Shaw M. Akula

Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Infection of Human Fibroblast Cells Occurs through Endocytosis

ABSTRACT Kaposis sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the α3β1 integrin via its glycoprotein gB, and virus binding studies suggest that α3β1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH4Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH4Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.

Kaposi's Sarcoma-Associated Herpesvirus Induces the Phosphatidylinositol 3-Kinase-PKC-ζ-MEK-ERK Signaling Pathway in Target Cells Early during Infection: Implications for Infectivity

ABSTRACT Human herpesvirus 8 (HHV-8) is implicated in the pathogenesis of Kaposis sarcoma. HHV-8 envelope glycoprotein B (gB) possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, by antibodies against α3 and β1 integrins, and by soluble α3β1 integrin (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Anti-gB antibodies immunoprecipitated the virus α3 and β1 complexes, and virus-binding studies suggest a role for α3β1 in HHV-8 entry. HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK), implicating a role for integrin and the associated signaling pathways in HHV-8 entry into the target cells. Immediately after infection, target cells exhibited morphological changes and cytoskeletal rearrangements, suggesting the induction of signal pathways. As early as 5 min postinfection, HHV-8 activated the MEK-ERK1/2 pathway. The focal adhesion components phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C-ζ (PKC-ζ) were recruited as upstream mediators of the HHV-8-induced ERK pathway. Anti-HHV-8 gB-neutralizing antibodies and soluble α3β1 integrin inhibited the virus-induced signaling pathways. Early kinetics of the cellular signaling pathway and its activation by UV-inactivated HHV-8 suggest a role for virus binding and/or entry but not viral gene expression in this induction. Studies with human α3 integrin-transfected Chinese hamster ovary cells and FAK-negative mouse DU3 cells suggest that the α3β1 integrin and FAK play roles in the HHV-8 mediated signal induction. Inhibitors specific for PI 3-kinase, PKC-ζ, MEK, and ERK significantly reduced the virus infectivity without affecting virus binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in HHV-8 entry into the target cells and a role for PKC-ζ, MEK, and ERK at a post-viral entry stage of infection. These findings implicate a critical role for integrin-associated mitogenic signaling in HHV-8s infection of target cells and suggest that, by orchestrating the signal cascade, HHV-8 may create an appropriate intracellular environment to facilitate the infection.

Human Herpesvirus 8 Envelope Glycoprotein B Mediates Cell Adhesion via Its RGD Sequence

ABSTRACT Human herpesvirus 8 (HHV-8) or Kaposis sarcoma-associated herpesvirus, implicated in the pathogenesis of Kaposis sarcoma, utilizes heparan sulfate-like molecules to bind the target cells via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8-gB possesses the Arg-Gly-Asp (RGD) motif, the minimal peptide region of many proteins known to interact with subsets of host cell surface integrins. HHV-8 utilizes α3β1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Since FAK activation is the first step in the outside-in signaling necessary for integrin-mediated cytoskeletal rearrangements, cell adhesions, motility, and proliferation, the ability of HHV-8-gB to mediate the target cell adhesion was examined. A truncated form of gB without the transmembrane and carboxyl domains (gBΔTM) and a gBΔTM mutant (gBΔTM-RGA) with a single amino acid mutation (RGD to RGA) were expressed in a baculovirus system and purified. Radiolabeled HHV-8-gBΔTM, gBΔTM-RGA, and ΔTMgpK8.1A proteins bound to the human foreskin fibroblasts (HFFs), human dermal microvascular endothelial (HMVEC-d) cells, human B (BJAB) cells, and Chinese hamster ovary (CHO-K1) cells with equal efficiency, which was blocked by preincubation of proteins with soluble heparin. Maxisorp plate-bound gBΔTM protein induced the adhesion of HFFs and HMVEC-d and monkey kidney epithelial (CV-1) cells in a dose-dependent manner. In contrast, the gBΔTM-RGA and ΔTMgpK8.1A proteins did not mediate adhesion. Adhesion mediated by gBΔTM was blocked by the preincubation of target cells with RGD-containing peptides or by the preincubation of plate-bound gBΔTM protein with rabbit antibodies against gB peptide containing the RGD sequence. In contrast, adhesion was not blocked by the preincubation of plate-bound gBΔTM protein with heparin, suggesting that the adhesion is mediated by the RGD amino acids of gB, which is independent of the heparin-binding domain of gB. Integrin-ligand interaction is dependent on divalent cations. Adhesion induced by the gBΔTM was blocked by EDTA, thus suggesting the role of integrins in the observed adhesions. Focal adhesion components such as FAK and paxillin were activated by the binding of gBΔTM protein to the target cells but not by gBΔTM-RGA protein binding. Inhibition of FAK phosphorylation by genistein blocked gBΔTM-induced FAK activation and cell adhesion. These findings suggest that HHV-8-gB could mediate cell adhesion via its RGD motif interaction with the cell surface integrin molecules and indicate the induction of cellular signaling pathways, which may play roles in the infection of target cells and in Kaposis sarcoma pathogenesis.

