitated KSHV-infected LR fractions with anti-α3β1 antibodies and analyzed them by mass spectrometry. The tyrosine kinase EphrinA2 (EphA2), implicated in many cancers, was identified in this analysis. EphA2 was activated by KSHV. EphA2 was also associated with KSHV and integrins (α3β1 and αVβ3) in LRs early during infection. Preincubation of virus with soluble EphA2, knockdown of EphA2 by shRNAs, or pretreatment of cells with anti-EphA2 monoclonal antibodies or tyrosine kinase inhibitor dasatinib significantly reduced KSHV entry and gene expression. EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. EphA2 shRNA ablated macropinocytosis-associated signaling events, virus internalization, and productive nuclear trafficking of KSHV DNA. Taken together, these studies demonstrate that the EphA2 receptor acts as a master assembly regulator of KSHV-induced signal molecules and KSHV entry in endothelial cells and suggest that the EphA2 receptor is an attractive target for controlling KSHV infection.

Interaction of c-Cbl with Myosin IIA Regulates Bleb Associated Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus

KSHV is etiologically associated with Kaposis sarcoma (KS), an angioproliferative endothelial cell malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that tyrosine phosphorylation of the adaptor protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the tyrosine phosphorylation of c-Cbl as early as 1 min post-infection and was recruited to the sites of bleb formation. Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl shRNA decreased the formation of KSHV induced membrane blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle myosin heavy chain IIA (myosin IIA), in bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-myosin light chain II in the interior of blebs of infected cells and this interaction was abolished by c-Cbl shRNA. Studies with the myosin II inhibitor blebbistatin demonstrated that myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of myosin IIA to the blebs during KSHV infection. Myosin II associates with actin in KSHV induced blebs and the absence of actin and myosin ubiquitination in c-Cbl ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and myosin IIA plays a crucial role in blebbing and macropinocytosis during viral infection and suggests that targeting c-Cbl could lead to a block in KSHV infection.

Kaposi’s Sarcoma Associated Herpesvirus Entry into Target Cells

Herpesvirus infection of target cells is a complex process involving multiple host cell surface molecules (receptors) and multiple viral envelope glycoproteins. Kaposi’s sarcoma associated herpesvirus (KSHV or HHV-8) infects a variety of in vivo target cells such as endothelial cells, B cells, monocytes, epithelial cells, and keratinocytes. KSHV also infects a diversity of in vitro target cells and establishes in vitro latency in many of these cell types. KSHV interactions with the host cell surface molecules and its mode of entry in the various target cells are critical for the understanding of KSHV pathogenesis. KSHV is the first herpesvirus shown to interact with adherent target cell integrins and this interaction initiates the host cell pre-existing signal pathways that are utilized for successful infection. This chapter discusses the various aspects of the early stage of KSHV infection of target cells, receptors used and issues that need to be clarified, and future directions. The various signaling events triggered by KSHV infection and the potential role of signaling events in the different stages of infection are summarized providing the framework and starting point for further detailed studies essential to fully comprehend the pathogenesis of KSHV.

Reactive oxygen species are induced by Kaposi's sarcoma-associated herpesvirus early during primary infection of endothelial cells to promote virus entry.

ABSTRACT The entry of Kaposis sarcoma-associated herpesvirus (KSHV) into human dermal microvascular endothelial cells (HMVEC-d), natural in vivo target cells, via macropinocytosis is initiated through a multistep process involving the binding of KSHV envelope glycoproteins with cell surface α3β1, αVβ3, and αVβ5 integrin molecules and tyrosine kinase ephrin-A2 receptor, followed by the activation of preexisting integrin-associated signaling molecules such as focal adhesion kinase (FAK), Src, c-Cbl, phosphoinositide 3-kinase (PI-3K), and Rho-GTPases. Many viruses, including KSHV, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells; however, the role of ROS in early events of viral entry and the induction of signaling has not been elucidated. Here we show that KSHV induced ROS production very early during the infection of HMVEC-d cells and that ROS production was sustained over the observation period (24 h postinfection). ROS induction was dependent on the binding of KSHV to the target cells, since pretreatment of the virus with heparin abolished ROS induction. Pretreatment of HMVEC-d cells with the antioxidant N-acetylcysteine (NAC) significantly inhibited KSHV entry, and consequently gene expression, without affecting virus binding. In contrast, H2O2 treatment increased the levels of KSHV entry and infection. In addition, NAC inhibited KSHV infection-induced translocation of αVβ3 integrin into lipid rafts, actin-dependent membrane perturbations, such as blebs, observed during macropinocytosis, and activation of the signal molecules ephrin-A2 receptor, FAK, Src, and Rac1. In contrast, H2O2 treatment increased the activation of ephrin-A2, FAK, Src, and Rac1. These studies demonstrate that KSHV infection induces ROS very early during infection to amplify the signaling pathways necessary for its efficient entry into HMVEC-d cells via macropinocytosis.

