Antibacterial efficacy of lytic phages against multidrug-resistant Pseudomonas aeruginosa infections in bacteraemia mice models.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that can cause a variety of infections in humans, such as burn wound infections and infections of the lungs, the bloodstream and surgical site infections. Nosocomial spread is often concurrent with high degrees of antibiotic resistance. Such resistant strains are difficult to treat, and in some cases, even reserved antibiotics are ineffective. A particularly promising therapy to combat infections of resistant bacteria is the deployment of bacteriophages, known as phage therapy. In this work, we evaluated the in vivo efficacy of two Pseudomonas phages in bacteremia mice models. For this study, non-neutropenic mice (BalB/C) were infected with P. aeruginosa AB030 strain and treated using two bacteriophages, AP025 and AP006. RESULTS: The results showed that a single dose of phages at higher concentrations, bacteria: phage at 1:10 and 1:100 were effective in eliminating the bloodstream infection and achieving 100% mice survival. CONCLUSION: This study highlights the efficacy of using a single dose of phages to restore mice from bacteremia.

Safety Assessment of Ubiquinol Acetate: Subchronic Toxicity and Genotoxicity Studies.

Coenzyme Q10 (CoQ10) is a lipid soluble, endogenous antioxidant present at highest levels in the heart followed by the kidney and liver. The reduced CoQ10 ubiquinol is well known for its chemical instability and low bioavailability. The present study was designed to synthesize ubiquinol acetate, which is more stable and biologically active, and further evaluate its safety and genotoxic potential. Synthesized ubiquinol acetate showed better stability than that of ubiquinol at the end of 3 months. In vitro genotoxicity studies (AMES test, in vitro micronucleus and chromosomal aberration) showed ubiquinol acetate as nongenotoxic with no clastogenic or aneugenic effects at high dose of 5000 and 62.5 µg/mL, respectively. In subchronic toxicity study, ubiquinol acetate was administered orally to Sprague Dawley rats at 150, 300, and 600 mg/kg/day for 90 days. No treatment related adverse effects were observed in males at 600 mg/kg/day; however, females showed treatment related increase in AST and ALT with small focal irregular white-yellow spots in liver on gross necropsy examination. Histopathological evaluation revealed hepatocellular necrosis in high dose females which was considered as adverse. Based on the results, the No-Observed-Adverse-Effect Level (NOAEL) of ubiquinol acetate in males and females was determined as 600 and 300 mg/kg/day, respectively.

Zebrafish as an in vivo high-throughput model for genotoxicity.

Compounds routinely used to increase the quality of life and combat disease undergo stringent potency and biosafety tests before approval. However, based on the outcome of ongoing research, new norms need to be effected to ensure that the compounds conform to biosafety at all target levels of activity. Whereas in vitro tests used to assess biosafety lack the potency and the translational attribute of a whole animal, mammalian preclinical models are expensive and time exhaustive. Zebrafish (Danio rerio) has emerged as an attractive alternative for biosafety studies due to its small size, genetics, breeding capabilities, and most importantly, similarity at the molecular and physiological levels with humans. It has been used extensively for testing various forms of toxicity, including developmental toxicity, cardiotoxicity, nephrotoxicity, and hepatotoxicity. We review here the utility of zebrafish as a powerful, sensitive, quantitative, noninvasive, and high-throughput whole-animal assay to screen for toxicity. Different forms of toxicity will be discussed briefly before we highlight the present state of genotoxicity study in zebrafish. This review, a first in this research area, will serve as a comprehensive introduction to the field of genotoxicity assay using zebrafish, a nascent but promising field that assays compounds for DNA damage. We also discuss possible approaches that could potentially be pursued to overcome some of the shortcomings in current genotoxic studies.

Validation of a human cell based high-throughput genotoxicity assay 'Anthem's Genotoxicity screen' using ECVAM recommended lists of genotoxic and non-genotoxic chemicals.

A novel high throughput-enabled human cell based screen, Anthem's Genotoxicity screen, was developed to achieve higher specificity for predicting in vivo genotoxins by an in vitro method. The assay employs engineered human colon carcinoma cell line; HCT116 cells that are stably engineered with three promoter-reporter cassettes such that an increased reporter activity reflects the activation of associated signaling events in a human cell. The current study focuses on the evaluation of sensitivity and specificity of Anthem's Genotoxicity screen using 62 compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM). The concordance of Anthem's Genotoxicity screen with in vivo tests was 95.5% with sensitivity of 95.2% and specificity of 95.7%. Thus Anthem's Genotoxicity screen, a high-throughput mechanism based genotox indicator test can be employed by a variety of industries for rapid screening and early detection of potential genotoxins.