Increased protein expression of the PTEN tumor suppressor in the presence of constitutively active Notch-1.

Mammalian Notch-1 is part of an evolutionarily conserved family of transmembrane receptorsbest known for involvement in cell fate decisions. Mutations that result in Notch-1 activation result inT-lineage oncogenesis. In other cell lineages, however, studies have indicated that cooperation withcellular signaling pathways, such as Ras, is necessary for Notch-mediated oncogenesis and in somesettings, Notch-1 has been reported to function as a tumor suppressor. In order to test the hypothesisthat the Notch-1 pathway exhibits cross-talk with Ras/Raf/MEK/ERK, the constitutively activecytoplasmic portion of Notch-1 was introduced into 293 HEK fibroblasts via retroviral transduction.ERK-1,-2 activation was markedly increased in cells expressing constitutively active Notch-1. Thesecells exhibited a more rounded morphology as compared to 293 cells transduced with an empty vectoror parental 293 cells. These observations correlated with decreased total and phosphorylated focaladhesion kinase protein (FAK). Subsequent examination of phosphatase and tensin homolog deletedon chromosome 10 (PTEN) revealed that total and phosphorylated PTEN protein was elevated in cellsexpressing constitutively active Notch-1. Loss of Akt phosphorylation was also observed in cellsbearing activated Notch-1. Two potential binding sites for the Notch effector CBF-1 were identified inthe human PTEN promoter sequence. A PTEN promoter luciferase reporter exhibited increasedactivity in the presence of Notch-1 signaling. These data indicate that Notch-1 can participate incross-talk with other signaling pathways such as Ras/Raf/MEK/ERK through the regulation of thePTEN tumor suppressor.

Raf promotes human herpesvirus-8 (HHV-8/KSHV) infection.

Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposis sarcoma (KS) and other tumors. Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway has been associated with a variety of tumors, including AIDS-related KS. The oncoprotein Raf is situated at a pivotal position in regulating the MAPK pathway. Hence, we analysed the effect of oncoprotein Raf on HHV-8 infectious entry into target cells. Here we report Raf expression to significantly enhance HHV-8 infection of target cells. These findings implicate a role for Raf not only in the infectious entry of HHV-8 but also in modulating KS pathogenesis.

Raf-Induced Vascular Endothelial Growth Factor Augments Kaposi's Sarcoma-Associated Herpesvirus Infection

ABSTRACT Recombinant green fluorescent protein encoding Kaposis sarcoma-associated herpesvirus (rKSHV.152) infection of β-estradiol stimulated human foreskin fibroblasts (HFF) or HFF/ΔB-Raf[FF]:ER (expressing a weaker form of B-Raf) could be enhanced to levels comparable to that of HFF/ΔB-Raf[DD]:ER cells by pretreating cells with soluble vascular endothelial growth factor (VEGF). Conversely, VEGF expression and infection efficiency typically observed in β-estradiol stimulated HFF/ΔB-Raf[DD]:ER cells could be lowered significantly by treating with VEGF small interfering RNA. In addition, we observed enhancement of the KSHV infection in HFF cells transfected with human VEGF121. These results confirm the ability of Raf-induced VEGF to augment KSHV infection of cells.