Angiogenin functionally interacts with p53 and regulates p53-mediated apoptosis and cell survival

Angiogenin, a 14-kDa multifunctional pro-angiogenic growth factor, is upregulated in several types of cancers. Anti-angiogenin monoclonal antibodies used as antagonists inhibited the establishment, progression and metastasis of human cancer cells in athymic mice (Olson et al., 1994). Silencing angiogenin and inhibition of angiogenins nuclear translocation blocked cell survival and induced cell death in B-lymphoma and endothelial cells latently infected with Kaposi sarcoma-associated herpesvirus (Sadagopan et al., 2009), suggesting that actively proliferating cancer cells could be inducing angiogenin for inhibiting apoptotic pathways. However, the mechanism of cell survival and apoptosis regulation by angiogenin and their functional significance in cancer is not known. We demonstrate that angiogenin interacts with p53 and colocalizes in the nucleus. Silencing endogenous angiogenin induced p53 promoter activation and p53 target gene (p53, p21 and Bax) expression, downregulated anti-apoptotic Bcl-2 gene expression and increased p53-mediated cell death. In contrast, angiogenin expression blocked pro-apoptotic Bax and p21 expression, induced Bcl-2 and blocked cell death. Angiogenin also co-immunoprecipitated with p53 regulator protein Mdm2. Angiogenin expression resulted in the inhibition of p53 phosphorylation, increased p53–Mdm2 interaction, and consequently increased ubiquitination of p53. Taken together, these studies demonstrate that angiogenin promotes the inhibition of p53 function to mediate anti-apoptosis and cell survival. Our results reveal for the first time a novel p53 interacting function of angiogenin in anti-apoptosis and survival of cancer cells and suggest that targeting angiogenin could be an effective therapy for several cancers.

c-Cbl-Mediated Selective Virus-Receptor Translocations into Lipid Rafts Regulate Productive Kaposi's Sarcoma-Associated Herpesvirus Infection in Endothelial Cells

ABSTRACT During target cell entry and infection, many enveloped and nonenveloped viruses utilize cell surface receptors that translocate into lipid rafts (LRs). However, the mechanism behind this translocation is not known. Kaposis sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3β1, αVβ3, and αVβ5, and the amino acid transporter x-CT protein and enters via c-Cbl-bleb-mediated macropinocytosis (Veettil et al., J. Virol. 82:12126-12144, 2008; Veettil et al., PLoS Pathog. 6:e1001238, 2010). Here we have demonstrated that very early during infection (1 min postinfection), c-Cbl induced the selective translocation of KSHV into the LR along with the α3β1, αVβ3, and x-CT receptors but not αVβ5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3β1 and αVβ3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVβ5 was polyubiquitinated, leading to clathrin entry that was targeted to lysosomes. c-Cbl knockdown blocked the macropinocytosis and receptor translocation and diverted KSHV to a clathrin-lysosomal noninfectious pathway. Similar results were also seen by LR disruption with MβCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3β1, -αVβ3, and -x-CT receptor translocations into the LRs and differential ubiquitination of receptors which are critical determinants of the macropinocytic entry route and productive infection of KSHV. Our studies suggest that interventions targeting c-Cbl and LRs are potential avenues to block KSHV infection of endothelial cells.

EphrinA2 regulates clathrin mediated KSHV endocytosis in fibroblast cells by coordinating integrin-associated signaling and c-Cbl directed polyubiquitination.

Kaposis sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2s tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.

CIB1 synergizes with EphrinA2 to regulate Kaposi's sarcoma-associated herpesvirus macropinocytic entry in human microvascular dermal endothelial cells.

KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2s association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Multifunctional Angiogenin To Utilize Its Antiapoptotic Functions

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative Kaposis sarcoma (KS). KSHV infection and the expression of latency-associated nuclear antigen (LANA-1) upregulates the angiogenic multifunctional 123-amino-acid, 14-kDa protein angiogenin (ANG), which is detected in KS lesions and in KSHV-associated primary effusion lymphoma (PEL) cells. ANG knockdown or the inhibition of ANGs nuclear translocation resulted in decreased LANA-1 gene expression and reduced KSHV-infected endothelial and PEL cell survival (Sadagopan et al., J. Virol. 83:3342–3364, 2009). Further studies here demonstrate that LANA-1 and ANG colocalize and coimmunoprecipitate in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and BC-3) cells. LANA-1 and ANG interaction occurred in the absence of the KSHV genome and other viral proteins. In gel filtration chromatography analyses of BC-3 cell lysates, ANG coeluted with LANA-1, p53, and Mdm2 in high-molecular-weight fractions, and LANA-1, p53, and Mdm2 also coimmunoprecipitated with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected cells, and colocalization between ANG and p53 was also observed in LANA-1-negative cells. The deletion constructs of ANG suggested that the C-terminal region of amino acids 104 to 123 is involved in LANA-1 and p53 interactions. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG-LANA-1, ANG-p53, and LANA-1-p53, the induction of p53, p21, and Bax proteins, the increased cytoplasmic localization of p53, the downregulation of Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. No such effects were observed in KSHV-negative BJAB cells. The phosphorylation of p53 at serine 15, which is essential for p53 stabilization and for p53s apoptotic and cell cycle regulation functions, was increased in BCBL-1 cells transduced with short hairpin RNA targeting ANG. Together, these studies suggest that the antiapoptosis observed in KSHV-infected cells and the suppression of p53 functions are mediated in part by ANG, and KSHV has probably evolved to utilize angiogenins multiple functions for the maintenance of its latency and cell survival. Thus, targeting ANG to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV infection and associated malignancies.