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

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

KSHV is etiologically associated with Kaposi's 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 Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency, Inflammation, Angiogenesis, Cell Survival and Invasion

Kaposi's sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS

Nipah Virus: Past Outbreaks and Future Containment

Viral outbreaks of varying frequencies and severities have caused panic and havoc across the globe throughout history. Influenza, small pox, measles, and yellow fever reverberated for centuries, causing huge burden for economies. The twenty-first century witnessed the most pathogenic and contagious virus outbreaks of zoonotic origin including severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV) and Nipah virus. Nipah is considered one of the world’s deadliest viruses with the heaviest mortality rates in some instances. It is known to cause encephalitis, with cases of acute respiratory distress turning fatal. Various factors contribute to the onset and spread of the virus. All through the infected zone, various strategies to tackle and enhance the surveillance and awareness with greater emphasis on personal hygiene has been formulated. This review discusses the recent outbreaks of Nipah virus in Malaysia, Bangladesh and India, the routes of transmission, prevention and control measures employed along with possible reasons behind the outbreaks, and the precautionary measures to be ensured by private–public undertakings to contain and ensure a lower incidence in the future

Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry

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 EphA2's 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-Induced Angiogenin Plays Roles in Latency via the Phospholipase Cγ Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle▿ †

During de novo infection of human dermal microvascular endothelial cells (HMVEC-d), Kaposi's sarcoma-associated herpesvirus (KSHV) induced the multifunctional angiogenin (ANG) protein, which entered the nuclei and nucleoli of infected cells and stimulated 45S rRNA gene transcription, proliferation, and tube formation, which were inhibited by blocking ANG nuclear translocation with the antibiotic neomycin (S. Sadagopan et al., J. Virol. 83:3342-3364, 2009). ANG was induced by KSHV latency protein LANA-1 (open reading frame 73 [ORF73]). Here we examined the presence and functions of ANG in KSHV-positive (KSHV+) primary effusion lymphoma (PEL/BCBL) cells. Significant ANG gene expression and secretion were observed in KSHV+ (BCBL-1 and BC-3) and KSHV+ and Epstein-Barr virus-positive (KSHV+ EBV+) (JSC-1) PEL cells and in BJAB-KSHV cells but not in EBV− KSHV− lymphoma cells (Akata, Loukes, Ramos, and BJAB), EBV+ lymphoma cells (Akata-EBV and Raji), and cells from an EBV+ lymphoblastoid cell line, thus suggesting a specific association of ANG in KSHV biology. Inhibition of nuclear translocation of ANG resulted in reduced BCBL-1 and TIVE-LTC (latently infected endothelial) cell survival and proliferation, while EBV− and EBV+ Akata cells were unaffected. Blocking nuclear transport of ANG inhibited latent ORF73 gene expression and increased lytic switch ORF50 gene expression, both during de novo infection and in latently infected cells. A greater quantity of infectious KSHV was detected in the supernatants of neomycin-treated BCBL-1 cells than 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Neomycin treatment and ANG silencing inhibited phospholipase Cγ (PLC-γ) and AKT phosphorylation, and in contrast, ANG induced ORF73 expression and PLC-γ and AKT phosphorylation. Further studies provided evidence that blockage of PLC-γ activation by neomycin appears to be mediating the inhibition of latent gene expression, since treatment with the conventional PLC-γ inhibitor U73122 also showed similar results. Silencing of ANG also resulted in reduced cell survival, reduced ORF73 gene expression, and lytic gene activation in BCBL-1 and TIVE-LTC cells and during de novo infection. Taken together, these studies suggest that KSHV has evolved to exploit ANG for its advantage via a so-far-unexplored PLC-γ pathway for maintaining its latency

Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells▿

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes clathrin-mediated endocytosis for its infectious entry into human foreskin fibroblast (HFF) cells (S. M. Akula, P. P. Naranatt, N.-S. Walia, F.-Z. Wang, B. Fegley, and B. Chandran, J. Virol. 77:7978-7990, 2003). Here, we characterized KSHV entry into primary human microvascular dermal endothelial (HMVEC-d) and human umbilical vein endothelial (HUVEC) cells. Similar to the results for HMVEC-d cells, KSHV infection of HUVEC cells also resulted in an initial high level and subsequent decline in the expression of the lytic switch gene, ORF50, while latent gene expression persisted. Internalized virus particles enclosed in irregular vesicles were observed by electron microscopy of infected HMVEC-d cells. At an early time of infection, colocalization of KSHV capsid with envelope was observed by immunofluorescence analysis, thus demonstrating endocytosis of intact enveloped virus particles. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and filipin (C35H58O11), a caveolar endocytosis inhibitor, did not have any effect on KSHV binding, entry (DNA internalization), or gene expression in HMVEC-d and HUVEC cells. In contrast to the results for HFF cells, virus entry and gene expression in both types of endothelial cells were significantly blocked by macropinocytosis inhibitors (EIPA [5-N-ethyl-N-isoproamiloride] and rottlerin [C30H28O8]) and by cytochalasin D, which affects actin polymerization. Inhibition of lipid raft blocked viral gene expression in HMVEC-d cells but not in HUVEC or HFF cells. In HMVEC-d and HUVEC cells, KSHV induced the actin polymerization and formation of lamellipodial extensions that are essential for macropinocytosis. Inhibition of macropinocytosis resulted in the distribution of viral capsids at the HMVEC-d cell periphery, and capsids did not associate with microtubules involved in the nuclear delivery of viral DNA. Internalized KSHV in HMVEC-d and HUVEC cells colocalized with the macropinocytosis marker dextran and not with the clathrin pathway marker transferrin or with caveolin. Dynasore, an inhibitor of dynamin, did not block viral entry into endothelial cells but did inhibit entry into HFF cells. KSHV was not associated with the early endosome marker EEA-1 in HMVEC-d cells, but rather with the late endosome marker LAMP1, as well as with Rab34 GTPase that is known to regulate macropinocytosis. Silencing Rab34 with small interfering RNA dramatically inhibited KSHV gene expression. Bafilomycin-mediated disruption of endosomal acidification inhibited viral gene expression. Taken together, these findings suggest that KSHV utilizes the actin polymerization-dependent, dynamin-independent macropinocytic pathway that involves a Rab34 GTPase-dependent late endosome and low-pH environment for its infectious entry into HMVEC-d and HUVEC cells. These studies also demonstrate that KSHV utilizes different modes of endocytic entry in fibroblast and endothelial cells

Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained NF-κB Activation during De Novo Infection of Primary Human Dermal Microvascular Endothelial Cells That Is Essential for Viral Gene Expression

In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, and induction of several cytokines, growth factors, and angiogenic factors. Since NF-κB is a key molecule involved in the regulation of several of these factors, here, we examined NF-κB induction during de novo infection of HMVEC-d and HFF cells. Activation of NF-κB was observed as early as 5 to 15 min postinfection by KSHV, and translocation of p65-NF-κB into nuclei was detected by immunofluorescence assay, electrophoretic mobility shift assay, and p65 enzyme-linked immunosorbent assay. IκB phosphorylation inhibitor (Bay11-7082) reduced this activation significantly. A sustained moderate level of NF-κB induction was seen during the observed 72 h of in vitro KSHV latency. In contrast, high levels of ERK1/2 activation at earlier time points and a moderate level of activation at later times were observed. p38 mitogen-activated protein kinase was activated only at later time points, and AKT was activated in a cyclic manner. Studies with UV-inactivated KSHV suggested a role for virus entry stages in NF-κB induction and a requirement for KSHV viral gene expression in sustained induction. Inhibition of NF-κB did not affect target cell entry by KSHV but significantly reduced the expression of viral latent open reading frame 73 and lytic genes. KSHV infection induced the activation of several host transcription factors, including AP-1 family members, as well as several cytokines, growth factors, and angiogenic factors, which were significantly affected by NF-κB inhibition. These results suggest that during de novo infection, KSHV induces sustained levels of NF-κB to regulate viral and host cell genes and thus possibly regulates the establishment of latent infection