Vascular endothelial growth factor augments human herpesvirus-8 (HHV-8/KSHV) infection.

Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposi’s sarcoma (KS) and other tumors. The Raf oncoprotein enhances HHV-8 infection of target cells. In addition, we have previously demonstrated that Raf induces vascular endothelial growth factor (VEGF) expression. VEGF is a growth factor that has autocrine growth activity and has been implicated in the formation of the spindle shape cell morphology characteristic of Kaposi’s sarcoma (KS). The aim of this study was to test the hypothesis that VEGF enhances infection of HHV-8. Herein, we demonstrate that the soluble VEGF enhanced green florescence protein encoding (GFP)-HHV-8 (rKSHV.152) infection of human foreskin fibroblasts (HFF) and not of 293 cells. We found this to be in part, due to the fact that HFF inherently produces significantly lower concentrations of VEGF when compared to 293 cells. Treating 293 cells (but not HFF) with a VEGF receptor (VEGFR) inhibitor significantly lowered infection. Furthermore, transfecting 293 cells with VEGF specific si-RNA did not alter the binding of HHV-8 to cells; but significantly lowered VEGF expression and thus GFP-HHV-8 infection. Interestingly, lowering VEGF expression in 293 cells wtih VEGF specific si-RNA did not completely inhibit GFP-HHV-8 infection. We conclude that VEGF is not a requirement for HHV-8 infection; but VEGF plays a major role in augmenting infection at a post binding stage of entry. These findings suggest that targeting VEGF/VEGFR may prove efficacious in controlling HHV-8 associated pathogenesis.

Disturbed Homeostasis and Multiple Signaling Defects in the Peripheral Blood B-Cell Compartment of Patients with Severe Chronic Sarcoidosis

ABSTRACT The presence of hypergammaglobulinemia, autoantibodies, and circulating immune complexes suggests that humoral immunity may contribute to the pathogenesis of sarcoidosis. However, little is known about the role played by B cells in the development of this disease. Here we investigated the subpopulation distribution, response to stimulation, and levels of the nuclear transcription factor NF-κB/p65 in peripheral blood B cells from patients with severe chronic sarcoidosis. Patients with severe chronic sarcoidosis had absolute B-cell lymphopenia and exhibited significantly decreased frequencies and total numbers of memory (CD19+ CD27+) B cells. The reduced numbers of memory B cells in these patients reflected a decrease in the total numbers of class-switched (CD19+ CD27+ IgD−) and unswitched (CD19+ CD27+ IgD+) memory B cells and coincided with an increased frequency of circulating (CD19+/− CD20− CD27++) plasmablasts. Polyclonal stimulation of sarcoid B cells resulted in reduced expression of activation markers (i.e., CD25, CD69, and CD86), decreased proliferation, and impaired plasma cell differentiation. Baseline expression of p65 in B cells was reduced in 65% of the patients. These results suggest disturbed homeostasis, intrinsic signaling defects, and anergy within the peripheral B-cell compartments of patients with severe chronic sarcoidosis.

Targeting the PI3K and MAPK pathways to treat Kaposi’s sarcoma-associated herpes virus infection and pathogenesis

Cells require the ability to appropriately respond to signals in their extracellular environment. To initiate, inhibit and control these processes, the cell has developed a complex network of signaling cascades. The phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways regulate several responses including mitosis, apoptosis, motility, proliferation, differentiation and many others. It is not surprising, therefore, that many viruses target the PI3K and MAPK pathways as a means to manipulate cellular function. Recently, Kaposi’s sarcoma-associated herpes virus (KSHV) has been added to the list. KSHV manipulates the PI3K and MAPK pathways to control such divergent processes as cell survival, cellular migration, immune responses, and to control its own reactivation and lytic replication. Manipulation of the PI3K and MAPK pathways also plays a role in malignant transformation. Here, the authors review the potential to target the PI3K and MAPK signaling pathways to inhibit KSHV infection and pathogenesis.

Disintegrin-like domain of glycoprotein B regulates Kaposi's sarcoma-associated herpesvirus infection of cells.

Kaposis sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoproteins interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.