Neelam Sharma-Walia

IFI16 Acts as a Nuclear Pathogen Sensor to Induce the Inflammasome in Response to Kaposi Sarcoma-Associated Herpesvirus Infection

Inflammasomes are cytoplasmic sensors of foreign molecules, including pathogens, and function to induce caspase-1 activation and IL-1β cytokine maturation. Whether such a mechanism exists in the nucleus and is effective against nuclear replicating pathogens is unknown. Nuclear replicating herpesvirus KSHV is associated with Kaposi Sarcoma, an angioproliferative tumor characterized by an inflammatory microenvironment including IL-1β. We demonstrate that during KSHV infection of endothelial cells, interferon gamma-inducible protein 16 (IFI16) interacts with the adaptor molecule ASC and procaspase-1 to form a functional inflammasome. This complex was initially detected in the nucleus and subsequently in the perinuclear area. KSHV gene expression and/or latent KSHV genome is required for inflammasome activation and IFI16 colocalizes with the KSHV genome in the infected cell nucleus. Caspase-1 activation by KSHV was reduced by IFI16 and ASC silencing. Our studies reveal IFI16 as a nuclear pathogen sensor and demonstrate that the inflammasome also functions in the nucleus.

ERK1/2 and MEK1/2 Induced by Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Early during Infection of Target Cells Are Essential for Expression of Viral Genes and for Establishment of Infection

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) in vitro target cell infection is characterized by the expression of the latency-associated genes ORF 73 (LANA-1), ORF 72, and K13 and by the transient expression of a very limited number of lytic genes such as lytic cycle switch gene ORF 50 (RTA) and the immediate early (IE) lytic K5, K8, and v-IRF2 genes. During the early stages of infection, several overlapping multistep complex events precede the initiation of viral gene expression. KSHV envelope glycoprotein gB induces the FAK-Src-PI3K-RhoGTPase (where FAK is focal adhesion kinase) signaling pathway. As early as 5 min postinfection (p.i.), KSHV induced the extracellular signal-regulated kinase 1 and 2 (ERK1/2) via the PI3K-PKCζ-MEK pathway. In addition, KSHV modulated the transcription of several host genes of primary human dermal microvascular endothelial cells (HMVEC-d) and fibroblast (HFF) cells by 2 h and 4 h p.i. Neutralization of virus entry and infection by PI-3K and other cellular tyrosine kinase inhibitors suggested a critical role for signaling molecules in KSHV infection of target cells. Here we investigated the induction of ERK1/2 by KSHV and KSHV envelope glycoproteins gB and gpK8.1A and the role of induced ERK in viral and host gene expression. Early during infection, significant ERK1/2 induction was observed even with low multiplicity of infection of live and UV-inactivated KSHV in serum-starved cells as well as in the presence of serum. Entry of UV-inactivated virus and the absence of viral gene expression suggested that ERK1/2 induction is mediated by the initial signal cascade induced by KSHV binding and entry. Purified soluble gpK8.1A induced the MEK1/2 dependent ERK1/2 but not ERK5 and p38 mitogen-activated protein kinase (MAPK) in HMVEC-d and HFF. Moderate ERK induction with soluble gB was seen only in HMVEC-d. Preincubation of gpK8.1A with heparin or anti-gpK8.1A antibodies inhibited the ERK induction. U0126, a selective inhibitor for MEK/ERK blocked the gpK8.1A- and KSHV-induced ERK activation. ERK1/2 inhibition did not block viral DNA internalization and had no significant effect on nuclear delivery of KSHV DNA during de novo infection. Analyses of viral gene expression by quantitative real-time reverse transciptase PCR revealed that pretreatment of cells with U0126 for 1 h and during the 2-h infection with KSHV significantly inhibited the expression of ORF 73, ORF 50 (RTA), and the IE-K8 and v-IRF2 genes. However, the expression of lytic IE-K5 gene was not affected significantly. Expression of ORF 73 in BCBL-1 cells was also significantly inhibited by preincubation with U0126. Inhibition of ERK1/2 also inhibited the transcription of some of the vital host genes such as DUSP5 (dual specificity phosphatase 5), ICAM-1 (intercellular adhesion molecule 1), heparin binding epidermal growth factor, and vascular endothelial growth factor that were up-regulated early during KSHV infection. Several MAPK-regulated host transcription factors such as c-Jun, STAT1α, MEF2, c-Myc, ATF-2 and c-Fos were induced early during infection, and ERK inhibition significantly blocked the c-Fos, c-Jun, c-Myc, and STAT1α activation in the infected cells. AP1 transcription factors binding to the RTA promoter in electrophoretic mobility shift assays were readily detected in the infected cell nuclear extracts which were significantly reduced by ERK inhibition. Together, these results suggest that very early during de novo infection, KSHV induces the ERK1/2 to modulate the initiation of viral gene expression and host cell genes, which further supports our hypothesis that beside the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with host cell receptor(s) create an appropriate intracellular environment facilitating infection.

