Selective switch between latency and lytic replication of Kaposi's sarcoma herpesvirus and Epstein-Barr virus in dually infected body cavity lymphoma cells.

The BC-1 cell line, derived from a body cavity-based, B-cell lymphoma, is dually infected with Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In these studies, the relationships between these two gammaherpesviruses and BC-1 cells were characterized and compared. Single-cell cloning experiments suggested that all BC-1 cells contain both genomes. In more than 98% of cells, both viruses were latent. The two viruses could be differentially induced into their lytic cycles by chemicals. EBV was activated into DNA replication and late-gene expression by the phorbol ester tetradecanoyl phorbol acetate (TPA). KSHV was induced into DNA replication and late-gene expression by n-butyrate. Amplification of both EBV and KSHV DNAs was inhibited by phosphonoacetic acid. Induction of the KSHV lytic cycle by n-butyrate was accompanied by the disappearance of host-cell beta-actin mRNA. Induction of EBV by TPA was not accompanied by such an effect on host-cell gene expression. Induction of the KSHV lytic cycle by n-butyrate was associated with the expression of several novel polypeptides. Recognition of one of these, p40, served as the basis of development of an assay for antibodies to KSHV in the sera of infected patients. BC-1 cells released infectious EBV; however, there was no evidence for the release of encapsidated KSHV genomes by BC-1 cells, even though n-butyrate-treated cells contained numerous intranuclear nucleocapsids. The differential inducibility of these two herpesviruses in the same cell line points to the importance of viral factors in the switch from latency to lytic cycle.     

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.     

Epstein-Barr virus recombinants from BC-1 and BC-2 can immortalize human primary B lymphocytes with different levels of efficiency and in the absence of coinfection by Kaposi's sarcoma-associated herpesvirus

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.     

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.     

Functional p53 signaling in Kaposi's sarcoma-associated herpesvirus lymphomas: implications for therapy

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.     

Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.     

Kaposi's sarcoma-associated herpesvirus open reading frame 50/Rta protein activates the entire viral lytic cycle in the HH-B2 primary effusion lymphoma cell line

Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.     

The Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Induced 5-Lipoxygenase-Leukotriene B4 Cascade Plays Key Roles in KSHV Latency,Monocyte Recruitment,and Lipogenesis

Kaposi''s sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi''s sarcoma (KS) and primary effusion lymphoma (PEL). KS lesions are characterized by endothelial cells with multiple copies of the latent KSHV episomal genome, lytic replication in a low percentage of infiltrating monocytes, and inflammatory cytokines plus growth factors. We demonstrated that KSHV utilizes inflammatory cyclooxygenase 2/prostaglandin E₂ to establish and maintain latency (Sharma-Walia, N., A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog 6:e1000777, 2010 [doi:10.1371/journal.ppat.1000777]). Here, we evaluated the role of 5-lipoxygenase (5LO) and its chemotactic metabolite leukotriene B4 (LTB4) in KSHV biology. Abundant staining of 5LO was detected in human KS tissue sections. We observed elevated levels of 5LO and high levels of secretion of LTB4 during primary KSHV infection of endothelial cells and in PEL B cells (BCBL-1 and BC-3 cells). Blocking the 5LO/LTB4 cascade inhibited viral latent ORF73, immunomodulatory K5, viral macrophage inflammatory protein 1 (MIP-1), and viral MIP-2 gene expression, without much effect on lytic switch ORF50, immediate early lytic K8, and viral interferon-regulatory factor 2 gene expression. 5LO inhibition significantly downregulated latent viral Cyclin and latency-associated nuclear antigen 2 levels in PEL cells. 5LO/LTB4 inhibition downregulated TH2-related cytokine secretion, elevated TH1-related cytokine secretion, and reduced human monocyte recruitment, adhesion, and transendothelial migration. 5LO/LTB4 inhibition reduced fatty acid synthase (FASN) promoter activity and its expression. Since FASN, a key enzyme required in lipogenesis, is important in KSHV latency, these findings collectively suggest that 5LO/LTB4 play important roles in KSHV biology and that effective inhibition of the 5LO/LTB4 pathway could potentially be used in treatment to control KS/PEL.     

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.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.     

Anti-HIV agent MAP30 modulates the expression profile of viral and cellular genes for proliferation and apoptosis in AIDS-related lymphoma cells infected with Kaposi's sarcoma-associated virus

The anti-HIV agent MAP30 (Momordica anti-HIV protein, 30 kDa) inhibits the proliferation of BC-2, an AIDS-related primary effusion lymphoma (PEL) cell line derived from an AIDS patient. BC-2 cells are latently infected with Kaposi's sarcoma-associated herpes virus (KSHV), also known as human herpes virus 8 (HHV8). We examined the effect of MAP30 on the expression of viral and cellular genes in BC-2 during latent and lytic states of the viral life cycle. By Northern analysis and RT-PCR, we found that MAP30 downregulates the expression of viral cyclin D (vCD), viral interleukin-6 (vIL-6), and viral FLIP (vFLIP), genes involved in cell cycle regulation, viral pathogenesis, and apoptosis. By pathway-specific cDNA microarray analysis, we found that BC-2 cells express high levels of egr-1, ATF-2, hsp27, hsp90, IkappaB, mdm2, skp1, and IL-2, cellular genes involved in mitogenesis, tumorigenesis, and inhibition of apoptosis in NFkappaB and p53 signaling pathways. These results define for the first time the specific cellular pathways involved in AIDS-related tumorigenesis and suggest specific novel targets for the treatment. Furthermore, we found that MAP30 downregulates the expression of egr-1, ATF-2, hsp27, hsp90, IkappaB, mdm2, and Skp1, while it upregulates the pro-apoptotic-related genes Bax, CRADD, and caspase-3. Thus, MAP30 modulates the expression of both viral and cellular genes involved in KS pathogenesis. These results provide valuable insight into the molecular mechanisms of MAP30 anti-KS action and suggest its utility as a therapeutic agent against AIDS-related tumors.     

KSHV-infected PEL cell lines exhibit a distinct gene expression profile

We analyzed the gene expression profiles of lymphocyte-originated tumor cell lines - primary effusion lymphoma (PEL) cell lines, T-cell leukemia (TCL) cell lines, Burkitt lymphoma (BL) cell lines - and two sets of normal peripheral blood mononuclear cells (PBMCs) - in order to determine characteristic gene expression profiles for each of the former three groups. And we found that these cell lines showed respective typical gene expression profiles and classified into clear four groups, PEL, TCL, BL, and normal PBMCs. Two B lymphocyte-originated tumor cell lines, PEL and BL cell lines, clearly exhibited distinct gene expression profiles, respectively. Even though there was only one line that was co-infected with both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), KSHV seemed to govern the gene expression profile of the co-infected line. These data suggested not only that established typical tumor cell lines show a distinct gene expression profile but also that this profile may be governed by certain viruses.