Kaposi's sarcoma-associated herpesvirus-positive primary effusion lymphoma tumor formation in NOD/SCID mice is inhibited by neomycin and neamine blocking angiogenin's nuclear translocation.

Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV(+) PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with multifunctional angiogenin to utilize its antiapoptotic functions.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative Kaposi's 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 ANG's 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 p53's 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 angiogenin's 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.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen and angiogenin interact with common host proteins, including annexin A2, which is essential for survival of latently infected cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection and latency-associated nuclear antigen (LANA-1) upregulate the multifunctional protein angiogenin (ANG). Our studies demonstrate that silencing ANG or inhibiting its nuclear translocation downregulates KSHV LANA-1 expression and ANG is necessary for KSHV latency, anti-apoptosis and angiogenesis (Sadagopan et al., J. Virol. 83:3342-3364, 2009; Sadagopan et al., J Virol. 85:2666-2685, 2011). Here we show that LANA-1 interacts with ANG and colocalizes in latently infected endothelial telomerase-immortalized human umbilical vein endothelial (TIVE-LTC) cells. Mass spectrometric analyses of TIVE-LTC proteins immunoprecipitated by anti-LANA-1 and ANG antibodies identified 28 common cellular proteins such as ribosomal proteins, structural proteins, tRNA synthetases, metabolic pathway enzymes, chaperons, transcription factors, antioxidants, and ubiquitin proteosome proteins. LANA-1 and ANG interaction with one of the proteins, annexin A2, was validated. Annexin A2 has been shown to play roles in cell proliferation, apoptosis, plasmin generation, exocytosis, endocytosis, and cytoskeleton reorganization. It is also known to associate with glycolytic enzyme 3-phosphoglyceratekinase in the primer recognition protein (PRP) complex that interacts with DNA polymerase α in the lagging strand of DNA during replication. A higher level of annexin A2 is expressed in KSHV+ but not in Epstein-Barr virus (EBV)+ B-lymphoma cell lines. Annexin A2 colocalized with several LANA-1 punctate spots in KSHV+ body cavity B-cell lymphoma (BCBL-1) cells. In triple-staining analyses, we observed annexin A2-ANG-LANA-1, annexin A2-ANG, and ANG-LANA-1 colocalizations. Annexin A2 appeared as punctate nuclear dots in LANA-1-positive TIVE-LTC cells. In LANA-1-negative TIVE-LTC cells, annexin A2 was detected predominately in the cytoplasm, with some nuclear spots, and colocalization with ANG was observed mostly in the cytoplasm. Annexin A2 coimmunoprecipitated with LANA-1 and ANG in TIVE-LTC and BCBL-1 cells and with ANG in 293T cells independent of LANA-1. This suggested that annexin A2 forms a complex with LANA-1 and ANG as well as a separate complex with ANG. Silencing annexin A2 in BCBL-1 cells resulted in significant cell death, downregulation of cell cycle-associated Cdk6 and of cyclin D, E, and A proteins, and downregulation of LANA-1 and ANG expression. No effect was seen in KSHV⁻ lymphoma (BJAB and Ramos) and 293T cells. These studies suggest that LANA-1 association with annexin A2/ANG could be more important than ANG association with annexin A2, and KSHV probably uses annexin A2 to maintain the viability and cell cycle regulation of latently infected cells. Since the identified LANA-1- and ANG-interacting common cellular proteins are hitherto unknown to KSHV and ANG biology, this offers a starting point for further analysis of their roles in KSHV biology, which may lead to identification of potential therapeutic targets to control KSHV latency and associated malignancies.

c-Cbl-mediated selective virus-receptor translocations into lipid rafts regulate productive Kaposi's sarcoma-associated herpesvirus infection in endothelial cells.

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. Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3ß1, αVß3, and αVß5, 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 α3ß1, αVß3, and x-CT receptors but not αVß5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3ß1 and αVß3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVß5 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 MßCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3ß1, -αVß3, 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.

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-1ß 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-1ß. 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.

Kaposi's sarcoma-associated herpesvirus-induced angiogenin plays roles in latency via the phospholipase C gamma 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.