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Envelope Glycoprotein gB Induces the Integrin-Dependent Focal Adhesion Kinase-Src-Phosphatidylinositol 3-Kinase-Rho GTPase Signal Pathways and Cytoskeletal Rearrangements

ABSTRACT Human herpesvirus 8 (HHV-8; Kaposis sarcoma-associated herpesvirus) envelope glycoprotein gB possesses an RGD motif, interacts with α3β1 integrin, and uses it as one of the entry receptors. HHV-8 induces the integrin-dependent focal adhesion kinase (FAK), a critical step in the outside-in signaling pathways necessary for the subsequent phosphorylation of other cellular kinases, cytoskeletal rearrangements, and other functions. As an initial step toward deciphering the role of HHV-8 gB-integrin interaction in infection, signal pathways induced by gB were examined. A truncated form of gB without the transmembrane and carboxyl domains (gBΔTM), a gBΔTM mutant form (gBΔTM-RGA) with an RGD-to-RGA mutation, and inhibitors of cellular kinases were used. HHV-8 gBΔTM, but not gBΔTM-RGA, induced FAK phosphorylation in target cells, which was in part dependent on the presence of α3β1 integrin. FAK was critical for the subsequent phosphorylation of Src by gBΔTM, and Src induction was essential for the phosphorylation of phosphatidylinositol 3-kinase (PI-3K). HHV-8 gBΔTM-induced PI-3K was essential for the induction of RhoA and Cdc42 Rho GTPases that was accompanied by the cytoskeletal rearrangements. These gB-induced morphological changes were inhibited by the PI-3K inhibitors. Ezrin, one of the essential elements required to cross-link the actin cytoskeleton with the plasma membrane and to induce the morphological changes, was induced by the Rho GTPases. Inhibition of cellular tyrosine kinases by the brief treatment of cells with 4′,5,7-trihydroxyisoflavone (genistein) blocked the entry of HHV-8 into target cells. These findings suggest that, independently of other viral glycoproteins and via its RGD motif, HHV-8 gB induces integrin-dependent pre-existing FAK-Src-PI-3K-Rho GTPase kinases. Since these signal pathways play vital roles in host cell endocytosis and movement of particulate materials in the cytoplasm, the early stages of HHV-8 gB interaction with host cells may provide a very conducive environment for the successful infection of target cells.

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

ABSTRACT Kaposis 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.

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.

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

Kaposis 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.

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

ABSTRACT In vitro Kaposis 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.

RhoA-GTPase Facilitates Entry of Kaposi's Sarcoma-Associated Herpesvirus into Adherent Target Cells in a Src-Dependent Manner

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8) binds to adherent target cell surface heparan sulfate molecules via its envelope glycoproteins gB and gpK8.1A, to integrins via gB, to the transporter CD98/xCT complex, and possibly to another molecule(s). This is followed by virus entry overlapping with the induction of preexisting host cell signal pathways, such as focal adhesion kinase, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, protein kinase C-ζ, and extracellular signal-regulated kinase 1/2. Here, using hemagglutinin-tagged plasmids expressing wild-type, dominant-positive, and dominant-negative forms of RhoA in HEK (human embryonic kidney) 293 cells, we investigated the role of RhoA-GTPase in virus entry. The dominant-negative form of RhoA GTPase and treatment of target cells with Clostridium difficile toxin B (CdTxB), a specific inactivator of Rho-GTPases, significantly blocked KSHV entry. KSHV infection induced closely similar levels of FAK and PI3-K in all three cell types. In contrast, very strong Src activation was observed in KSHV-infected dominant-positive RhoA cells compared to wild-type cells, and only moderate Src activation was seen in dominant-negative cells. Inhibition of Src activation by CdTxB and reduction of RhoA activation by Src inhibitors suggest that KSHV-induced Src is involved in RhoA activation, which in turn is involved in a feedback-sustained activation of Src. Since the decreased entry in RhoA dominant-negative cells may be due to inefficient signaling downstream of RhoA, we examined the induction of RhoA-activated Dia-2, which is also known to induce Src. Dia-2 coimmunoprecipitated with activated Src, which was inhibited by Src inhibitors, in the infected cells. Together with the reduced virus entry in RhoA dominant-negative cells, these results suggest that activated RhoA-dependent Dia-2 probably functions as a link between RhoA and Src in KSHV-infected cells, mediating the sustained Src activation, and that KSHV-induced Src and RhoA play roles in facilitating entry into adherent target cells.

Focal Adhesion Kinase Is Critical for Entry of Kaposi's Sarcoma-Associated Herpesvirus into Target Cells

ABSTRACT Kaposis sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface α3β1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK−/−) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK−/−) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.

Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and α3β1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with α3β1 and αVβ3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-αV and -β1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against αVβ3 and αVβ5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble α3β1, αVβ3, and αVβ5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that αVβ3 and αVβ5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin αVβ5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas α3β1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and αVβ3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of αVβ5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with α3β1 and KSHV. Preincubation of KSHV with soluble heparin and α3β1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (αVβ3, α3β1, and αVβ5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.