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-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.     

Targeting KSHV/HHV-8 latency with COX-2 selective inhibitor nimesulide: a potential chemotherapeutic modality for primary effusion lymphoma

The significance of inflammation in KSHV biology and tumorigenesis prompted us to examine the role of COX-2 in primary effusion lymphoma (PEL), an aggressive AIDS-linked KSHV-associated non-Hodgkin's lymphoma (NHL) using nimesulide, a well-known COX-2 specific NSAID. We demonstrate that (1) nimesulide is efficacious in inducing proliferation arrest in PEL (KSHV+/EBV-; BCBL-1 and BC-3, KSHV+/EBV+; JSC-1), EBV-infected (KSHV-/EBV+; Raji) and non-infected (KSHV-/EBV-; Akata, Loukes, Ramos, BJAB) high malignancy human Burkitt's lymphoma (BL) as well as KSHV-/EBV+ lymphoblastoid (LCL) cell lines; (2) nimesulide is selectively toxic to KSHV infected endothelial cells (TIVE-LTC) compared to TIVE and primary endothelial cells (HMVEC-d); (3) nimesulide reduced KSHV latent gene expression, disrupted p53-LANA-1 protein complexes, and activated the p53/p21 tumor-suppressor pathway; (4) COX-2 inhibition down-regulated cell survival kinases (p-Akt and p-GSK-3β), an angiogenic factor (VEGF-C), PEL defining genes (syndecan-1, aquaporin-3, and vitamin-D3 receptor) and cell cycle proteins such as cyclins E/A and cdc25C; (5) nimesulide induced sustained cell death and G1 arrest in BCBL-1 cells; (6) nimesulide substantially reduced the colony forming capacity of BCBL-1 cells. Overall, our studies provide a comprehensive molecular framework linking COX-2 with PEL pathogenesis and identify the chemotherapeutic potential of nimesulide in treating PEL.     

Kaposi's sarcoma-associated herpesvirus LANA-1 interacts with the short variant of BRD4 and releases cells from a BRD4- and BRD2/RING3-induced G1 cell cycle arrest

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA-1) is required for the replication of episomal viral genomes. Regions in its N-terminal and C-terminal domains mediate the interaction with host cell chromatin. Several cellular nuclear proteins, e.g., BRD2/RING3, histones H2A and H2B, MeCP2, DEK, and HP1alpha, have been suggested to mediate this interaction. In this work, we identify the double-bromodomain proteins BRD4 and BRD3/ORFX as additional LANA-1 interaction partners. The carboxy-terminal region of the short variant of BRD4 (BRD4S) containing the highly conserved extraterminal domain directly interacts with an element in the LANA-1 carboxy-terminal domain. We show that ectopically expressed BRD4S and BRD2/RING3 delay progression into the S phase of the cell cycle in epithelial and B-cell lines and increase cyclin E promoter activity. LANA-1 partly releases epithelial and B cells from a BRD4S- and BRD2/RING3-induced G1 cell cycle arrest and also promotes S-phase entry in the presence of BRD4S and BRD2/RING3. This is accompanied by a reduction in BRD4S-mediated cyclin E promoter activity. Our data are in keeping with the notion that the direct interaction of KSHV LANA-1 with BRD4 and other BRD proteins could play a role in the G1/S phase-promoting functions of KSHV LANA-1. Further, our data support a model in which the LANA-1 C terminus contributes to a functional attachment to acetylated histones H3 and H4 via BRD4 and BRD2, in addition to the recently demonstrated direct interaction (A. J. Barbera, J. V. Chodaparambil, B. Kelley-Clarke, V. Joukov, J. C. Walter, K. Luger, and K. M. Kaye, Science 311:856-861, 2006) of the LANA-1 N terminus with histones H2A and H2B.     

Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma and several other malignancies. The lack of an efficient infection system has impeded the understanding of KSHV-related pathogenesis. A genetic approach was used to isolate infectious KSHV. Recombinant bacteria artificial chromosome (BAC) KSHV containing hygromycin resistance and green fluorescent protein (GFP) markers was generated by homologous recombination in KSHV-infected BCBL-1 cells. Recombinant KSHV genomes from cell clones that were resistant to hygromycin, expressed GFP, and produced infectious virions after induction with tetradecanoyl phorbol acetate (TPA) were rescued in Escherichia coli and reconstituted in 293 cells. Several 293 cell lines resulting from infection with recombinant virions induced from a full-length recombinant KSHV genome, named BAC36, were obtained. BAC36 virions established stable latent infection in 293 cells, harboring 1 to 2 copies of viral genome per cell and expressing viral latent proteins, with approximately 0.5% of cells undergoing spontaneous lytic replication, which is reminiscent of KSHV infection in Kaposi's sarcoma tumors. TPA treatment induced BAC36-infected 293 cell lines into productive lytic replication, expressing lytic proteins and producing virions that efficiently infected normal 293 cells with a approximately 50% primary infection rate. BAC36 virions were also infectious to HeLa and E6E7-immortalized human endothelial cells. Since BAC36 can be efficiently shuttled between bacteria and mammalian cells, it is useful for KSHV genetic analysis. The feasibility of the system was illustrated through the generation of a KSHV mutant with the vIRF gene deleted. This cellular model is useful for the investigation of KSHV infection and pathogenesis.     

Effect of extremely low frequency electromagnetic fields (ELF-EMF) on Kaposi's sarcoma-associated herpes virus in BCBL-1 cells

Association between extremely low frequency electromagnetic fields (ELF-EMF) and human cancers is controversial, and few studies have been conducted on their influence on oncogenic viruses. We studied the effects of 1 mT, 50 Hz sine waves, applied for 24-72 h, on Kaposi's sarcoma (KS)-associated herpesvirus (KSHV or HHV-8) in BCBL-1, a latently infected primary effusion lymphoma (PEL) cell line. ELF-EMF exposure did not affect the growth and viability of BCBL-1 cells, either stimulated or not with TPA. The total amount of KSHV DNA detected in ELF-EMF exposed cultures not stimulated with TPA did not differ from that of the unexposed controls (P = ns). However, in the presence of TPA stimulation, total KSHV DNA content was found higher in ELF-EMF exposed than in control BCBL-1 cultures (P = .024) at 72 h exposure, but not earlier. Viral DNA increase significantly correlated with increased mean fluorescence intensity/cell for the lytic antigen gp K8.1A/B (P < .01), but not with percentage of gp K8.1A/B-positive cells or of cells containing virions. Viral progeny produced under ELF-EMF exposure consisted mainly of defective viral particles.     

Exploitation of the Complement System by Oncogenic Kaposi's Sarcoma-Associated Herpesvirus for Cell Survival and Persistent Infection

During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi''s sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi''s sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.     

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen reduces interleukin-8 expression in endothelial cells and impairs neutrophil chemotaxis by degrading nuclear p65

Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is the major viral latent protein and functions as a multifaceted protein. Here, we report that LANA-1 attenuates the endothelial response to tumor necrosis factor alpha (TNF-α) stimulation and inhibits consequent neutrophil chemotaxis. Reporter assays showed that LANA-1 constantly repressed nuclear factor (NF)-κB transactivity upon TNF-α stimulation. We also found that LANA-1 decreased nuclear p65 protein levels through enhancement of polyubiquitylation-mediated p65 degradation and that an elongin B/elongin C-cullin 5-LANA-1-p65 complex assembled by LANA-1 was responsible for this enhanced p65 degradation. In telomerase-immortalized human umbilical vein endothelial cells, LANA-1 was demonstrated to repress interleukin-8 expression, which was involved in neutrophil recruitment to the inflammatory site. Through an in vitro transmigration assay, we determined a suppressive effect of LANA-1 on neutrophil chemotaxis. Our work suggests that KSHV LANA-1 is a negative modulator of acute inflammation and sheds light on a new mechanism by which KSHV during the latent life cycle evades the host innate immune response.     

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.     

Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus interacts with origin recognition complexes at the LANA binding sequence within the terminal repeats

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.     

Surface downregulation of major histocompatibility complex class I,PE-CAM,and ICAM-1 following de novo infection of endothelial cells with Kaposi's sarcoma-associated herpesvirus

Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.