Phosphorylation and polyubiquitination of transforming growth factor beta-activated kinase 1 are necessary for activation of NF-kappaB by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) protein has been shown to induce several signaling pathways leading to the modulation of host gene expression. The hijacking of these pathways facilitates the viral life cycle and leads to tumorigenesis. In the present work, we show that transforming growth factor ß (TGF-ß)-activated kinase 1 (TAK1) is an important player in NF-κB activation induced by vGPCR. We observed that the expression of an inactive TAK1 kinase mutant (TAK1M) reduces vGPCR-induced NF-κB nuclear translocation and transcriptional activity. Consequently, the expression of several NF-κB target genes normally induced by vGPCR was blocked by TAK1M expression, including interleukin 8 (IL-8), Gro1, IκBα, COX-2, cIAP2, and Bcl2 genes. Similar results were obtained after downregulation of TAK1 by small interfering RNA (siRNA) technology. The expression of vGPCR recruited TAK1 to the plasma membrane, and vGPCR interacts with TAK1. vGPCR expression also induced TAK1 phosphorylation and lysine 63-linked polyubiquitination, the two markers of the kinase's activation. Finally, inhibition of TAK1 by celastrol inhibited vGPCR-induced NF-κB activation, indicating this natural compound could be used as a potential therapeutic drug against KSHV malignancies involving vGPCR.

Characterization of entry and infection of monocytic THP-1 cells by Kaposi's sarcoma associated herpesvirus (KSHV): role of heparan sulfate, DC-SIGN, integrins and signaling.

KSHV effectively binds, enters and establishes infection in THP-1 cells with initial concurrent expression of latent ORF73 and lytic ORF50 genes and subsequent persistence of ORF73. KSHV genome persisted for 30 days and lytic cycle could be activated. KSHV utilized heparan sulfate for binding to THP-1 cells and primary monocytes. Blocking DC-SIGN did not inhibit KSHV binding; however, virus entry in THP-1 cells and in primary monocytes was reduced. In addition to the previously identified integrins alpha3beta1, alphavbeta3 and alphavbeta5, integrin alpha5beta1 was also utilized for infection. KSHV entered THP-1 cells via clathrin and caveolin mediated endocytosis and did not utilize macropinocytosis as in human dermal endothelial cells, and required an endosomal acidification. Infection also induced phosphorylation of FAK, Src, PI3K, NF-kappaB and ERK1/2 signaling molecules, and entry was blocked by tyrosine kinase inhibitors. These findings suggest that THP-1 cells are highly useful model for studying KSHV infection of monocytes.

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.

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 sarcoma-associated herpes virus (KSHV) G protein-coupled receptor (vGPCR) activates the ORF50 lytic switch promoter: a potential positive feedback loop for sustained ORF50 gene expression.

KSHV vGPCR, a lytic cycle associated protein, induces several signaling pathways leading to the activation of various transcription factors and consequently the expression of cellular and viral genes. Though the role of vGPCR in KSHV tumorigenicity has been well studied, its function related to the viral life cycle is poorly understood. Reduction in vGPCR by RNA interference also resulted in the reduction in KSHV lytic switch ORF50 gene and protein expression. Induction of vGPCR by doxycycline in BC3.14 cells also resulted in more KSHV production. When this was explored, induction of the ORF50 promoter by vGPCR expression was observed. Further examination of the molecular mechanisms by which vGPCR regulates the ORF50 promoter, using various ORF50 promoter constructs, revealed that induction of ORF50 promoter by vGPCR did not involve AP1 but was dependent on Sp1 and Sp3 transcription factors. vGPCR signaling led to an increase in Sp1 and Sp3 DNA binding activity and a decrease in histone deacetylase (HDAC) activity. These activities were pertussis toxin independent, did not involve Rho and Rac-GTPases and involved the heterotrimeric G protein subunits Galpha12 and Galphaq. Studies using pharmacologic inhibitors and dominant-negative proteins identified phospholipase C, the novel protein kinase C (novel PKC) family and protein kinase D (PKD) as part of the signaling initiated by vGPCR leading to ORF50 promoter activation. Taken together, this study suggests a role for vGPCR in the sustained expression of ORF50 which could lead to a continued activation of lytic cycle genes and ultimately to successful viral progeny formation.

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 (C(35)H(58)O(11)), 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 [C(30)H(28)O(8)]) 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 upregulates angiogenin during infection of human dermal microvascular endothelial cells, which induces 45S rRNA synthesis, antiapoptosis, cell proliferation, migration, and angiogenesis.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is associated with the angioproliferative KS lesions characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth factors, and angiogenic factors. De novo KSHV infection of human microvascular dermal endothelial cells results in increased secretion of several growth factors, cytokines, chemokines, and angiogenic factors, and the multifunctional angiogenic protein angiogenin is one of them. KS tissue sections were positive for angiogenin, highlighting the importance of angiogenin in KS pathogenesis. Examination of KSHV-mediated angiogenin upregulation and secretion and potential outcomes revealed that during infection of primary endothelial cells, KSHV induced a time- and dose-dependent increase in angiogenin gene expression and protein secretion beginning as early as 8 h postinfection and lasting until the fifth day of our observation period. TIVE latently transformed cells (TIVE-LTC) latently infected with KSHV secreted high levels of angiogenin. Angiogenin was also detected in BCBL-1 cells (human B cells) carrying KSHV in a latent state. Significant induction of angiogenin was observed in cells expressing KSHV ORF73 (LANA-1; latent) and ORF74 (lytic) genes alone, and moderate induction was seen with the lytic KSHV ORF50 gene. Angiogenin bound to surface actin, internalized in a microtubule-independent manner, and translocated into the nucleus and nucleolus of infected cells. In addition, it increased 45S rRNA gene transcription, antiapoptosis, and proliferation of infected cells, thus demonstrating the multifunctional nature of KSHV-induced angiogenin. These activities were dependent on angiogenin nuclear translocation, which was inhibited by neomycin. Upregulation of angiogenin led to increased activation of urokinase plasminogen activator and generation of active plasmin, which facilitated the migration of endothelial cells toward chemoattractants, including angiogenin, and chemotaxis was prevented by the inhibition of angiogenin nuclear translocation. Treatment of KSHV-infected cell supernatants with antiangiogenin antibodies significantly inhibited endothelial tube formation, and inhibition of nuclear translocation of angiogenin also blocked the expression of KSHV-induced vascular endothelial growth factor C. Collectively, these results strongly suggest that by increasing infected endothelial cell 45S rRNA synthesis, proliferation, migration, and angiogenesis, KSHV-induced angiogenin could be playing a pivotal role in the pathogenesis of KSHV infection, including a contribution to the angioproliferative nature of KS lesions. Our studies suggested that LANA-1 and vGPCR play roles in KSHV-induced angiogenesis and that the angiogenic potential of vGPCR might also be due to its ability to induce angiogenin.

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and alpha3beta1 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 alpha3beta1 and alphaVbeta3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-alphaV and -beta1 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 alphaVbeta3 and alphaVbeta5 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 alpha3beta1, alphaVbeta3, and alphaVbeta5 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 alphaVbeta3 and alphaVbeta5 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 alphaVbeta5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas alpha3beta1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and alphaVbeta3-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 alphaVbeta5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with alpha3beta1 and KSHV. Preincubation of KSHV with soluble heparin and alpha3beta1 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 (alphaVbeta3, alpha3beta1, and alphaVbeta5) 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.

Kaposi's sarcoma-associated herpesvirus induces sustained levels of vascular endothelial growth factors A and C early during in vitro infection of human microvascular dermal endothelial cells: biological implications.

Kaposi's sarcoma (KS), a vascular tumor associated with human immunodeficiency virus type 1 infection, is characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth and angiogenic factors, and angiogenesis. KS spindle cells are believed to be of the lymphatic endothelial cell (LEC) type. Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) is etiologically linked to KS, and in vitro KSHV infection of primary human dermal microvascular endothelial cells (HMVEC-d) 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, sustained induction of NF-kappaB and several cytokines, and growth and angiogenic factors. KSHV induced robust vascular endothelial growth factor A (VEGF-A) and VEGF-C gene expression as early as 30 min postinfection (p.i.) in serum-starved HMVEC-d, which was sustained throughout the observation period of 72 h p.i. Significant amounts of VEGF-A and -C were also detected in the culture supernatant of infected cells. VEGF-A and -C were also induced by UV-inactivated KSHV and envelope glycoprotein gpK8.1A, thus suggesting a role for virus entry stages in the early induction of VEGF and requirement of KSHV viral gene expression for sustained induction. Exogenous addition of VEGF-A and -C increased KSHV DNA entry into target cells and moderately increased latent ORF73 and lytic ORF50 promoter activation and gene expression. KSHV infection also induced the expression of lymphatic markers Prox-1 and podoplanin as early as 8 h p.i., and a paracrine effect was seen in the neighboring uninfected cells. Similar observations were also made in the pure blood endothelial cell (BEC)-TIME cells, thus suggesting that commitment to the LEC phenotype is induced early during KSHV infection of blood endothelial cells. Treatment with VEGF-C alone also induced Prox-1 expression in the BEC-TIME cells. Collectively, these studies show that the in vitro microenvironments of KSHV-infected endothelial cells are enriched, with VEGF-A and -C molecules playing key roles in KSHV biology, such as increased infection and gene expression, as well as in angiogenesis and lymphangiogenesis, thus recapitulating the microenvironment of early KS lesions.